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Green Synthesis of Nanomaterials for Bioenergy Applications
Green Synthesis of Nanomaterials for Bioenergy
Applications

Edited by
Neha Srivastava
Department of Chemical Engineering and Technology, IIT (BHU),
Varanasi, Uttar Pradesh, India

Manish Srivastava
Department of Chemical Engineering and Technology, IIT (BHU),
Varanasi, Uttar Pradesh, India

P. K. Mishra
Department of Chemical Engineering and Technology, IIT (BHU),
Varanasi, Uttar Pradesh, India

Vijai Kumar Gupta

ERA Chair of Green Chemistry, Department of Chemistry and Biotechnology,
School of Science, TALLINN University of Technology, Tallinn, Estonia
This edition first published 2021
© 2021 John Wiley & Sons Ltd

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Library of Congress Cataloging-in-Publication Data

Names: Srivastava, Neha, 1981– editor.
Title: Green synthesis of nanomaterials for bioenergy applications / edited
by Neha Srivastava, Department of Chemical Engineering and Technology,
IIT (BHU) Varanasi, India, Manish Srivastava, Department of Chemical
Engineering and Technology, IIT (BHU) Varanasi, India, P.K. Mishra,
Department of Chemical Engineering and Technology, IIT (BHU) Varanasi,
India, Vijai Kumar Gupta, ERA Chair of Green Chemistry, Department of
Chemistry and Biotechnology School of Science, TALLINN University Of
Technology, Estonia.
Description: First edition. | Hoboken, NJ : Wiley-Blackwell, 2020. |
Includes bibliographical references and index.
Identifiers: LCCN 2020029279 (print) | LCCN 2020029280 (ebook) | ISBN
9781119576815 (cloth) | ISBN 9781119576808 (adobe pdf) | ISBN
9781119576792 (epub)
Subjects: LCSH: Biomass energy. | Green chemistry. | Organic
compounds–Synthesis–Technological innovations. | Nanostructured
materials–Synthesis. | Nanostructured materials–Industrial
applications.
Classification: LCC TP339 .G753 2020 (print) | LCC TP339 (ebook) | DDC
660.028/6–dc23
LC record available at https://lccn.loc.gov/2020029279
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Cover Design: Wiley
Cover Image: © Chokniti Khongchum/Shutterstock

Set in 9.5/12.5pt STIXTwoText by SPi Global, Pondicherry, India

10 9 8 7 6 5 4 3 2 1
 v

Contents

List of Contributors vii

Foreword xi
Acknowledgements xiii
About the Editors xv

1 Nanocatalysts and Biofuels: Applications and Future Challenges 1

Desikan Ramesh, Thangavelu Kiruthika, Balasubramaniam Prabha,
Maduraimuthu Djanaguiraman, and Subburamu Karthikeyan

2 Nanomaterials: Types, Synthesis, and Characterization 23

Zahra Vaseghi and Ali Nematollahzadeh

3 Recent Advances on Classification, Properties, Synthesis, and

Characterization of Nanomaterials 83
Veer Singh, Priyanka Yadav, and Vishal Mishra

4 Synthesis of Metallic and Metal Oxide Nanomaterials 99

Ayse Demirbas, Tuna Karaytuğ, Nihan Arabacı, Ebru Sebnem Yilmaz,
and Ismail Ocsoy

5 Analysis of Green Methods to Synthesize Nanomaterials 125

Pavlos Nikolaidis

6 Biosynthesis of Silver Nanoparticles from Acacia nilotica

(L.) Wild. Ex. Delile Leaf Extract 145
Karishma I. Sheikh and Kalpesh B. Ishnava

7 Nanomaterials for Enzyme Immobilization 165

Nihan Arabacı, Tuna Karaytuğ, Ayse Demirbas, Ismail Ocsoy,
and Ahmet Katı
vi Contents

8 Nanomaterial Biosynthesis and Enzyme Immobilization:

Methods and Applications 191
Indu, Ankush Yadav, Mrinal Kanti Mandal, and Kashyap Kumar Dubey

9 Carbon Nanotubes for Hydrogen Purification and Storage 211

Pietro Bartocci, Giovanni Russo, Haiping Yang, Song Hu,
Øyvind Skreiberg, Liang Wang, Fausto Gallucci, Gianni Bidini,
and Francesco Fantozzi

Index 239
 vii

List of Contributors

Nihan Arabacı Central University of Haryana

Department of Biology Mahendergarh
Faculty of Arts and Sciences Çukurova Haryana
University India
Adana
Turkey Francesco Fantozzi
Department of Engineering
Pietro Bartocci University of Perugia
Department of Engineering Perugia
University of Perugia Italy
Perugia
Italy Fausto Gallucci
Inorganic Membranes and Membrane
Gianni Bidini Reactors
Department of Engineering Department of Chemical Engineering and
University of Perugia Chemistry
Perugia Eindhoven University of Technology
Italy Eindhoven
the Netherlands
Ayse Demirbas
Faculty of Fisheries and Aquatic Sciences Song Hu
Recep Tayyip Erdogan University State Key Laboratory of Coal
Rize Combustion
Turkey Huazhong University of Science and
Technology
Maduraimuthu Djanaguiraman Wuhan
Department of Crop Physiology Hubei
Tamil Nadu Agricultural University China
Coimbatore China-EU Institute for Clean and
Tamil Nadu Renewable Energy
India Huazhong University of Science and
Technology
Kashyap Kumar Dubey Wuhan
Bioprocess Engineering Laboratory Hubei
Department of Biotechnology China
viii List of Contributors

Indu Agricultural Engineering College and

Bioprocess Engineering Laboratory Research Institute
Department of Biotechnology Tamil Nadu Agricultural University
Central University of Haryana Coimbatore
Mahendergarh Tamil Nadu
Haryana India
India
Mrinal Kanti Mandal
Kalpesh B. Ishnava Department of Chemical Engineering
Assistant professor NIT Durgapur
Ashok and Rita Patel Institute of Integrated Durgapur
Studies and Research in Biotechnology and West Bengal
Allied Sciences (ARIBAS) India
Anand
Gujarat Vishal Mishra
India School of Biochemical Engineering
IIT (BHU)
Tuna Karaytuğ Varanasi
Department of Biology Uttar Pradesh
Institute of Natural and Applied Sciences India
Çukurova University
Adana Ali Nematollahzadeh
Turkey Chemical Engineering Department
University of Mohaghegh Ardabili
Subburamu Karthikeyan Ardabil
Department of Renewable Energy Iran
Engineering
Agricultural Engineering College and Pavlos Nikolaidis
Research Institute Department of Electrical Engineering
Tamil Nadu Agricultural University Cyprus University of Technology
Coimbatore Limassol
Tamil Nadu Cyprus
India
Ismail Ocsoy
Ahmet Katı Department of Analytical Chemistry
Department of Detergent and Chemical Faculty of Pharmacy
Technologies Erciyes University
Hayat Kimya Research and Development Kayseri
Center Turkey
Kocaeli
Turkey Balasubramaniam Prabha
Department of Renewable Energy
Thangavelu Kiruthika Engineering
Department of Renewable Energy Agricultural Engineering College and
Engineering Research Institute
 List of Contributors ix

Tamil Nadu Agricultural University Liang Wang

Coimbatore SINTEF Energy Research
Tamil Nadu Trondheim
India Norway

Desikan Ramesh Priyanka Yadav

Horticultural College and Research School of Biochemical Engineering
Institute for Women IIT (BHU)
Tamil Nadu Agricultural University Varanasi
Tiruchirappalli Uttar Pradesh
Tamil Nadu India
India
Ankush Yadav
Giovanni Russo Bioprocess Engineering
Department of Engineering Laboratory
University of Perugia Department of Biotechnology
Perugia Central University of Haryana
Italy Mahendergarh
Haryana
Karishma I. Sheikh India
Ashok and Rita Patel Institute of Integrated
Studies and Research in Biotechnology and Haiping Yang
Allied Sciences (ARIBAS) State Key Laboratory of Coal
Anand Combustion
Gujarat Huazhong University of Science and
India Technology
Wuhan
Veer Singh Hubei
School of Biochemical Engineering China
IIT (BHU) China-EU Institute for Clean and
Varanasi Renewable Energy
Uttar Pradesh Huazhong University of Science and
India Technology
Wuhan
Øyvind Skreiberg Hubei
SINTEF Energy Research China
Trondheim
Norway Ebru Sebnem Yilmaz
Department of Biology
Zahra Vaseghi Faculty of Arts and Science
Chemical Engineering Department Hatay Mustafa Kemal University
University of Mohaghegh Ardabili Antakya
Ardabil Hatay
Iran Turkey
 xi

­Foreword

Bioenergy is a potential option to replace fossil fuels effectively and in a sustainable

manner. Various known bioenergy options such as biohydrogen, biogas, biomethane,
bioethanol, biomethanol, biobutenol, algal biofuels, and biodiesel are supposed to be
very promising alternative renewable energy options for eliminating severe environ-
mental issues. Significant efforts have been made to explore various bioenergy options
and related technologies in practice. However, its commercial viability and symmetri-
cal distribution are still a long way from practical implementation of bioenergy tech-
nologies. This book series explores the use of nanotechnology, which is grabbing the
attention of the biofuels sector by playing the role of enhancer, to improve bioenergy
production technology. Application of nanotechnology is emerging as new area for bio-
energy production through its contribution as catalyst, enzyme, and microbial immo-
bilizer. Nanomaterials have enormous potential for commercial markets and the
industrial market is expected to grow and become more flexible in coming decades.
Therefore, with an accelerating demand for viable and sustainable economic bioenergy
production, the potential combination of bioenergy and nanotechnology area must be
explored.
Green Synthesis of Nanomaterials for Bioenergy Applications is much needed contribution
to this series and I am happy to write this positive and satisfactory message. The book con-
tains nine chapters covering green synthesis and characterization of nanomaterials for
cost-effective bioenergy applications. The current world scenario of bioenergy and applica-
tion of nanotechnology in bioenergy production, different immobilization methods for
enhancing bioenergy production, synthesis, and mechanism of nanomaterial for economic
bioenergy production with green approach are presented and discussed in detail. The book
presents a new horizon of advancement and sustainable solutions for the improvement of
bioenergy production in the form of nanotechnology. These chapters suggest that the appli-
cation of nanotechnology will play a major role in bioenergy production and they will serve
as gems for those working in the relevant fields including scientists, researchers, teachers,
and students.
I am taking the opportunity to congratulate Dr. Neha Srivastava [IIT (BHU) Varanasi], Dr.
Manish Srivastava [IIT (BHU) Varanasi]), Prof. (Dr.) P.K. Mishra [IIT (BHU) Varanasi], and
Dr. Viaji Kumar Gupta for their significant efforts in bringing about this publication in order
to fulfill the needs of scientists, teachers, researchers, and students. My congratulations to
xii ­Forewor

all the editors for their contribution, devotion, and dedication in this endeavor. All the
authors and editors of this book deserve sincere appreciation for their commendable
achievements.

Date: 10.06.2019 Dr. Anthonia O’Donovan

Applied Biology and Biopharmaceutical Science,
School of Science and Computing,
Galway-Mayo Institute of Technology,
Galway, Ireland
 xiii

­Acknowledgements

The editors are thankful to all the academicians, scientists and researchers whose contributions
have supplemented this book presentation effectively. We are also thankful to our parents and
loved once whose blessings & constant support pumped academic activities deeply. It is natural
that some mistakes might have tiptoed in text involuntarily and for these we owe responsibility.
Moreover, we are very grateful to all contributors for their contribution in present book. We are
also thankful to Wiley for giving this opportunity to editors and Department of Chemical
Engineering & Technology, IIT (BHU) Varanasi, U.P., India for all technical support. We thank
them from the core of our heart. Editor Manish Srivastava acknowledges the Science and
Engineering Research Board for SERB-Research Scientist Award-2019 and also to DST, Govt of
India for the DST-INSPIRE Faculty Award [IFA13-MS-02] 2014.
 xv

About the Editors

Dr. Neha Srivastava

Institute Post-Doctral Fellow, Department of Chemical Engineering and Technology

IIT (BHU) Varanasi, Varanasi-221 005, U.P., India
Mobile no. +91-9 988 062 681, Email: [email protected]
Field of Expertise: Biofuels production, microbial bioprocessing, and enzyme technologies

Neha Srivastava is currently working as a post-doctorate fellow in the Department of

Chemical Engineering and Technology, IIT (BHU) Varanasi, India. She has published 26
research articles in peer-reviewed journals and has filed three patents. She completed her
PhD from Department of Molecular and Cellular Engineering, SHIATS, India in 2016 in
the area of bioenergy. Neha has been received six Young Scientist Awards. Presently, she is
working on biofuels production (cellulase enzymes; production and enhancement; biohy-
drogen production from waste biomass; bioethanol production).
xvi About the Editors

Dr. Manish Srivastava

SERB-Research Scientist
Department of Chemical Engineering and Technology
IIT (BHU) Varanasi, Varanasi-221 005, U.P., India
E-mail: [email protected], [email protected]
Contact no: + 91-7 503 757 601
Field of Expertise: Synthesis of nanomaterials and their application as catalysts for
­development of electrode materials in energy storage, biosensors, and biofuels
production.

Manish Srivastava has worked as DST INSPIRE faculty in the Department of Physics
and Astrophysics, University of Delhi, India during June 2014 to June 2019. Currently
he is working as SERB-Research Scientist in the Department of Chemical Engineering
and Technology IIT (BHU), Varanasi, India. He has published 46 research articles in
peer-reviewed journals, authored several book chapters, and filed one patent. He
worked as a post doctorate fellow in the Department of BIN Fusion Technology,
Chonbuk National University from August 2012 to August 2013. He was an Assistant
Professor in the Department of Physics, DIT School of Engineering, Greater Noida,
from July 2011 to July 2012. He received his PhD in Physics from the Motilal Nehru
National Institute of Technology, Allahabad, India in 2011. Presently, he is working on
the synthesis of graphene-based metal oxide hybrids and their applications as catalysts.
His area of interest is synthesis of nanostsructured materials and their applications as
catalysts for development of electrode materials in energy storage, biosensors, and
­biofuels production.
 About the Editors xvii

Professor P.K. Mishra

Department of Chemical Engineering and Technology

IIT (BHU) Varanasi, Varanasi-221 005, India
Mobile no. 9415301462
Email: [email protected]
Field of Expertise: Biofuels production, microbial bioprocessing, and enzyme technologies.

P. K. Mishra is currently Professor and Head in the Department of Chemical Engineering &
Technology, Indian Institute of Technology (BHU), Varanasi, India. He obtained his PhD in
Chemical Engineering from Institute of Technology, Banaras Hindu University in 1995. He has
authored/co-authored over 60 technical papers published in reputed national/international
journals and supervised more than 20 doctoral students. He has received several awards and
honors and has five patents. He is a Fellow of the Institution of Engineers India. He has received
several awards and honors at national/international levels. He has also made significant
­contribution toward development of entrepreneurship ecosystem in the eastern part of the
country. He is coordinator for Technology Business Incubator at the Institute and member
Executive committee NISBUD, Ministry of Skill Development, Government of India.
xviii About the Editors

Dr. Vijai Kumar Gupta

ERA Chair of Green Chemistry

Department of Chemistry and Biotechnology
School of Science,
Tallinn University of Technology
Akadeemia tee 15, 12 618 Tallinn, Estonia
Phone +372 620 2833
Mobile +372 5671 1014
Email: [email protected]; [email protected]

Dr. Vijai Kumar Gupta, ERA Chair of Green Chemistry, Department of Chemistry and
Biotechnology, School of Science, Tallinn University of Technology, Tallinn, Estonia, is one
of the leading experts in the area of microbial biology and biotechnology. He is a member
of the International Sub-commission on Trichoderma and Hypocrea, Austria; International
Society for Fungal Conservation, UK; and Secretary of European Mycological Association.
Dr. Gupta is a Fellow of the prestigious Linnaean Society, London, UK; Fellow of Indian
Mycological Association; and Fellow of Mycological Society of India. He has been honored
with several awards in his career including Indian Young Scientist Award for his advanced
research achievements in the field of fungal biology and biotechnology. He is the editor of
a number of leading scientific journals of high repute and has many publications, with
h-index 21. He has edited many books for publishers of international renown such as CRC
Press, Taylor & Francis, USA; Springer, USA; Elsevier Press, The Netherlands; Nova Science
Publisher, USA; DE Gruyter, Germany; and CABI, UK.
 1

Nanocatalysts and Biofuels

Applications and Future Challenges
Desikan Ramesh1, Thangavelu Kiruthika2, Balasubramaniam Prabha2,
Maduraimuthu Djanaguiraman3, and Subburamu Karthikeyan2
1
Horticultural College and Research Institute for Women, Tamil Nadu Agricultural University, Tiruchirappalli, Tamil Nadu, India
2
Department of Renewable Energy Engineering, Agricultural Engineering College and Research Institute, Tamil Nadu
Agricultural University, Coimbatore, Tamil Nadu, India
3
Department of Crop Physiology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India

1.1 ­Introduction

The economy of the developing countries is entirely based on fossil fuels and variation in the
price of fossil fuels. On the one hand, the demand for and consumption of fossil fuels are
increasing every year because of an increase in population, rapid growth of the automobile
sector, and industrialization. Energy consumption, economic growth, and population are
interlinked. A recent estimate shows that crude oil, gas, and coal resources will be exhausted
in the next five decades if production continues at current resource extraction rates (Behera
and Varma 2019). On the other hand, increasing fuel demand, fluctuating fuel prices, uncon-
trolled population growth, global warming, and ill effects of environmental pollution will
force us to search for an alternate ecofriendly fuel to fossil fuels. Among the renewable
energy sources, biomass sources—namely plants, oils, and fats—are considered as feedstock
to produce a variety of biofuels as future resources (Martini and Schell 2012).
Biomass feedstocks include all types of residues from the agricultural field and process-
ing operations, wood processing industry wastes, forestry residues and branches, lignocel-
lulosic feedstocks, organic fraction of municipal solid waste, and animal wastes, etc. The
estimated annual global biomass production is 104.9 billion metric tons of carbon (Field
et al. 1998). The annual photosynthesis yield in the world is ca. 720 billion tons of organic
raw cellulose materials (Tong 2019) that have potential for conversion to biofuels.
Generally, biomass resources are playing an influential role in supplying food or fuel.
Originally, the raw biomass materials were used for the production of heat and other energy
requirements, which can make an essential contribution to satisfying the energy needs of
society (Ruiz-Altisent 1994). Recently, biofuels production from biomass feedstocks is get-
ting more attraction in developed/developing countries. The reasons for this interest are due
to the reduction of foreign currency/crude oil imports, reduced dependence on crude oil,

Green Synthesis of Nanomaterials for Bioenergy Applications, First Edition. Edited by Neha Srivastava,
Manish Srivastava, P. K. Mishra and Vijai Kumar Gupta.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
2 1 Nanocatalysts and Biofuels

emissions from burning of fossil fuels, and their impact on the environment, i.e. air ­pollution
as well as global warming, etc. To overcome the abovementioned environmental issues,
­biofuels can be promoted to replace conventional commercial diesel and petrol fuels in the
transport sector. There are several biofuel technology pathways of production from various
biomass feedstocks. To mitigate the greenhouse gas emissions, we have to start avoiding fos-
sil fuels and/or promote the use of biofuels. Today, the biofuel industries are facing several
challenges: specifically, poor supply chain and logistics, more expensive raw materials,
higher costs for processing and production compared to petrofuels, low efficiency of the
conversion process, and lack of supporting biofuel policies for promotions. Researchers are
focused on improving the conversion efficiency of different biomass conversion methods,
which can indirectly reduce the process cost and biofuel price. In conclusion, the economi-
cally viable biomass conversion technologies will reach commercial scales.
Recently, nanotechnology has been attempted to improve the overall performance of
­different biomass conversion systems, which, although in the research stage, have the
potential to address the problems currently faced by the biofuel industries. In this chapter,
the current research on application of nanocatalysts in the field of biofuels production is
presented and their impact on product yield is also discussed.

1.2 ­Biofuels Production

Biofuel is a solid or liquid or gaseous fuel that can be generated from biomass feedstocks,
which can replace (partially or wholly) conventional petrofuels. The biofuel production
from feedstocks may be produced through biomass conversion methods. The biofuels can
be produced in the form of liquid or gaseous or solid (Figure 1.1).
The kind of biofuel mainly depends on the process conditions used in the technology and
nature of feedstock materials. The biofuel production technologies for biomass feedstocks
have reached the fourth generation, depending on conversion methods and feedstocks
used. The first generation deals with the production of biofuels using food crops, and tech-
nologies under this category are commercialized for biodiesel and bioethanol production.
Feedstocks used for this generation include various carbohydrate and lipid sources for
bioethanol and biodiesel production. The second generation deals with non-food crops for
biofuel production. This generation’s target is to produce bioethanol from all types of ligno-
cellulosic feedstocks. The third generation focusses on production of biofuels (biodiesel/
bioethanol) from microalgae. The fourth generation aims to produce biodiesel/bioethanol
from genetically modified crops or microbial lipids. Among them, only the first generation
for biofuel production from food crops is commercialized. Other generation technologies
are still at the research and development stage.
Generally, there are three major biofuel production routes: thermochemical, biochemical,
and chemical conversion methods. The thermochemical conversion technologies (TCCTs)
deal with the conversion of feedstocks into biofuels using heat with or without air/oxygen,
whereas biochemical conversion technologies (BCCTs) use microorganisms under aerobic
or anaerobic conditions. The comparison of the BCCT and TCCTs on biofuels is presented
in Table 1.1. The chemical conversion technologies (CCTs) are used to produce biodiesel
from vegetable oil feedstocks. Biofuels are facing difficulty in selling at a commercial level;
conventional fossil fuels are of higher calorific value and cheaper than biofuels. It is very
 1.2 ­Biofuels Productio 3

Biofuels

Solid biofuels Liquid biofuels Gaseous biofuels

Fresh form Processed form Biooil Biodiesel Bioethanol

Biomass briquettes Charcoal

Fuelwood/biomass

Biohydrogen Biogas Producer gas

Hydrogen Syngas Biomethane

Figure 1.1 Types of biofuels production from various biomass feedstocks via different biomass
energy conversion methods.

Table 1.1 Comparison of BCCTs and TCCTs for biofuel production.

S. No. Parameters BCCTs TCCTs

1 Mode of action Microorganisms Heat

2 Maximum reaction temperature < 60 °C Up to 1200 °C
3 Products from biomass
a. Solid fuels Not possible Possible (e.g. charcoal)
b. Liquid fuels Possible (e.g. Possible (e.g. biooil,
bioethanol) biomethanol)
c. Gaseous fuels Possible (e.g. biogas) Possible (e.g. syngas)
4 Suitable technology available for Anaerobic digestion Hydrothermal process
feedstocks with higher moisture content
5 End products like multiple products Acetone-Butanol- Biocrude
Ethanol (ABE)
6 Chemicals production from biomass Possible Possible
feedstocks
7 Secondary products as soil amendments Biodigested slurry Biochar
to maintain health
8 Reaction time h to days sec. to days
4 1 Nanocatalysts and Biofuels

challenging to enhance the calorific value of biofuels and make them on par with fossil
fuels. This may result in an increase of the production cost, which is a major challenge for
scaling-up to a commercial level. Hence, the production costs should be brought down
through technological breakthrough or government policies that provide support in the
form of incentives and tax benefits to promote biofuels and protect the environment.

1.3 ­Role of Catalysts in Biomass Conversion

The biomass composition is one determining factor that prescribes the biofuels and bio-
chemicals that can be produced from the biomass feedstocks via TCCTs, BCCTs, or CCTs.
The yield of end products is varied when it comes to biomass types and reaction conditions
used. The process conditions are determined by the catalyst types and quantity, reaction
temperature, reaction pressure, reaction time, biomass compositions, and its properties.
The catalyst has a significant influence in speeding up the reactions in the process and
thus, the product yield. The catalysts are classified into four categories viz., homogeneous,
heterogeneous, biocatalyst, and hetero-homogenized types (Philippot and Serp 2013). The
strength and weakness of homogeneous and heterogeneous catalysts used in the chemical
reactions are presented in Table 1.2. The catalyst selection for the biomass conversion

Table 1.2 Comparison of homogeneous and heterogeneous catalysts (Miessler and Spessard
1991; Farnetti et al. 2009; Chen 2014).

S. No Parameters Homogeneous Heterogeneous

1 Nature of catalyst The reaction occurs between This catalysis uses the
the same type of catalyst and different type of catalyst
reactants to that of the reactants
2 Examples Soluble organometallic or Bulk metal or metal on
coordination compounds a solid support
3 The type of catalyst used Usually liquid Mostly solid
4 Stability and degrading Low Comparatively high
nature
Thermal stability Poor Good
5 Reaction mechanisms Easy to understand Unknown and difficult
to understand
6 Separation of catalyst from Difficult Easy
end products
7 Applicability Limited Wide
8 Selectivity High Low
9 Active site Well-defined Poorly designed
10 Reutilization of catalyst Difficult and costly Simple and cheap
11 Neutralization More amount of water required Less in this case
12 Continuous processes Limited Possible
13 Corrosion More Less
 1.4 ­Application of Nanocatalyst 5

­ rocess is based on different parameters such as low cost, high reactivity, efficiency and
p
ecofriendliness, and reusability (Liu 2005). Limitations of heterogeneous catalysts used for
biomass conversion are long reaction rates and low efficiency due to poor mass transfer or
diffusion between the heterogeneous catalyst and reactants (Klaewkla et al. 2011).

1.4 ­Application of Nanocatalysts
The biomass conversion technologies are subjected to frequent changes with updating of
latest conversion technologies in this field. Nanotechnology is one of new emerging sci-
ences, which has application in different fields—namely biomedical applications, optic
and electronic, sorbents, sensors, and catalysis—due to its merits over conventional cata-
lytic conversion technologies (Ali Sinag 2018). Through nanotechnology nanocatalysts
were developed by combining characteristics such as higher catalytic activities and easy
recovery for homogeneous and heterogeneous catalysts respectively, which also has the
higher specific surface area (Zuliani et al. 2018). Nanocatalysts can be made from low-cost
metals, which must fulfill important properties such as high metal dispersion and stability
(Chen et al. 2015). The properties of nanoscale materials can exhibit different from that of
macroscale materials, and this offers unique applications for nanomaterials (Chaturvedi
et al. 2012). The nanosized materials as nanocatalysts can be used directly or as solids sup-
ported with nanoparticles (Tong 2019). The properties of nanoparticles may be modified
according to the requirements of varying conditions of the nanoparticle synthesis process
(Pélisson et al. 2012; Akia et al. 2014). The usage of nanocatalysts can minimize the mass
transfer resistance due to its large surface to volume ratios (Zuliani et al. 2018). Nanocatalysts
also have a more comprehensive scope in the area of biomass conversion technologies for
biofuel production from different biomass feedstocks.

1.4.1 Biomass Pretreatment

Lignocellulosic biomass feedstocks (LCB) are one of the possible candidates for promoting
the bioeconomy for sustainable development. Pretreatment is one of the most crucial pro-
cesses involved for turning LCB into liquid biofuels via the biochemical route. Downstream
process selection is mainly based on biomass pretreatment used and by-products produced.
The major obstacles in LCB-based liquid biofuels by larger scale units are costly, energy
intensive, and complex processes involved in the pretreatment method. The barriers of
first-generation biofuels can be partially overcome by effective utilization of LCB, which
are inexpensive and readily available as waste. Cellulosic ethanol is much more cost-effec-
tive and has a higher net energy ratio than that of grain ethanol. Release of fermentable
sugars from LCB for further processing remains challenging due to complex binding
between lignin, cellulose, and hemicellulose compounds, which are closely linked with
each other.
In comparison with sugar and starch crops, the lignin acts as a shield to protect carbohy-
drates in LCB and prevent the enzymatic hydrolysis and releasing fermentable sugars. Several
biomass pretreatment methods such as physical, chemical, biochemical, or combined
approaches have been tried for different LCB materials and are currently in the research and
development stage. Pretreatment can be a costly process in LCB into biofuels conversion; it
6 1 Nanocatalysts and Biofuels

holds significant potential for efficiency improvement through advanced technologies.

To achieve economic and environmental sustainability, the ideal pretreatment process should
handle high solid loadings with minimal use of chemicals and energy. Existing catalysts have
a number of problems such as inhibitors production, higher catalyst cost (enzyme), degrada-
tion of sugars, corrosion, low conversion efficiency, and biomass loading rates.
Recent studies have shown that the nanoparticles are performing better than conven-
tional catalysts used in the biomass pretreatment (Pan et al. 2012; Duque and Eugenia
2013; Koo et al. 2017). Silica-coated magnetic nanoparticles (SiM NPs) with perfluoroalkyl
sulfonic/alkylsulfonic were used to pretreat wheat straw. Ten percent of wheat straw hemi-
cellulose was solubilized by nanoparticles; higher than that of the control (Duque and
Eugenia 2013). Pan et al. (2012) reported that the titanium dioxide nanotube/leadoxide
electrode performed better at treating kraft lignin due to its higher oxidative and increased
surface area available for the reactions. Magnetite nanoparticles (Fe3O4 NPs) are also used
to convert LCB to sugar. The enzymatic digestibility was enhanced by 177% and 87% for
reed stem and paddy straw, respectively, under optimal conditions of H2O2 and Fe3O4 NPs.
Advantages of the method are that NPs can be quickly recovered and recycled (Koo et al.
2017). Paramagnetic-based ­nanocatalysts are an attractive choice for depolymerization of
cellulose into glucose monomer due to simplified catalyst separation using magnetic field
(Guo et al. 2012; Lee et al. 2014).

1.4.2 Biochemical Conversion Route

In the case of biochemical conversion method, the microorganism of specific species or a con-
sortium of microorganisms is used to convert the raw materials into biofuels. The two main
methods falling under this category are anaerobic digestion and fermentation process. In the
case of anaerobic digestion, the organic matter present in the biomass can be utilized by micro-
organisms to yield the biogas and biodigested slurry. The bioethanol can be produced by the
fermentation process using different feedstocks. Bioethanol can then be added to gasoline to
run a petrol engine. The problems associated with existing biochemical methods are higher
production cost and low yield per raw materials used, i.e. low conversion efficiency. In the case
of anaerobic digestion, the conversion rate for organic matter into biogas ranged from 30% to
40% (Faisal et al. 2019). This indicates maximum efficiency of conversion of biomass through
present anaerobic digestion without catalyst, which can be further improved by adopting
advanced technologies with suitable nanocatalysts. To increase the performance and yield of
existing practices, the nanocatalysts can be introduced in the BCCTs.

1.4.2.1 Anaerobic Digestion

The moisture content of raw materials plays a significant role in the selection of appropri-
ate biofuel production technology. In the case of biomass feedstocks with higher moisture
content, anaerobic digestion is a preferable method than TCCTs. The biogas can be used for
lighting, cooking, engine fuel, and electricity generation. The calorific value of the biogas
depends on methane content (average CH4:60%) and other impurities in the biogas, which
can be improved by the removal of these impurities. The biogas with more than 90% meth-
ane content is called as biomethane. The details of nanocatalysts’ applications to anaerobic
digestion of different feedstocks for enhancing biogas and methane productions are shown
in Table 1.3. In a recent study, it was observed that use of nanoparticles in the anaerobic
 1.4 ­Application of Nanocatalyst 7

Table 1.3 Applications of nanocatalysts to enhance biogas production.

Biomass Targeted
S. No. feedstock used Nanocatalyst used biofuel Yield Reference

1 Mixed liquor Fe nanocatalyst Biogas 0.345 (l/g VS Thiruselvi

volatile reduction) et al. (2018)
suspended
solids
2 Cattle manure Nanostructured Biogas 499 ml/g TS Li et al. (2018)
SiC
3 Rice straw Fe3O4 Methane 129% Khalid et al.
nanoparticle (2019)
4 Slaughterhouse Biosynthesized Methane 45% Yazdani et al.
wastewater iron NPs (2019)
5 Poultry litter 12 mg/l Ni NPs Methane 368 ml/g VS Hassanein
et al. (2019)
6 Waste-activated Fe0 Methane 217.16 ml/g Wang et al.
sludge VSS (2016)
7 Waste-activated Fe2O3 Methane 217.16 ml/g Wang et al.
sludge VSS (2016)
8 Waste-activated Fe0 Methane 70.6% Su et al.
sludge (2013)
9 Sewage sludge Ni (100 nm, Methane Increased Tsapekos
5–10 mg/kgVS) up to 10% et al. (2018)
10 Domestic Zero valent iron Methane 88% Amen et al.
sludge (2017)
11 Raw manure 2 mg/l Ni NPs Biogas, 614.5 ml/g Abdelsalam
(feces and Methane VS, et al. (2017a)
urine) 361.6 ml/g
VS
12 Manure 20 mg/l Fe3O4 Biogas, 584 ml/g VS, Abdelsalam
magnetic NPs Methane 351.8 ml/g et al. (2017b)
VS
13 Cattle dung Ni NPs Biogas, 1190.8 ml, Abdelsalam
slurry Methane 707.1 ml et al. (2016)
14 Cattle dung Co NPs (1 mg/l) Biogas, 1142.1 ml, Abdelsalam
slurry Methane 653.1 ml et al. (2016)
15 Cattle dung Fe NPs (20 mg/l) Biogas, 985.2 ml, Abdelsalam
slurry Methane 545.1 ml et al. (2016)
16 Cattle dung 20 mg/l Fe3O4 Biogas, 1154 ml, Abdelsalam
slurry NPs Methane 703.3 ml et al. (2016)
17 Dairy cattle 500 mg/l TiO2 Biogas, 336.25, Farghali et al.
manure NPs Methane 192.31 ml/ (2019)
gVS
18 Wastewater Fe3O4 (7 nm, Biogas, 180%, 234% Casals et al.
sludge 100 ppm) methane (2014)
19 Microalgae Fe3O4 NPs, Ni Biogas, 624 ml, Zaidi et al.
Enteromorpha NPs Biohydrogen 51.42% (v/v) (2018)
8 1 Nanocatalysts and Biofuels

digestion had increased the biogas production from 40% to 200% compared to the control
(UAB 2014; Faisal et al. 2019).

1.4.3 Thermochemical Conversion Methods

Thermochemical conversions of biomass are a convenient way to produce biofuels. TCCTs
employ heat to degrade the organic matter with/without air or oxygen to produce biofuels.
TCCTs include combustion, gasification, liquefaction, and carbonization (Mattucci et al.
1989). These technologies can be used to produce gaseous or solid or liquid biofuels. The
classification of these three methods is based on the amount of oxygen or air utilized in the
thermochemical reaction. The main goals for catalyst usage in TCCTs are to intensify the
reaction, increase the yield of the product, and reduce reaction temperature (Lv et al. 2007;
Di Felice et al. 2009; Xie et al. 2012). The nanocatalysts used in TCCTs can improve the
quantity and quality of the biofuels.

1.4.3.1 Gasification
The producer gas is produced by thermal degradation of biomass in a gasifier unit, and this
process uses a limited amount of oxygen or air for partial combustion. The gasification pro-
cess converts the majority of the bound chemical energy of the solid fuel into gas to yield
producer gas or syngas. The producer gas can be used as fuel in the combustors or dual fuel
engine to produce heat or power. The purpose of catalyst used in the biomass gasification is
to increase the syngas production, hydrogen content, tar reduction, conversion efficiency,
and gas quality. Also, the catalyst affects reaction conditions, and overall energy efficiency
was increased by lowering reaction temperature and time than non-catalytic gasification pro-
cess (Gao et al. 2017). The catalyst must have excellent properties like efficient intar removal,
resistance to deactivation, and quick regeneration and also produce hydrogen or syngas
(Wilcoxon 2012; Akia et al. 2014). The biomass tars that are formed during the gasification
process can be removed from the gasifier unit in two ways. In the first method, the char can
be further cracked with the help of a catalyst to enhance the gas yield. The second method
deals with cleaning producer gas outside of the gasifier unit by removing tar and other impu-
rities. Two kinds of catalysts used for tar conversion are mineral (e.g. calcined rocks, olivine,
clay minerals, and ferrous metal oxides) and synthetic (e.g. activated alumina, alkali metal
carbonates) catalysts (Sutton et al. 2001; Akia et al. 2014). Even though the catalysts are oper-
ative in tar removal and increased conversion efficiency, quick deactivation of catalysts due
to sintering effect and coke formation is the main limiting factor for large-scale applications
of catalytic gasification process (Palma 2013; Shen and Yoshikawa 2013; Chan and Tanksale
2014). Alternatively, the tar containing water used for cleaning gasifier plants can be remedi-
ated using NPs or microbial catalysts. The abovementioned problems associated with con-
ventional catalysts can be solved by introducing nanocatalyst in the gasification process.

1.4.3.2 Pyrolysis
Pyrolysis involves the thermal decomposition of the biomass in the absence of air/oxygen
to yield useful end products. Slow and fast pyrolysis (or hydrothermal process) of biomass
can yield the charcoal/biochar and biooil respectively. In the case of the biomass liquefac-
tion process, the primary role of the catalyst application is to enhance the biooil yield and
 1.4 ­Application of Nanocatalyst 9

its quality (Tong 2019). Homogenous catalysts (NaHCO3, Na2CO3, Ca(OH)2, KOH, Ba(OH)2,
FeSO4) and heterogeneous catalysts (Pd/C, Pt/C, Ni/SiO2-Al2O3, zeolite, Fe, Ru/C, CoMo/γ-
Al2O3,) are used in biomass liquefaction (Duan and Savage 2010; Tong 2019). Nanocatalyst
has limited applications in the case of charcoal making, whereas it has broad applications
for biooil production and upgradation. The maximum amount of biooil yield was achieved
as 35.5 wt% under fast pyrolysis without a catalyst. Use of the nanocatalysts (MCM-22 and
ITQ-2) would result in a decrease of the biooil yield due to catalytic cracking of biooil vapor
in catalyst pores (Uemura et al. 2017). The properties of biooil are higher water content,
low calorific value, corrosive nature, and chemical instability, which can be mitigated
through complete removal of oxygen from biooil (Uemura et al. 2017). The different types
of nanocatalysts used in TCCTs for biomass conversion are presented in Table 1.4.

1.4.4 Biodiesel
Generally, the liquid biofuels are free from nitrogen and sulfur, which contains oxygen and
supports for complete burning (Mattucci et al. 1989). Biodiesel falls under the liquid biofu-
els category, which is derived from animal fat/raw or used fried oils. Generally, oil and
alcohols are reacted in the transesterification process to yield two products such as bio-
diesel and glycerol. The biodiesel may be in the form of fatty acid methyl esters (FAME), or
fatty acid ethyl esters (FAEE) produced from plant or animal fat oils by using methanol or
ethanol in the reaction. The biodiesel production can be produced with or without a cata-
lyst to speed up the reaction. The catalyst used may be alkaline, acid, or biocatalyst.
Generally, a homogeneous catalyst is used for the transesterification process (Dias et al.
2008; Helwani et al. 2009). Major drawbacks of homogeneous catalysts are problems in
products separation, uses huge amounts of water for neutralization and environmental
pollution due to wastewater disposal (Vicente et al. 2004; Meher et al. 2006; Sharma et al.
2008). The heterogeneous catalysts offer solutions to problems associated with homogene-
ous catalysts in biodiesel production (Granados et al. 2007; Martyanov and Sayari 2008). If
the heterogeneous catalyst is used, the production can be reduced and there is the possibil-
ity of continuous biodiesel production. At industrial-level biodiesel production, heteroge-
neous catalysts have more constraints than homogeneous catalysts. However, heterogeneous
catalysts also have several drawbacks such as being mass transfer resistant and having a
longer reaction time (Kouzu et al. 2008). In order to overcome these issues and enhance the
overall performance of the process, nanocatalysts can be used for biodiesel production.
Improved biodiesel yield was achieved by creating more contact between nanocatalysts
and reactants through a high specific surface area of nanostructured materials (Tong 2019).
The different nanocatalysts used for biodiesel are:
Zn-doped CaO
Co-doped ZnO
Ni-doped ZnO
Magnetic MgO/MgFe2O4
Ca(30)/Al-MCM-41(U)
MgO/MgAl2O4
CaO–AuNPs
Table 1.4 Applications of nanocatalysts in biofuel production via thermochemical conversion method.

S. No. Biomass Nanocatalyst Biomass conversion method Targeted biofuel Results Reference

1 Rice husk Si-Ni0 nanoparticles Pyrolytic gasification Syngas Conversion efficiency: Shen et al. (2014)
96.5%
2 Sawdust Nano-ZnO Gasification Producer gas Conversion rate: 92.4% Akia et al. (2014)
3 Sawdust Nano-SnO2 Gasification Producer gas Conversion rate: 84.2% Akia et al. (2014)
4 Cellulose Nano-NiO Pyrolysis Char 5.64 wt.% Li et al. (2007)
5 Microalgae Nano-Ni/SiO2 Hydrothermal liquefaction Biooil 30.0 wt% Saber et al. (2016)
6 Rice straw NiO Hydrothermal liquefaction Biooil 30.4% Younas et al. (2017)
7 Chlorella sp. Na+ZSM-5 nanocrystals Hydrodenitrogenation of Upgraded biooil 75.0 wt% Hosseinpour et al.
microalgae crude biooil (2017)
8 Rice straw Ni/CeO2 Hydroliquefaction Biooil 66.7% Chen et al. (2018)
9 Sugarcane bagasse Unpromoted Cu2O/g- Near-critical water media Hydrogen 10 mmol/g Tavasoli et al. (2015)
Al2O3-MgO catalyst
10 Wood sawdust Fe-Zn/Al2O3 Gasification Hydrogen 9.65 mmol H2/g Chen et al. (2015)
11 Municipal solid Ni-Cu/γ-Al2O3 Steam gasification Hydrogen 34 g/kg fuel Gao et al. (2017)
waste
12 Municipal solid Cu/γ-Al2O3 Steam gasification Hydrogen 32 g/kg fuel Gao et al. (2017)
waste
13 Sawdust Nano-Ni–La–Fe/γ-Al2O3 Steam gasification Hydrogen 12.1 wt.% Arregi et al. (2018)
14 Wood sawdust Nano Fe-Zn/Al2O3 Catalytic steam reforming Hydrogen 1.9 wt.% Chen et al. (2015)
15 Wood sawdust NiO-ZnO-Al2O3 Pyrolysis and steam Hydrogen 8.2–20.1 mmol H2/g Dong et al. (2017)
reforming
16 Bagasse Ni-Fe/γ- Al2O3 Steam reforming Hydrogen 35.9% Jafarian et al. (2017)
nanocatalysts
S. No. Biomass Nanocatalyst Biomass conversion method Targeted biofuel Results Reference

17 Pinewood and Nano-Ni Hydrothermal water Hydrogen 2.8–5.8 mmol/g Nanda et al. (2016)
wheat straw gasification
18 Enteromorpa Ru promoted Hydrothermal gasification Hydrogen 12.28 (mmol H2/g of Norouzi et al. (2017)
intestinalis Fe–Ni/c-Al2O3 biomass)
biomass
19 Sugarcane bagasse K2O Near-critical water media Alcohols and 83.3 mmol/g Tavasoli et al. (2015)
promoted ethers
Cu/g-Al2O3-MgO
20 Sugarcane bagasse Ni/CNT catalysts + Cu Supercritical water Hydrogen 14.7% (21.8 mmol H2/g Rashidi and Tavasoli
gasification of biomass) (2015)
21 Spirulina Ni/TiO2 Hydrothermal liquefaction biooil 32.5–43.5 wt% Tian et al. (2018)
22 Rice husk char Ni Nano catalytic pyrolysis Syngas H2: 49.46% & CO: Prabhahar et al.
32.32% (2019)
12 1 Nanocatalysts and Biofuels

Cs–Ca/TiO2–SiO2
CaO/CuFe2O4
Silica doped with zinc oxide
Nano CaO
Mn-ZnO nanocomposite capped with polyethylene glycol
Lipase immobilized functionalized magnetic nanocatalyst.
Their optimal conditions for better biodiesel yield from various feedstocks are presented
in Table 1.5. The nanocatalyst has unique properties like high catalytic reactions and more
specific surface area, which can perform better than heterogeneous catalysts to produce
biodiesel (Akia et al. 2014).

1.4.5 Future Challenges

The criteria used for selection of nanocatalysts in biofuel production are the biomass energy
conversion method employed, reaction conditions (reaction temperature, reaction time,
mode of reactivity) used, and biomass type. The principles and synthesis of nanocatalysts
may be different from conventional methods (Changwei et al. 2011) but will always be effi-
cient. It is necessary to design and develop an exclusively designer nanocatalyst according to
targeted biofuel production from biomass feedstocks. For instance, the nanoparticles used at
a higher temperature in the case of thermochemical conversion process have problems such
as sintering, changes in morphology, homogeneity, and changes in selectivity; deactivation
also occurs (Sharma et al. 2016), which results in restricted applications at low-temperature
range and residence time. These issues can be conquered by developing ligands or porous
coating to nanoparticles (Joo et al. 2009; Zuliani et al. 2018). The metal nanoparticles coated
with a mesoporous silica shell can withstand up to 727 °C with no sintering effects and
maintaining morphology and shape in the reactions (Joo et al. 2009). The materials used for
ligands or coating include carbon, inorganic components, and proper metals to overcome
the sintering problems in the thermochemical conversion process (Sharma et al. 2016). The
catalyst mixed well with solid residue in the case of pyrolysis would lead to difficulty in sepa-
ration as well as recycling and the catalyst being deactivated due to carbon deposition
(Williams and Horne 1995; Horne and Williams 1996; Vitolo et al. 2001; Changwei et al.
2011). The catalyst development for biofuel production should focus on catalyst stability,
selection of precious metals, improvement in reaction kinetics, rapid diffusion, high selec-
tivity, less deactivation, and continuous production (Shivhare et al. 2018).
On the other hand, the manufacturing of nanomaterials or nanoproducts can produce
nanowastes—wastes produced from the use or production of nanomaterials, which con-
tains nanoparticles (Figure 1.2). The waste management plan should be framed to monitor
the nanowastes. Fast growing nanotechnology industries would lead to the uncontrollable
release of nanoproducts into the environment, which may have adverse effects on humans
and the environment (Musee 2011). According to Musee (2010, 2011), nanowastes are clas-
sified into three categories based on their toxic effects in humans and other ecological sys-
tems: Class I nanowaste (very low or no toxic effects); Class II nanowaste (harmful or toxic
effects); and Class II nanowaste (toxic to very toxic effects). Hence, these nanowastes must
be effectively managed for safe disposal.
Table 1.5 Details of nanocatalysts used for biodiesel production.

Optimal conditions for biodiesel production

S. No. Feedstock Nanocatalyst used Temperature, oC Catalyst amount Methanol:oil ratio Time Yield, % Reference

1 Waste cooking oil Zn-doped CaO (0.5–2 wt.% Zn2+) 65 5 wt% 20:1 4h 96.74 Borah et al. (2019a)
2 Cooking oil Ti(SO4)O 75 1.5 wt% 9:1 3h 97.10 Gardy et al. (2016)
3 Non-edible oil Co-doped ZnO — 2.5 wt% 9:1 — 98.03 Borah et al. (2019b)
4 Bombax ceiba oil CaO NPs 65 1.5 wt.% 10.37:1 70 min 96.20 Hebbar et al. (2018)
5 Castor oil Ni-doped ZnO 55 11% 8:1 1h 95.20 Baskar et al. (2018)
6 Castor oil Ferromagnetic ZnO 55 14 wt % 12:1 50 min 91.00 Baskar and Soumiya
(2016)
7 Canola oil ZnO/BiFeO3 65 4 wt.% 15 : 1 6h 95.43 Salimi and Hosseini
(2019)
8 Cottonseed oil 5-Na/ZnO 65 12 wt% 24:1 4h > 98 Malhotra and Ali
(2019)
9 Sunflower oil Magnetic MgO/MgFe2O4 110 4 wt.% 12:1 4h 91.20 Alaei et al. (2018)
10 Sunflower oil Ca(30)/Al-MCM-41(U) 70 10 wt. % 12:1 — 84.20 Vardast et al. (2019)
11 Sunflower oil MgO/MgAl2O4 90 3 wt.% 12:1 3h 92.70 Yousefi et al. (2018)
12 Sunflower oil CaO–AuNPs 65 3% 9:1 3h 90–97 Bet-Moushoul et al.
(2016)
13 Soyabean oil Chlorocholine 60 4.13 11:1 4.4 h 95.20 Hosseini et al.
hydroxide - Boehmite (2019)
14 Soybean oil Mesoporous super-acid NCs 140 3 wt. % 20:1 5h >85 Wang et al. (2019)
15 Soybean oil Fe(III) doped ZnO — 10 wt % 10:1 3h 98.00 Saxena et al. (2019)
16 Soybean oil CaO 60 4 wt.% 6:1 80 min 89.89 Badnore et al. (2018)

(Continued)
Table 1.5 (Continued)

Optimal conditions for biodiesel production

S. No. Feedstock Nanocatalyst used Temperature, oC Catalyst amount Methanol:oil ratio Time Yield, % Reference

17 Soybean oil Ni0.5Zn0.5Fe2O4 NPs 180 2 wt.% 12 : 1 1h 99.38 Dantas et al. (2018)
18 Chicken fat CaO/CuFe2O4 70 3.0% 15:1 4h 94.52 Seffati et al. (2019)
19 Ulva lactuca, Silica doped with zinc oxide, 55 8 0.0% — 50 min 97.43 Kalavathy and
macroalgae Baskar (2019)
20 Microalgae Nano CaO — 1.7% 9:1 3.6 h 86.41 Pandit and Fulekar
(2017)
21 Nannochloropsis Mn-ZnO with PEG 60 3.5% 15:1 4h 87.50 Raj et al. (2019)
oculata
22 Aspergillus niger Lipase Immobilized — — 4:1 — 84.00 Jambulingam et al.
Functionalized Magnetic (2019)
Nanocatalyst
23 Chlorella vulgaris CaO 70 1.39 wt % 10:1 3h 92.03 Pandit and Fulekar
oil (2019)
24 Calophyllum Zn-doped CaO 55 6% (w/v) 9:1 80 min 89.00 Naveenkumar and
inophyllum oil Baskar (2019)
25 Cordiamyxa BaMoO4-Ce2O3 NPs 65 0.5 wt.% 16:1 30 min 87.50 Karthikeyan (2019)
biooil
26 Dairy Waste CaO 65 2.4 wt% 6:1 3h 96.00 Kavitha et al. (2019)
27 Rapeseed oil Fe3O4-CeO2 65 4.5 wt % 7:1 2h 96.13 Ambat et al. (2019)
28 Rapeseed oil HPA/ZIF-8 70 4 wt.% 10:1 2h 98.02 Jeon et al. (2019)
29 Refined Cs–Ca/TiO2–SiO2 60 — 12:1 2h 98.00 Feyzi and Shahbazi
vegetable oil (2015)
30 Acidic oil Mg/ACM-U (Si/Ce = 25) 70 5 wt. % 9:1 6h 95.40 Dehghani and
Haghighi (2019)
 ­Reference 15

Raw materials

Nanocatalyst synthesis Nano-waste

Nanocatalyst

Applications in biomass conversion

Recoverable nanocatalysts Non-recoverable nanocatalysts

Reuse Nano-waste

Figure 1.2 Nanocatalyst synthesis and nanowaste generation.

1.5 ­Conclusion

The alternative usage of the biomass feedstocks is to be explored to increase the income for
plant growers. The biomass conversion methods do not entirely exploit the effective utilization
of the biomass feedstocks without catalyst due to low conversion efficiency and end products
yield. Generally, the catalyst used in the process would improve the overall reaction and sys-
tem performance. The recent studies show that nanocatalysts are more effective than conven-
tional catalysts for biofuels production from the biomass feedstocks via biochemical,
thermochemical, or chemical conversion methods. Since the biomass composition varied
from one source to another, much attention has to be given to the development of designer
nanocatalysts through suitable synthesis methods to achieve a more efficient conversion rate
and effective utilization of biomass feedstocks. This approach will support the biorefinery
industries to produce multiple products and fulfill the future fuel and chemicals requirement.
Also, advanced biorefineries used for multiple bioproducts production systems should have a
sophisticated quality testing laboratory to assess and monitor nanowaste generation in their
industries. It is also advised to test the toxicity of nanoparticles or nanocatalysts before use in
the biorefinery industry because the excess quantity will cause metal toxicity to the environ-
ment. These are necessary steps to follow for safe disposal of nanowastes.

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 23

Nanomaterials
Types, Synthesis, and Characterization

Zahra Vaseghi and Ali Nematollahzadeh

Chemical Engineering Department, University of Mohaghegh Ardabili, Ardabil, Iran

2.1 ­Introduction

Nanotechnology is composed of two words: nano and technology. The prefix “nano” comes
from an ancient Greek word meaning dwarf or extremely small. This prefix is used to show a
reduction factor of 109 times in the International System of Units (SI). Combining the prefix
nano with technology yields the word “nanotechnology,” meaning manipulation of materials at
the tiny scale to gain the advantage of outstanding characteristics exhibited by the large surface
area of nanoscale materials. Since materials in the size range 1–100 nm show ­outstanding
­properties, the nanometer scale in the range of above molecular dimension and below macro-
scopic ones (i.e. larger than 1 nm and smaller than 100 nm) lies in the nanotechnology field of
study (Pal et al. 2011). Nanotechnology and nanoscience allow the use of nanoscale materials
in different technologies and industries such as pharmaceuticals (Somanathan et al. 2016),
­cosmetics (Lu et al. 2015), wastewater treatment (Vaseghi et al. 2018a), environmental protection
(Yirsaw et al. 2016), catalysis/photocatalysis (Ong et al. 2018; Suchomel et al. 2018), etc.
Inspired by the abovementioned explanations, this chapter firstly aims to introduce and
probe into different types of nanomaterials and their corresponding applications in various
areas of nanotechnology. The discussion is followed by investigating two main approaches
for nanomaterial synthesis—namely top–down and bottom–up approaches—along with
introducing multiple subcategories for each approach. Finally, different methods for char-
acterization of nanomaterials are explored comprehensively.

2.2 ­Nanomaterials in Different Formats

2.2.1 Dimensionality-Confined Nanomaterials

Dimensionality is a major feature that can be used to segregate different structures of
­nanomaterials (Tiwari et al. 2012). Although different classifications may be chosen for nano-
materials (e.g. according to morphology, dimensionality, and composition), ­categorizing

Green Synthesis of Nanomaterials for Bioenergy Applications, First Edition. Edited by Neha Srivastava,
Manish Srivastava, P. K. Mishra and Vijai Kumar Gupta.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
24 2 Nanomaterials

them based on the dimensions of confinement could be useful (Figure 2.1). According to this
definition, in zero-dimensional nanomaterials, all dimensions (x, y, and z) are in the nanoscale
(i.e. < 100 nm), whereas for one-dimensional and two-dimensional nanomaterials, two and
one of the dimensions are in nanoscale, respectively. Similarly, three-dimensional nanostruc-
tures are made up of zero-dimensional, one-dimensional, and two-dimensional nanomateri-
als as building blocks. Solid state physics and optoelectronics has devoted the names quantum
wells, quantum wires, and quantum dots to two-dimensional, one-dimensional, and zero-
dimensional structures, respectively (García-Calzón and Díaz-García 2012; Mino et al. 2013).
Herein, examples of zero-dimensional, one-dimensional, two-dimensional, and three-
dimensional nanomaterial structures are reported.

2.2.1.1 Zero-Dimensional Nanomaterials

2.2.1.1.1 Nanoparticles Although synthetic nanoparticles have been explored and used
during the recent decades, natural nanoparticles have existed on the planet earth for
billions of years. The term nanoparticle and nanoscale came into existence and use since
the early 1990s. Till then, nanoparticles were introduced by other terms such as ultrafine
and submicron particles (Boskovic and Agranovski 2010; Jovanović 2018). Nanoparticles
have large surface area per unit mass compared to their bulk counterparts making giving
them unique mechanical, optical, electric and chemical properties (Mattsson et al. 2018).

2.2.1.1.2 Nanoclusters Nanoclusters are considered as a classification of nanoparticles

with at least one of their dimensions between 1 and 100 nm and possessing a narrow size
distribution. Nanoclusters may be made up of a single atom or a combination of different
atoms that are bound together through covalent, metallic, van der Waals, metallic, ionic

3D

Nano-structured:
2D Fullerites, heteolayers
fiber composites

1D
Thin films, layered films, quantum wells

0D
Nanowires, nanotubes, quantom wires

Nanospheres, quantom wires

Figure 2.1 Dimensionality classification of nanostructures. Source: García-Calzón and Díaz-García

(2012). Copyright © 2012, Elsevier.
 2.2 ­Nanomaterials in Different Format 25

forces as well as hydrogen bonding. The world of nanoclusters makes a bridge between the
atomic and molecular world and that of the bulk materials. Bulk materials usually possess
constant physical properties; while for nanoclusters the physical properties may vary based
on changes in conditions. The reason lies in the difference in electron distribution of these
two categories. In bulk materials, the electrons are located in continuous energy bands while
in individual atoms and molecules the energy bands are distinct. Nanoclusters are placed
between these two positions concerning electron distribution (Philip 2001; Sinha et al. 2017).

2.2.1.1.3 Quantum Dots Quantum dots are fluorescent semiconductor nanocrystals with a
diameter of as small as 1–10 nm. They are composed of a semiconductor core, which is coated
by a semiconducting shell. The most commonly used shells include cadmium selenide,
cadmium telluride, zinc cadmium, lead selenide, and zinc sulfide, which are used to enhance
the optical property of quantum dots and make a covering to facilitate aqueous solubility,
especially regarding biological and biomedical applications (Bose and Wong 2015). These
nanocrystals were explored by Louis Brus and primarily employed for biomedical applications
(Steigerwald and Brus 1989). The motivation to use the name “quantum dot” for these
materials is their experience of quantum confinement, meaning that the size of the particle
restricts the electron wavefunction. Owing to this property, quantum dots have a band gap
energy being dependent on size (Suri et al. 2013). Quantum dots have been used in a broad
range of applications such as chemical sensing, targeted delivery, biosensing, bioimaging,
nanomedicine, photocatalysis, and electrocatalysis (Lim et al. 2015).

2.2.1.2 One-Dimensional Nanomaterials

2.2.1.2.1 Nanotubes The discovery of carbon nanotubes (CNTs) in 1991 (Zheng et al. 2004)
has significantly motivated researchers around the world to study one-dimensional
nanostructures. Nanotubes are considered to be microscopic tubes with diameters of less
than 100 nm, while their length is out of nanoscale. Nanotubes usually have a hollow
structure. Due to the unique properties proposed by nanotube geometry, it has been of
particular interest to researchers during recent decades. Nanotubes are recognized as
prevalent nanomaterials possessing one-dimensional structure, meaning that one of their
dimensions is beyond the nanoscale (Yoo et al. 2018). Nanotubes are effective in various fields
such as heavy metal removal (Abbas et al. 2016), human healthcare (Kumar et al. 2017), food
packaging (Makaremi et al. 2017), energy, and biosensing applications (Muhulet et al. 2018).

2.2.1.2.2 Nanowires Nanowires are nanometric materials with a few nanometers in

diameter and a much longer length (Waqar et al. 2015). Electrons in nanowires are free to
travel along their length but their motion toward the other two dimensions is restricted by
quantum mechanics, which results in variation in their characteristics. Nanowires exhibit
unique properties that make them outstandingly different from their bulk counterparts.
For instance, semiconducting nanowires with electronically switchable characteristics
have been applied successfully as sensing agents. In this sense, assemblies of copper
nanowire electrodes have been applied as sensitive electrochemical nitrate sensors with the
capabilities to detect at the micromolar level (Stortini et al. 2015). Zhang et al. (2015a) have
fabricated a novel TiO2 modified Co3O4 nanowire array with the potential to be applied for
ethanol detection at the considerably low temperature of 160 °C with a detection limit of
26 2 Nanomaterials

10 ppm. Nanowires have also been applied as piezoelectric materials where they have been
shown to enhance the piezoelectric effect. In research performed by Xue et al. (2015), the
piezoelectric field was created inside the ZnO nanowire. The nanowires under piezoelectric
field were efficiently used to enhance the photocatalytic degradation of methylene blue. In
another study, nanowires of zinc oxide and gallium nitride were evaluated the piezoelectric
coefficients based on the size of the mentioned nanowires. They have found that both ZnO
and GaN nanowires have exhibited a huge piezoelectric size effect with a relatively stronger
performance of GaN nanowire (Agrawal and Espinosa 2011).

2.2.1.3 Two-Dimensional Nanomaterials

2.2.1.3.1 Nanosheets Nanosheets that are known to possess two-dimensional geometry
morphologies are created as a result of continuous nanolayers break up. Some of their distinct
features include large surface area, highly flat structure, enhanced electron mobility, and
semiconductor properties. Due to these characteristics, a variety of applications are defined
for nanosheets (Zhang et al. 2015b). For example, TiO2 nanosheets are synthesized using
several methods including hydrothermal, solvothermal, alcohothermal, CVD, and sol-gel based
techniques (Sajan et al. 2016). Metal/metal oxide nanosheets have numerous applications such
as precursors for supercapacitors (CoS1.097/Nitrogen-doped carbon nanosheets) (Cao et al. 2016),
humidity sensors (black phosphorus nanosheets) (Late 2016), photocatalysts (gC3N4 nanosheets)
(Dong and Cheng 2015), lithium-ion battery (metal–organic framework (MOF) nanosheets)
(Fang et al. 2016), hydrogen evolution catalysts (gallium sulfide nanosheets) (Harvey et al. 2015),
self-powdered photodetectors (black phosphorus nanosheets) (Ren et al. 2017), etc.

2.2.1.4 Three-Dimensional Nanomaterials

Three-dimensional nanomaterials are bulk nanomaterials which are beyond nanoscale
dimensions in all three directions. Three-dimensional nanomaterials mainly contain zer-
dimensional, one-dimensional, and two-dimensional nanomaterials as building blocks
(García-Calzón and Díaz-García 2012). Recently, the design and fabrication of three-
dimensional nanostructures have been the focus of researchers. For instance, in one study,
3D Ni3S2 superstructures were utilized for overall water splitting through electrocatalysis
(Zhu et al. 2016). Graphene is another example of a three-dimensional structure with
numerous applications due to its large surface area, proper electrical conductivity, low den-
sity, and good mechanical strength (Fan et al. 2015).

2.2.2 Inorganic, Organic, and Hybrid Nanomaterials

Nanomaterials may be formed by inorganic elements (inorganic nanomaterials). Inorganic
nanomaterials including metal nanoparticles (gold, silver, copper, nickel, zinc, etc.), metal
oxide nanoparticles (copper oxide, zinc oxide, magnesium oxide, nickel oxide, silica, mag-
netite, etc.), graphene, quantum dots, etc. have become one of the most prominent research
areas in different industries and sciences such as biotechnology, biomedicine, electrochem-
istry, sensors/biosensors, catalysis/photocatalysis, etc.
Organic nanomaterials are composed of natural or synthetic organic molecules. Some
examples of prevalent organic nanoparticles include liposomes, albumin, dendrimers, carbon
nanomaterials, and polymeric micelles (López-Dávila et al. 2012). Organic nanoemulsions are
found to participate in the formulation of many industries such as food and cosmetics. Being
 2.3 ­Nanomaterials Synthesi 27

made up of organic or natural ingredients, along with the ability to encapsulate compounds,
has made these nanomaterials suitable and efficient candidates for drug delivery and biomedi-
cal applications (Jesus and Grazu 2012).
Organic nanoparticles are different from inorganic nanoparticles in the following aspects:
●● Since the holding strength between organic nanoparticles is not intense enough, most of
the organic nanoparticles have a dynamic feature.
●● Due to unspecified geometry of molecules that take part in the production of organic
nanoparticles, unlike the inorganic nanoparticles, nanometric size may not truly exist for
organic nanoparticles.
●● The formation of inorganic nanoparticles usually occurs by precipitation of inorganic precursor
salts. Different binding forces may exist between atoms such as metal, covalent, etc. However,
organic nanoparticles may be self-assembled or brought together by chemical binding.
The third class of nanomaterials is called hybrid nanomaterials. Hybrid organic–inorganic
nanocomposites are a class of nanomaterials that take advantage of both organic and inor-
ganic phases as well as their interfaces (Pinna 2007). Nowadays, the most industrially applied
hybrid materials consist mainly of metal oxides or metal-oxo polymers (inorganic part) and
organic biopolymers or macromolecules (Sanchez et al. 2011). Due to versatile sources of syn-
thesis for hybrid nanomaterials, their area of application is outstandingly extensive. Hybrid
nanomaterials are applied in solar/fuel cells (Bi et al. 2015; Hou et al. 2016), biosensors (Bai
et al. 2016), photocatalysis/(bio)catalysis (Ben-Shahar et al. 2016; Sharma et al. 2015),
­controlled drug release (Kesavan et al. 2018), dental products (Najafi et al. 2019), protective/
decorative/corrosion coatings (Mohan Das Gandhi et al. 2018), smart textiles, anticorrosion
coatings, contrast agents for MRI, flame retardants, anti-cancerous agents, etc.
Based on the different application of hybrid nanomaterials and the unique properties,
different synthesis methods may be applied. The most prominent techniques for obtaining
hybrid nanomaterials include laser-by-laser (i.e. merging individual layers of oppositely
charged nanomaterials in a specifically studied substrate), self-assembly, and electrospin-
ning (i.e. production of nanofibers with distinct materials in its bulk or a dispersed phase
absorbed onto the nanofiber surface) techniques (Andre et al. 2018).
Interestingly, the nature of hybrid interfaces puts this class of nanomaterials into two
categories. In class 1, the weak bonds including van der Waals, hydrogen, and ionic bonds
are exchanged by organic/inorganic components; while in class 2, overall or part linkage
between the two phases is observed by strong chemical bonds such as covalent or ion-
covalent bonds (Sanchez et al. 2011).

2.3 ­Nanomaterials Synthesis

Nanomaterials are particularly of great importance owing to numerous applications in daily
life. Thus, different synthesis techniques have been developed based on the specific applica-
tion of the nanomaterials. The two main issues that should be taken into consideration in
selecting nanomaterial synthesis methods are the properties of the desired nanomaterials
and the potential of their application on large scales. Although many synthesis techniques
may be efficient in laboratory settings, their usage on larger scales might encounter several
obstacles. Nevertheless, all the methods for nanomaterial synthesis generally lie in two main
28 2 Nanomaterials

categories—bottom–up and top–down approaches—each of which encompasses several

methods. In the top–down approach, synthesis starts with the bulk materials and ends in
fine nanoparticles through step-by-step grinding of the material. Some of the commonly
used top–down techniques include mechanical milling, photolithography, electron beam
lithography, anodization, and plasma etching. In contrast, the bottom–up technique involves
assembling the nanomaterials from the bottom meaning atom by atom, molecule by mole-
cule and cluster by cluster. Many nanomaterial synthesis protocols including various chemi-
cal methods such as chemical precipitation, sol-gel methods, CVD, and also biological
synthesis routes, are included in this category (Arole and Munde 2014). Schematic represen-
tation of top–down and bottom–up approaches is depicted in Figure 2.2.

2.3.1 Top-Down Methods

2.3.1.1 Mechanical Milling
Mechanical milling, which was developed for the first time by John Benjamin (1970), is
classified as a prominent top–down approach. This synthesis technique is of particular
interest when the synthesis procedure starts with a micrometer scale. In an ordinary pro-
cess, a mixture of elements as powder charge is placed into the mill with an appropriate
milling medium, with the objective of blending as well as the size reduction of the ele-
ments. The main parameters affecting the size of nanoparticles obtained from the milling
process include charge ratio, drum rotation speed, brittleness of primary materials, milling
period, size and size distribution, and construction materials of the balls (Bello et al. 2015).
Furthermore, the milling temperature can significantly influence diffusivity. At higher
temperatures, the phases with atomic mobility such as intermetallics are expected to form,

Top-Down Figure 2.2 Schematic representation of (a) top–down and

(b) bottom–up approaches for nanomaterial synthesis.

Bulk

Powder

Nanoparticle

Clusters

Atoms

Bottom-Up
 2.3 ­Nanomaterials Synthesi 29

while lower temperatures mainly result in amorphous structures (Yadav et al. 2012). As an
example of ball milling process for nanomaterial fabrication, Ban et al. (2011) synthesized
copper-nickel alloy nanoparticles to be applied in magnetic hyperthermia using the
mechanical milling technique. They have optimized the milling process based on the
desired alloy composition. The optimized parameters to obtain Cu27.5Ni72.5 with 10 nm
crystallite size included ball-to-power mass ratio of 20 and a Curie temperature of 45 °C.

2.3.1.2 Nanoimprint Lithography

During recent years, considerable research has been devoted to nanoimprint lithography due
to a plethora of features including having high resolution, being regarded as high throughput
technology, and its cost-effectiveness. Application of nanoimprint lithography has been
observed in various sectors such as molecular electronics (Jung et al. 2004; Wu et al. 2005),
biosensors (Lee et al. 2011; Martinez-Perdiguero et al. 2013), etch mask patterning (Yang et al.
2007), and photonics (Espinha et al. 2018; Mele et al. 2005). It is worth noting that application
of nanoimprint lithography is not limited to the one-dimensional and two-dimensional envi-
ronment. Several types of research are performed to explore the potential of this technique in
three-dimensional printing (Landis et al. 2013; Li et al. 2001; Ofir et al. 2010; Saive et al. 2017).
The process of nanoimprinting is composed of two steps. In the first step, the master stamp
and the polymer to be printed are placed in parallel stages (1. PVA release layer and PEGDA
ae applied to BARC coated silicon surface. 2. The quartz template is pressed onto PEGDA and
exposed to UV light.3. The template is removed to reveal particles with thin residual layer.
4. Brief oxygen plasma etch is performed to remove the residual layer. 5. Particles are harvested
directly in water or buffer by the one-step dissolution of the PVA layer) followed by heating
to approximately 100 °C above the glass transition temperature (Tg) of the polymer. The next
step provides physical contact between the stamp and the thin film while simultaneously
exerting external pressure. After complete compression of the polymer, the formed viscous
polymer is forced to flow into the cavities of the mold. In this way, the features of the stamp
are imparted onto the polymer. Viscoelasticity of the polymer is the essential factor that dic-
tates the time and temperature required for proper imprinting behavior. After the completion
of the imprinting process, the polymer is cooled to below Tg with subsequent demounting
and separation from the stamp. In the second stage of the nanoimprinting process, the
imprinted features are transferred into a hard medium. Depending on the special target
application, the pattern is imparted onto the material (Kam and Torres 2019). During recent
research, stimuli-responsive, mono-disperse nanoparticles with a precise size of 50 nm were
synthesized using step and flash imprint lithography (S-FIL). During this nano-modeling
process, the topography of a quartz template was used to mold ultraviolet crosslinkable mac-
romers including poly(ethylene glycol (PEG) diacrylates and dimethacrylates (PEGDA or
PEGDMA) on a silicon wafer (Figure 2.3) (Glangchai et al. 2008).

2.3.1.3 Laser Ablation

Laser ablation is a thermal or non-thermal process in which removal or ablation of atoms
from a solid body occurs through irradiation from a focused pulsed laser beam or a continu-
ous wave. The main difference between a pulsed laser beam and a continuous wave is that in
the latter the output is continuous as the laser is powered, while in the former, despite a
shorter time the more intense output is observed resulting in the same average output power
30 2 Nanomaterials

1 2

5 3

Figure 2.3 Step and flash imprint lithography (S-FIL) method: 1. Poly Vinyl Alcohol (PVA) release layer
and PEGDA is applied to Bottom layer Anti-Reflective Coating (BARC) coated silicon surface; 2. The
quartz template is pressed onto PEGDA and exposed to UV light; 3. The template is removed to reveal
particles with thin residual layer; 4. Brief oxygen plasma etch is performed to remove residual layer;
5. Particles are harvested directly in water or buffer by one-step dissolution of the PVA layer.
Source: Glangchai et al. (2008). Copyright © 2008, Elsevier.

as a continuous wave. Production of nanoparticles and thin films is one of the key achieve-
ments of this technique. Another application of laser ablation include drilling of a very
small hole in hard materials such as metals and diamond (Zhang and Guan 2014).
Materials that do not evaporate when heated or metal composites with different constitu-
ent properties at the vapor phase are good candidates for the laser ablation technique. In
this sense, the products of this process would be CNTs, fullerenes, and metals. In laser
ablation, the laser beam is directed at the target surface, which is a solid material. The solid
material responds to the laser beam by releasing atoms/molecules, ions, etc., resulting in
the formation of a plasma plume. As the release of the mentioned moieties continues, the
plasma plume grows until it reaches supersaturation. In the next step, the plasma plume
experiences rapid cooling through gas expansion leading to the state of condensation
nucleation. Furthermore, the formation of one-dimensional structures such as nanotubes
and nanowires is likely to happen for deposited materials. It is worth mentioning that the
fulfillment of the laser ablation process is also feasible when the target material is sub-
merged in the liquid phase resulting in the formation of much smaller particles. Since the
stoichiometry of elements is not influenced by ablation, conducting this process at liquid
phase is ideal for nanoparticles with complex compositions (Virji and Stefaniak 2014).

2.3.1.4 Electrospinning
One of the prevalent top–down approaches employing electrostatic forces to drive a con-
ductive fluid so as to fabricate fibrous materials ranging from several nanometers to tens
 2.3 ­Nanomaterials Synthesi 31

of micrometers with high surface area (10–100 m2/g) is called electrospinning. The mate-
rials with the potential to be electrospun include diverse kinds of substances such as poly-
mers, organic molecules, sol-gels, metals, ceramics, and even hybrid materials (Chen
et al. 2011; Imaizumi et al. 2011). The fibrous materials produced through electrospinning
can be engineered to be core-shell (Khalf et al. 2015), hollow (He et al. 2016), anisotropic
(Hu et al. 2016), or biphasic (Son et al. 2018) according to the specific application field of
the product.
The electrospinning technique finds its roots in 1902 when Morton and Cooley reported
the first electrospun polymeric fibers in separate patents. In a typical experimental proce-
dure, a polymer solution is pushed through a narrow bore syringe needle called a spin-
neret. Using a syringe pump, the process is carried out at a constant flow rate. A high
voltage supply (5–25 kV) is connected to the spinneret along with a ground collector placed
at its vicinity (10–20 cm). The flow rate of the feed within the experimental set up of elec-
trospinning would be in the range of 1–5 ml/h. The ions that are formed as a result of
charge transfer by the polymer or the solvent repel one another, which subsequently leads
to the formation of narrow jets that decrease the repulsion. Schematic representation of
three-dimensional-printed electrospinning set up for the preparation of loratadine nanofib-
ers is depicted in Figure 2.4.
The parameters affecting the fiber diameters can be classified into two separate catego-
ries. The first category deals with the experimental conditions such as applied voltage
(Deitzel et al. 2001), the distance between the spinneret and the collector (Wang and Kumar
2006), and the flow rate of the feed (Li and Xia 2003). Characteristic of the electrospinning
solution including surface tension and viscosity (Haghi and Zaikov 2011) is the other vital
parameter influencing the diameter of the produced nanofibrous materials.

Rotating collector
Electrospinning nozzle (grounded)
(high voltage)

Air

Loratadine
nanofibers

Figure 2.4 Schematic representation of three-dimensional electrospinning set up. Source: Ambrus
et al. (2019). Copyright © 2019, Elsevier.
32 2 Nanomaterials

2.3.2 Bottom-Up Methods

The bottom–up approach starts from the atomic level and leads to the formation of nano-
structures with further self-assembly of the atoms/molecules. The growth and self-assem-
bly of the atoms and molecules as building blocks leads to the formation of nanomaterials
with well-defined size, morphology, and chemical composition. Bottom–up methods for
nanomaterial synthesis are generally categorized as solid-phase and liquid-phase methods
(Figure 2.5). The solid phase methods can be either chemical (CVD and thermal decompo-
sition) or physical (PVD). However, most of the bottom–up methods for nanomaterial syn-
thesis including liquid/liquid methods (chemical reduction, indirect reduction, biological
reduction, spray drying, spray pyrolysis, and solvothermal) and sedimentation methods
(sol-gel, co-precipitation, alkaline precipitation, and hydrolysis) are carried out in the liq-
uid phase. The aforementioned methods will now be explained in more detail.

2.3.2.1 Chemical Vapor Deposition

CVD is widely used in processing materials. Its main application involves the production of
solid thin film coatings to surfaces. Nevertheless, this method is also applied in producing
bulk materials, powders, and composite materials with high purity. The major parameters
affecting the quality of the deposited material include type of reactor (i.e. hot wall and cold
wall reactors) reaction temperature (ranging from 200 to 1600 °C), reaction pressure (from
sub-torr to above atmospheric pressure), reaction rate, and concentration of precursors.
In a typical CVD process, a precursor gas flows into a chamber containing one or more
materials to be heated for surface coating. Once the chemical reactions occur on the hot
surfaces, thin films are formed on them. Usually, the volatile byproducts produced during
the process, along with unreacted precursor gases, are removed by gas flow inside the

Bottom-Up
methods

Solid-phase methods Liquid-phadse methods

Chemical Physical Liquid/Liquid Sedimentation

Thermal Physical vapor

Chemical vapor
deposition
deposition decomposition
Sol-gel
Chemical reduction Co-precipitation
Photoreduction
Biological reduction
Spray drying
Spray pyrolysis
Solvothermal

Figure 2.5 Overview of bottom-up methods for nanomaterial synthesis.

 2.3 ­Nanomaterials Synthesi 33

reaction chamber. In advanced CVD processes, plasmas, ions, photons, lasers, hot fila-
ments, and combustion reactions are applied to enhance the deposition rate of the thin
films and lower the reaction temperature.
In CVD processes with hot wall reactors, the system containing multiple objects is heated by
a furnace surrounding it. In such a system, different parts are loaded into the system, it is
heated to desirable temperature, and then reactive gases are introduced. Hot wall reactor sys-
tems are often operated at high temperatures and low pressures. The opposite of hot wall reac-
tors is cold wall reactors. In these systems, the materials within the chamber are heated but the
walls are cooled. Cold wall reactors usually operate under relatively high pressures ranging
from several hundreds of torr to atmospheric pressures (Kim et al. 2004; Park and Sudarshan
2001). In Figure 2.6, the schematic representation of the two types of CVD reactors is depicted.

2.3.2.2 Physical Vapor Deposition

PVD is a type of vacuum deposition method, mainly applied in semiconductor industries,
that is used to produce thin films and coatings. In this method, the target has an evapora-
tion source acting as a cathode in which the material is vaporized from a solid or liquid
source. Heating with an electron beam results in evaporation of the particles in atomic size.
The clash between the released particles and the gas molecules introduced into the cham-
ber accelerates the particles. This forms plasma that passes through the deposition cham-
ber then a vacuum pump to the surface where it condenses, leading to the formation of a
thin film. PVD can be used to produce thin films as well as multilayered coatings (Baptista
et al. 2018). The thickness of these films could be in the range of few to thousands of
nanometers (Mattox 2010). A schematic of the PVD process is represented in Figure 2.7.
Both PVD and CVD are used to form thin films on the substrate. The main difference
between these two techniques—as can be implied by their names-—is that PVD uses physi-
cal forces to form thin films, whereas in CVD chemical reactions generate the formation of
thin films. No chemical reaction occurs during the course of the PVD process. On the other
hand, CVD uses a source material with much less purity due to it being mixed with volatile
precursors. Once this mixture is injected into the chamber containing the substrate to be
coated, the deposition process occurs.

(a) (b) Gas shower

Fe/Quartz
Thermocouple Furnace Quartz tube Graphite heater
Chamber
Thermocouple

Gases Pump

Figure 2.6 Schematic pictures of (a) hot wall CVD furnace and (b) cold wall CVD chamber.
Source: Mu et al. (2016). Copyright © 2016, Elsevier.
34 2 Nanomaterials

Vacuum Chamber

Gas
Inlet Substrate

Particles Power
Supply
Plasma

Evaporation
Source
Vacuum
Target System

Figure 2.7 Schematic illustration of physical vapor deposition (PVD) process. Source: Baptista
et al. 2018). Copyright © 2018, Elsevier.

2.3.2.3 Thermal Decomposition

The thermal decomposition (thermolytic) method for the synthesis of nanomaterials is a
kind of chemical decomposition method that is caused by heat and leads to the production
of nanoparticles with high crystallinity and narrow size distribution. In this method,
decomposition of organometallic compounds occurs at high refluxing temperatures in sol-
vents with high boiling points and with the aid of stabilizing surfactants. Thermal decom-
position is extensively used for the synthesis of metal oxide nanomaterials including ZnO
(Saravanan et al. 2013), Fe3O4 (Amara et al. 2009), Fe2O3 (Darezereshki 2011), NiO (Ramesh
et al. 2018), CuO (Ibrahim et al. 2018), Co3O4 (Momeni et al. 2018), metal nanoparticles
such as Fe (Simeonidis et al. 2007), Ni (He et al. 2013), and Co (Shao et al. 2006), and alloys
such as CoPst3 (Tzitzios et al. 2005) and FePt (Nandwana et al. 2007). In this approach,
control over size, morphology, and polydispersity of the produced nanoparticles would be
feasible by controlling the ratios of starting materials such as precursors, solvents, and
surfactants (Khan and Khan 2017; Unni et al. 2017).
The typical experimental apparatus used for the synthesis of nanomaterials through
chemical decomposition method is depicted in Figure 2.8. The set up consists of a round-
bottomed three-necked flask placed onto a heating stirrer. The system is also equipped with
a glass water condenser. A thermometer immersed in a beaker containing silicon oil, which
is mounted on the heating stirrer plate, is used to monitor the running temperature.
Furthermore, in order to maintain the inert atmosphere, argon or nitrogen gases flow con-
tinuously within the system during the course of the reaction.

2.3.2.4 Chemical Reduction

Chemical reduction is the most versatile technique for nanomaterial production due to
facile operation and the requirement for simple equipment. Most of the chemical reduction
 2.3 ­Nanomaterials Synthesi 35

Outlet (N2 or Ar Gas)

Precursors solution

Thermometer
Inlet (N2 or Ar Gas)

Silicon Oil

Heating/Stirring Plate

Figure 2.8 Schematic diagram of thermal decomposition process. Source: Khan and Khan (2017).
Copyright © 2017, Springer.

methods obey the Brust–Schiffrin two-phase process during which chemical reduction
occurs in the oil–water interface. In the next step, which happens immediately after the
first step, the thiolated molecules are adsorbed and stabilized in the organic phase (Li et al.
2011; Perala and Kumar 2013).
In the chemical reduction method, an aqueous solution containing the metal ions is used
as the precursor. The ions are reduced to their metallic form by adding chemical reducing
agents such as sodium borohydride (Mehr et al. 2015), sodium citrate (Piella et al. 2016),
sodium ascorbate (Sun et al. 2016) and hydrazine hydrate (Gurusamy et al. 2017) in a vola-
tile organic solvent such as toluene (Zhai et al. 2016), and chloroform (Shankar and Rhim
2016) to the reaction mixture. This process leads to agglomeration and formation of clus-
ters followed by the formation of nanoparticles. Sodium borohydride is a well-known and
strong reductant that is extensively utilized in the synthesis of nanomaterials. The use of
such chemical reducing agents often necessitates the application of capping agents to avoid
colloidal aggregation of nanoparticles and stabilize them. Otherwise, size control of the
produced nanoparticles may not be feasible (Pandey and Manivannan 2015). The most
commonly used stabilizing agents include amines and thiols such as dodecylamine (Morán-
Lázaro et al. 2016), dodecanethiol (Mancini et al. 2016), and alkanethiolates (San et al.
36 2 Nanomaterials

2017), as well as surfactants (Sun et al. 2017), polymers (Pourmasoud et al. 2018), carbox-
ylic acids (Shombe et al. 2016), and organic compounds (Sun et al. 2015).

2.3.2.5 Photoreduction
The chemical reduction method, despite being versatile, has some limitations such as the
use of relatively large amounts of reducing and stabilizing agents, which should be sepa-
rated from the final product (i.e. nanoparticles) in the last step. For nanoparticles exhibit-
ing optical properties such as silver and gold nanoparticles, photoreduction is a useful
method to overcome this problem. In this method, irradiation of reaction mixture is done
using a light source such as a laser or lamp along with the introduction of photoreducing
agents without the need to use stabilizing/capping agents (Zaarour et al. 2014). One of the
benefits of the photoreduction method is that the unwanted byproducts are mostly elimi-
nated, and the method can be carried out in both the solid and liquid phases (Takenaga and
Yamamoto 2009).

2.3.2.6 Biological Reduction

In the chemical reduction method, the reducing and stabilizing agents are toxic chemicals that
impose significant risks and hazards on the environment and humans. The biological synthe-
sis route—often referred to as green synthesis—is based on the green chemistry principles. In
the aforementioned method, three main criteria of nanoparticle synthesis (i.e. ­solvent, reduc-
ing agent, and capping/stabilizing agent) ought to be green. In this respect, water is used as the
main solvent in the reaction mixture. However, in some cases, methanol and ethanol may be
applied instead. One of the merits of the green approach is that the reducing agent acts simul-
taneously as the capping agent, too. Thus, the requirement for an external capping agent to be
added to the reaction mixture is eliminated (Vaseghi et al. 2018b).
The reducing and capping agents in the green synthesis method fall into two main cate-
gories: microorganisms (i.e. bacteria, fungi, yeasts, algae) and plant extracts (Figure 2.9).
Diverse species of bacteria (e.g. Bacillus subtilis (Reddy et al. 2010), Bacillus mycoides
(Lampis et al. 2014), Bacillus methylotrophicus (Wang et al. 2016), Escherichia coli
(Gurunathan et al. 2009), Lactobacillus acidophilus (Namasivayam et al. 2010), Enterococcus
faecalis (Shoeibi and Mashreghi 2017), etc.), fungi (e.g. Aspergillus sp. (Bhainsa and
D’souza, 2006; Li et al. 2012; Vigneshwaran et al. 2007), Fusarium sp. (Ahmad et al. 2003;
Ingle et al. 2008, 2009), Penicillium sp. (Hemath Naveen et al. 2010; Honary et al. 2013;
Nayak et al. 2011), Trichoderma sp. (Ahluwalia et al. 2014; Elgorban et al. 2016; Tripathi
et al. 2013), etc.), yeasts (Saccharomyces cerevisiae (Mala and Rose 2014), Saccharomyces
boulardii (Korbekandi et al. 2014), Yarrowia lipolytica (Agnihotri et al. 2009), Candida sp.
(Mishra et al. 2011), Rhodotorula sp. (Cunha et al. 2018), Pichia pastoris (Chen et al. 2014),
etc.), and algae (Sargassum wightii (Shanmugam et al. 2014), Spirulina platensis (Sayadi
et al. 2018), Phormidium tenue (Roopan and Elango 2016), Chlorococcum humicola (Jena
et al. 2013), etc.) have been used as a source for nanoparticle synthesis through biological
reduction of the metal precursors.
Phyto-synthesis of nanomaterials using plants offers several advantages in comparison
to the microbial synthesis. For instance, rapid synthesis, more stable nanoparticles, elimi-
nation of complex treatment after nanoparticle synthesis, and increase in monodispersity
of the produced nanoparticles are among some valuable merits of this synthesis method.
 2.3 ­Nanomaterials Synthesi 37

Cu+
Zn2+
Cr3+ Mn2+ Fe2+
2+
Fe2+ Co Cu2+
Ni2+ Ag+

Figure 2.9 Schematic representation of the biogenic reducing agents participating in the
bioreduction process of metal ions.

Although the actual mechanism of bioreduction of the metal ions using plant extracts and
subsequent formation of nanoparticles has not yet been fully explored, it has been declared
that the reducing potential of plants is based on the individual plant’s phytochemicals such
as terpenoids, alkaloids, polysaccharides, polyphenols, reducing sugars, amino acids, and
proteins. Furthermore, the extract may be elucidated from different plant organs such as
stems, leaves, flowers, and seeds (Vaseghi et al. 2018b, 2019).
The number of plants being used in the biological synthesis of nanoparticles is outstand-
ingly great and research covering this field is increasing to fill the gaps, especially concern-
ing the exact mechanism through which the nanoparticles are formed. Nevertheless,
Eryngium campestre (leaf extract) (Vaseghi et al. 2019), Froriepia subpinnata (leaf extract)
(Vaseghi et al. 2018c), Jatropha curcas (latex extract) (Hudlikar et al. 2012), Hibiscus rosa
sinensis (leaf extract) (Philip 2010), Memecylon edule (leaf extract) (Elavazhagan and
Arunachalam 2011), Mimosa pudica (root extract) (Niraimathee et al. 2016), Trachyspermum
ammi and Papaver somniferum (seed extract) (Vijayaraghavan et al. 2012), Syzygium aro-
maticum (bud extract) (Raghunandan et al. 2010), Amaranthus polygonoides (leaf extract)
(Muthukumar and Matheswaran 2015), Coriandrum sativum (seed extract) (Nazeruddin
et al. 2014), etc. are some of the plants being worked on for nanoparticle synthesis.

2.3.2.7 Spray Drying

Spray drying is defined as a rather simple technique in which an aqueous solution of the
precursors is atomized; followed by subsequent evaporation of the solvent in a countercur-
rent flow regime. The starting material in this method is an aqueous solution that should be
homogenously mixed to obtain a well-distributed solution. In the next step, a suspension of
proper droplets could be prepared via different methods such as rotary disks, spray nozzle,
38 2 Nanomaterials

ultrasonic nebulizer, etc., depending on the required particle size. The product is dried in the
next step, resulting in the production of solid particles. Overall, it should be pointed out that
the particle size and distribution of the final product in spray drying strongly depends on the
concentration and fine atomization of the applied primary solution. After the process is
completed, the powder is collected using a precipitator (cyclone, filter bag, or electric field)
(Okuyama et al. 2006; Sharma et al. 2010). A schematic diagram of a typical spray drying
apparatus is depicted in Figure 2.10.
Spray drying has numerous advantages in comparison with other methods for nanoma-
terial synthesis. It is quite a versatile technique being compatible with many materials and
substances. Recently, high purity, spherical nanoparticles are produced based on this facile
approach (Nandiyanto and Okuyama 2011).
Spay drying is mostly applied in manufacturing dried food such as dried eggs (Katekhong
and Charoenrein 2018), animal feed supplements (Van Dijk et al. 2001), cake mixtures
(Rocha et al. 2012), food flavorings (Reineccius 2004), fish oil (Encina et al. 2016), starch
derivatives (Murúa-Pagola et al. 2009), yogurt (Francisco et al. 2018), juices (Tontul and
Topuz 2017), infant formula (Masum et al. 2019), etc. In the pharmaceuticals industry,
spray drying is efficiently utilized to produce drugs with well-defined physical and chemi-
cal properties (Arzi and Sosnik 2018). In materials processing, spray drying is used to pro-
duced powder metals, metal oxides, and semi-conductors with homogeneous distribution
and spherical shape, which prevents agglomeration of the produced particles. In addition,
the spherical morphology of the produced powders has made them suitable agents in rheo-
logical applications compared to the classic powders (Okuyama et al. 2006). The particles
produced with the spray drying technique have also been reportedly utilized in the ferti-
lizer industry (Nasri et al. 2015) and oxide ceramics production (Bertrand et al. 2005).

Exhaust Figure 2.10 Schematic diagram of spray drying set up. Source:
Okuyama et al. (2006). Copyright © 2006, Elsevier.
Particles
Collector

Furnace

Carrier gas

Droplets
Atomizer
 2.3 ­Nanomaterials Synthesi 39

2.3.2.8 Spray Pyrolysis

Spray pyrolysis is a liquid to particle conversion method that takes place in the vapor phase
at a moderately high temperature. In this technique, a solution containing dissolved pre-
cursor is sprayed into a vector gas in the form of fine droplets onto the surface of a heated
substrate. Selecting suitable chemical reactants is very important so that the unwanted
products evaporate at the temperature of deposition. Since, in this technique, solutions or
sols are used, it is classified as a liquid phase method (Lévy-Clément 2006; Okuyama and
Lenggoro 2003). Spray pyrolysis is extensively used in industrial applications because it is
simple, cost-effective, versatile, and can be developed efficiently for large-scale deposition.
Being a non-contact process, multiple layers or films can be deposited in spray pyrolysis
without destroying the underlying layers (Escoubas et al. 2013). Spray pyrolysis set up has
two main constituents, namely a furnace to provide heat to the substrate and a spray gun to
spray the solution onto the substrate (Caglar et al. 2008).
The technique is economical due to the fact that it is done in a continuous manner under
atmospheric pressure, unlike other techniques such as sol-gel and CVD, which are carried
out using multiple steps and under vacuum pressure. In addition, the chemical flexibility
of this method means the production of advanced ceramics and films would be feasible.
For performing a spray pyrolysis process, four major steps are anticipated:
1) Droplets are generated from a precursor solution.
2) Shrinkage of droplet size occurs as a result of evaporation.
3) Precursors are converted into oxides.
4) Finally, the solid particles are formed.
Droplets are usually generated either with the aid of air (two-fluid atomization) or with-
out air interference (ultrasonic atomization). The advantage of the two-fluid atomization is
being high throughput; whereas ultrasonic atomization does not possess this precious
merit. However, ultrasonic atomization leads to narrow size distribution, in contrast to the
former, which results in broad size distribution (Tsai et al. 2004).

2.3.2.9 Solvothermal
A variety of nanomaterials that have specific chemical and physical properties with poten-
tial applications in various industries could be synthesized through the solvothermal
method. The solvothermal method often takes place at the supercritical regime at tempera-
tures above 300 °C. However, in recent years, some studies have considered this method for
nanocrystal synthesis under rather mild conditions (i.e. under 300 °C). Various nanomate-
rials including metals, metal oxides (Zare et al. 2018), semi-conductors (Šutka et al. 2018),
alloys (Lin et al. 2018), composites (Song et al. 2018), hydroxides (Huang et al. 2018), zeo-
lites, and ceramics with different morphologies such as nanoparticles (Ramakrishnan et al.
2018), nanospheres (Wang et al. 2019), nanosheets (Cai et al. 2015), nanotubes (Mukherjee
et al. 2018), nanorods (Lu et al. 2017), etc. have been synthesized through the solvothermal
technique so far.
In the solvothermal method, in addition to water, various organic solvents with different
features may serve as a reaction medium. Thus, the potential of creating new materials in
this synthesis approach is relatively high. Since in the solvothermal method the organic
solvent has the dual role of supplying the reaction medium and forming a solvent–reactant
40 2 Nanomaterials

complex, the selection of an appropriate solvent is exceptionally important. Several param-

eters should be taken into consideration when selecting the solvent for this method effi-
ciently including molecular weight, density, melting point, boiling point, the heat of
evaporation, dipole moment, polarity, and dielectric constant. Among these parameters,
solvation of the solvent is mostly dependent on the solvent polarity, which is defined as the
sum of interaction forces between the solvent and the solute. The prevalent organic sol-
vents used in the solvothermal technique include methyl alcohol, ethyl alcohol, propanol,
butanol, heptanol, benzyl alcohol, Ethylene glycol, etc. (Feng and Li 2017).

2.3.2.10 Sol-Gel
Sol-gel is relatively a new method used particularly for the synthesis of metal oxide nano-
particles as well as oxide composites. The term aqueous sol-gel is used when the reaction
medium in the sol-gel process is water. Otherwise (when using organic solvents), the pro-
cess is called the non-aqueous sol-gel method. The non-aqueous sol-gel method offers
some superiorities, especially for metal nano-oxide synthesis, compared to the aqueous
sol-gel method. The metal oxides prepared by the former method using organic solvents
such as alcohols, ketones, aldehydes, etc. are more qualified in terms of surface properties,
nanoparticle size, morphological features, and chemical composition. Furthermore, prop-
erties of the solvent and precursor metal salt used in the sol-gel route play a vital role in the
characteristics of the products.
The sol-gel method for the synthesis of metal oxides is often carried out in three steps
including hydrolysis, condensation, and drying processes:
●● Step 1: the precursor salt undergoes hydrolysis process leading to the formation of a
hydroxide solution.
●● Step 2: the hydroxide solution is readily condensed so that three-dimensional gel struc-
tures are formed.
●● Step 3: the prepared gel is subjected to the drying process resulting in the formation of
xerogel or aerogel, based on the methodology applied for the drying process.
Figure 2.11 schematically shows the consecutive steps involved in the reaction pathway
of the sol-gel method.

2.3.2.11 Co-Precipitation
The co-precipitation method is one of the oldest wet chemical methods used for the synthe-
sis of nanomaterials. In this method, a specific ion is precipitated fractionally, followed by
the precipitation of other ions located in the vicinity of the specified ion. The most com-
monly used precipitants in the aforementioned method include hydroxides, chlorides, sul-
fates, carbonates, and oxalates. Some typical examples of the co-precipitation method for
the synthesis of various nanomaterials are given in what follows.
In the case of magnetite nanoparticles, co-precipitation method leads to the formation of
spherical nanoparticles approximately less than 20 nm in size. Petcharoen et al. (Petcharoen
and Sirivat 2012) have synthesized magnetite nanoparticles using ammonium hydroxide as
the precipitating agent, while hexanoic acid and oleic acid served as a coating or capping
agents at the beginning of the crystallization phase of magnetite. The co-precipitation reac-
tion is shown by Eqs. (2.3).
 2.3 ­Nanomaterials Synthesi 41

Precursor
solution

Hydrolysis
Stirring
Condensation

Gel formation

Evaporative Supercritical
drying drying
Drying process

Xerogel Aerogel
Final
compound

Figure 2.11 Schematic representation of the typical reaction pathway in the sol-gel method.
Source: Rao et al. (2017). Copyright © 2017, Elsevier.

Fe2 2 OH Fe OH 2
(2.1)

Fe3 3 OH Fe OH 3
(2.2)

Fe OH 2
2 Fe OH 3
Fe3O4 4 H2O (2.3)

In case the ferric or ferrous ions participate separately in the formation of magnetite
nanoparticles, the reaction mechanism would be different from the mentioned case as fol-
lows (Eqs. (2.4)–(2.9)):

Fe2 2 OH Fe OH 2
(2.4)

3Fe OH 2
0.5O2 Fe OH 2
2 FeOOH H2O (2.5)

Fe OH 2
2 FeOOH Fe3O4 2 H2O (2.6)

Fe3 3 OH Fe OH 3
(2.7)
42 2 Nanomaterials

Fe OH 3
FeOOH H2O (2.8)

12 FeOOH N 2 H4 4 Fe3O4 6 H2O N 2 (2.9)

The authors have declared that the synthesized magnetite nanoparticles reached a larger size
when individual ferrous and ferric ions were applied compared with the primary case where
the mixture of the mentioned ions was applied. The reason lies in the fact that, in the second
case, the transformation of ferrous or ferric ions and subsequently the formation of intermedi-
ates takes more time compared to the first case, in which both ions took part in the reaction
simultaneously, leading to the formation of relatively larger sized magnetite nanoparticles.

2.4 ­Nanomaterial Characterization

Many more types of nanomaterials are explored nowadays than decades ago. This necessi-
tates the implication of advanced techniques for their characterization. Several techniques
have been used to characterize different physical and chemical properties of nanomaterials
such as size, morphology, crystal structure, elemental composition, and other properties of
nanostructures. Also, there is a possibility of evaluating one particular physical property of
nanomaterials by more than one technique. In other words, the results obtained from differ-
ent characterization techniques may interconnect or support each other. Thus, choosing the
most suitable approach for nanomaterial characterization undoubtedly is of great impor-
tance. Herein, we have tried to explain different methods for nanomaterial characterization.
Microscopy-based techniques such as transmission electron microscopy (TEM), scanning
electron microscopy (SEM), atomic force microscopy (AFM), etc. suggest valuable informa-
tion regarding the size and morphology of nanomaterials. For gaining information about
chemical and elemental composition, optical properties and other prevalent characteristics
of nanomaterials X-ray, spectroscopy, scattering, and similar techniques are used. Some of
the techniques are specific to particular types of nanomaterials. For example, vibrating sam-
ple magnetometer (VSM), magnetic force microscopy (MFM), and superconducting quan-
tum interference device (SQUID) are regarded as common techniques to measure the
magnetic properties of the nanomaterials. In the following sections, various common analyti-
cal techniques yet to be used for characterization of nanomaterials are described in detail.

2.4.1 Fourier Transform Infrared Spectrometer

Until the invention of the Fourier transform infrared (FT-IR) spectrometer, three generations
of IR spectrometers existed. The invention of the first IR spectrometer dates back to 1950s.
This spectrometer operated using the prism optical splitting system made of NaCl. The limi-
tations of this generation of spectrometers include the requirement of water in the sample,
narrow scan range, and relatively poor repeatability. Thus, the first generation of IR spec-
trometers is not valid anymore. The application of the second generation of IR spectrometers,
which use gratings as monochrometer, started in the 1960s. Although the performance of the
second generation was much better than the first one, its application still encountered several
obstacles such as low sensitivity, low scan speed, and weak accuracy of wavelengths, which
 2.4 ­Nanomaterial Characterizatio 43

limits its use after the invention of the third generation of IR spectrometers. In the FT-IR
spectrometers, which are considered as the third generation, the interferometer was replaced
by monochrometers. Some of the superiorities of FT-IR spectrometers compared to the previ-
ous generations include a higher signal-to-noise ratio of the spectrum, higher wavenumber
accuracy, shorter scan time for all frequencies (around 1 second), higher resolution (0.1–
0.005 cm−1), and wider scan range (1000–10 cm−1). Due to these valuable advantages, FT-IR
spectrometers readily replaced previous generations of spectrometers (Rodriguez-Saona and
Allendorf 2011; Starsinic et al. 1983). A typical FT-IR spectrometer is composed of different
sections such as a sample compartment, source, interferometer, detector, amplifier, analog to
digital (A/D) converter, and a computer. Radiation produced by the source passes the sample
through the interferometer until it reaches the detector. Using the amplifier and A/D con-
verter the signal is amplified and converted into a digital signal followed by its transfer into a
computer where FT-IR data are documented (Bini et al. 1997; Joffre et al. 1996).
Scientists are able to use FT-IR spectrometers to explore the molecular structure because
molecules absorb infrared radiation. When sample molecules are exposed to infrared radia-
tion they selectively absorb those wavelengths, which cause a change in the dipole moment
of the sample molecules resulting in the transfer of vibrational energy of the molecules
from the ground to excited states. The variations in the dipole moment and the possibility
of energy level transitions are parameters affecting the intensity of absorption peaks, while
the number of absorption peaks is strongly dependent on the vibrational freedom of the
molecules. Except for O2, N2, and Cl2, which experience no dipole change during vibration
and radiation, all of the molecules are infrared active. All three modes of matter (i.e. liquid,
solid, and gas) could be analyzed by FT-IR spectra often in the region between 4000 and
400 cm−1.
The FT-IR spectrum of Cu/Cr/Ni ternary nanoparticles synthesized in a green method
using E. campestre as well as FT-IR spectra of the plant extract before the bioreduction
process and the solution remained after completion of the reaction from centrifugation is
depicted in Figure 2.12. It can be inferred from this figure that the FT-IR spectrum of the
plant extract before and after the bioreduction process has changed in some peak locations.
Some of the intense peaks have disappeared after the bioreduction process and emerged in
the FT-IR spectrum of the nanoparticles. For example, the peak at 2920 cm−1 has disap-
peared from the FT-IR spectrum of E. campestre after the bioreduction but has emerged
with slight change in the peak location (2930 cm−1) in the FT-IR spectrum of the ternary
nanoparticles. The bands at 2920 cm−1 are mainly attributed to the aliphatic and aldehyde
amine groups, indicating that these groups may participate in capping ligand leading to
stabilizing the produced nanoparticles. Furthermore, FT-IR spectra revealed that phenolic
compounds from E. campestre were most probably responsible for the bioreduction of
metal ions and ternary Cu/Cr/Ni production (Vaseghi et al. 2019).

2.4.2 Raman Scattering Spectroscopy

Raman scattering (RS) spectroscopy is a type of vibrational spectroscopy complementary to
the common infrared absorption spectroscopy. Unlike FT-IR spectroscopy, which functions
based on the absorption process, RS spectroscopy is based on an inelastic scattering developed
by a beam of high-intensity laser light (Lindon et al. 2016). In RS spectroscopy, frequencies
44 2 Nanomaterials

1172.72
2370.51

2086.99
(a)

2852.72
2920.23

511.14
1645.28
2370.51

2081.19

1114.86

518.85
3446.79

(b)
Transmittance

1392.61

814

454
(c)

568
1637.56
3446.79

1052
2860

1264
2930

1416
3332

1526
1666

4000 3400 2800 2200 1600 1000 400

Wavenumber (cm–1)

Figure 2.12 FT-IR spectrum of (a) E. campestre aqueous leaf extract before bioreduction process,
(b) solution obtained by centrifugation after the bioreduction process, and (c) ternary Cu/Cr/Ni
nanoparticles with plant capping. Source: Vaseghi et al. (2019). Copyright © 2019, Springer.

associated with the vibrational states of molecules are directly connected to the energy shift of
the photons that are inelastically scattered. Hence, the probed vibrational information of the
molecules is not identical using the two analytical techniques. In other words, vibrations in RS
are detected based on polarizability change, whereas in FT-IR spectroscopy changes in the
dipole moment are monitored. Thus, it can be inferred that due to differences in the disciplines
of the two spectroscopies, it is possible that an intense peak in FT-IR spectroscopy is recog-
nized as a weak one in RS spectroscopy and vice versa. Furthermore, sample preparation in RS
spectroscopy is much easier than FT-IR spectroscopy (Lindon et al. 2016).
It is worth mentioning that the laser beam has the potential to focus on diffraction-limited
spots from the order of 0.5 μm. Thus, RS spectroscopy is capable of reporting a combination
of molecular and crystalline information with high resolution leading to the analysis of
micrometric to nanometric scale samples (Lewandowska and Liu 2010). RS spectroscopy
 2.4 ­Nanomaterial Characterizatio 45

has yet been extensively used to assess the vibrations and local atomic arrangements of
­nanomaterials, which provide routes to study the structural nature of nanomaterials. It is
proved that, as the particle size decreases, the Raman shift and bandwidth vary. Also, with
an increase in grain size sharper and stronger peaks appear in RS spectra with a slight shift
toward higher wavenumber (Bhaumik et al. 2014). For instance, the spectrum can be used
for determination of oxygenated groups in graphene oxide (Badrinezhad et al. 2018) or, as
an example, Raman spectra of ZnO nanoparticles synthesized via spin coating by annealing
at different temperatures (i.e. 300 °C, 350 °C, 400 °C, 450 °C, 500 °C, and 550 °C) is depicted
in Figure 2.13(a–e) (Kaviyarasu et al. 2017). It is clear that in all of the deposited thin films

1062.1
698.5

718.9
438.9

541.0
579.0
408.1

590.1
203.2

331.9

(e)

1051.4
583.7
438.9

695.1
205.1

330.8

(d)
438.9
Intensity (arb. units)

583.3
330.9
204.4

(c)
438.9

1054.4
537.3
584.7
203.2

332.4

(b)
438.6

1051.3
537.7
203.0

588.4
332.4

(a)

200 400 600 800 1000 1200

Raman shift (cm–1)

Figure 2.13 a–e Raman spectra of ZnO nanoparticles at different temperatures. Source:
Kaviyarasu et al. (2017). Copyright © 2017, Elsevier.
46 2 Nanomaterials

significant bands of ZnO are evident. In the RS spectra of ZnO, the E2 (high) mode at the
band at 439 cm−1 is indicative of crystal quality of ZnO. The bands at 537 cm−1 and 588 cm−1
are assigned to E1 (LO) mode contributed to oxygen atoms. The peaks at 698.5 cm−1 and
718.9 cm−1 are attributed to the 2E1 (LO) mode. Finally, the appeared bands in the range of
1050 cm−1–1200 cm−1 are considered as overtones or combination bands in ZnO.

2.4.3 Ultraviolet-Visible Spectroscopy

Ultraviolet-visible (UV-Vis) spectroscopy is a quantitative analytical technique extensively
used in nanotechnology-based sciences for studying the optical properties of nanomateri-
als. In this technique, the absorption of near ultraviolet (180–390 nm) or visible (390–
780 nm) radiation by chemical moieties in the liquid or gas phase is investigated. The
radiations in the mentioned regions cause electronic transitions of the molecules. As a
simple explanation, the amount of the absorbed radiation is directly proportional to the
analyte concentration in the solution (Beer’s law).
In organic compounds, the radiation energy is usually absorbed in the near ultraviolet
region generally as a result of σ→ σ*, n→ σ, n→ π*, and π→ π* transitions. These transitions
commonly occur on molecules such as aromatic and conjugated aliphatic species. Also,
some of the inorganic compounds such as transition metal ions and their aqueous com-
plexes, which are generally colored, are known to be detected with relatively sharp absorp-
tion peaks in the visible region (Worsfold et al. 2019).
UV-Vis apparatus mainly consists of a radiation source, sample compartment, wave-
length selection device, detector, and output device. UV-Vis spectrophotometers are usu-
ally classified as single-beam or double-beam devices. Double-beam instruments are not
as simple as single-beam ones but have some advantages such as the capability to adapt
themselves in the conditions where the light is stray, the intensity of the source radiation
fluctuates, or some solid bodies exist in the sample solution. Another popular configura-
tion is known as diode array spectrophotometers, in which the polychromatic coming
from the source crosses through the sample followed by its dispersion into a linear diode
array detector.
UV-Vis spectroscopy is usually the first analysis used for the confirmation of nanoparti-
cle synthesis based on the surface plasmon resonance (SPR) of the free electrons. For exam-
ple, UV-Vis spectra of colloidal solution containing trimetallic oxide Cu/Cr/Ni nanoparticles
synthesized using the leaf extracts of E. campestre, F. subpinnata, and their 50:50 mixture,
is shown in Figure 2.14. Maximum absorption peak at around 220 nm was observed for the
nanoparticles synthesized using the three types of leaf extracts showing the SPR of the
trimetallic oxide nanoparticles. Also, the extract solutions turned into colloidal solutions
after the formation of nanoparticles.

2.4.4 X-Ray Diffraction

X-ray diffraction (XRD) is a prominent and non-destructive technique for identifying
the crystalline structure, chemical composition, and physical properties of nanomateri-
als (Sima et al. 2016). Using XRD, one can get valuable information on structures, phases,
preferred crystalline orientations, average crystallite/grain size, etc. (Kohli 2012).
 2.4 ­Nanomaterial Characterizatio 47

(a)
3

NPs from E. campestre Extract

2.5
NPs from F. subpinnata Extract
NPs from Mixed Extract
2
Absorbance

1.5

0.5

0
190 290 390 490 590
Wavelength (nm)

(b) (c)

Figure 2.14 (a) UV-Vis spectra of trimetallic oxide Cu/Cr/Ni NPs obtained from E. campestre leaf
extract (dark black line), F. subpinnata leaf extract (black line) and mixed extract (grey line), (b and c)
representation of plant extracts and biosynthesized trimetallic oxide NPs; EC represents for E.
campestre, FS represents for F. subpinnata, and M represents for mixed plant extract. Source: Vaseghi
et al. (2018c). Copyright © 2018, Elsevier.

The phenomenon that mainly occurs during XRD is the elastic scattering of X-ray pho-
tons by crystal planes in a periodic lattice. Lattice spacings can be derived from Bragg’s
law as ­follows (Eq. (2.10)):

n 2 d sin (2.10)
48 2 Nanomaterials

where n is the order of reflection, λ is the X-ray wavelengths, d is the defined spacing
between crystal planes of a sample and θ is the angle between the incident or the reflected
beam and the crystal plane (Kacher et al. 2009). In addition, the crystalline grain size is
determined by the Scherrer equation through broadening the most intense peak in the
XRD pattern of the sample as follows (Eq. (2.11)):

k
dc (2.11)
B cos

where dc is the crystallite diameter, λ is the X-ray wavelength (λ = 0.1542 nm), k is the shape
factor (0.9–1), θ is the Bragg angle (in radian) and B is the full width at half maximum (in
radian) (Vaseghi et al. 2019).
Also, the composition of the unknown nanomaterial can be identified by comparing the
position and intensity of the intense peaks in the XRD pattern of the sample with the refer-
ence patterns from the International Center for Diffraction Data (ICDD) database. In addi-
tion, for a particular type of synthesized nanomaterial, comparing with the existing valid
publications would be a great help.
As an example, the XRD pattern of the synthesized Cu/Cr/Ni nanoparticles by Vaseghi
et al. (2019) is depicted in Figure 2.15. The presence of all three metals is confirmed at the
intense peak locations (36.73° (A), 43.48° (B) and 63.14° (C)). Moreover, by interpreting
XRD pattern using X’Pert HighScore plus software they have found that the peak locations
were attributed to Cr2O3 and CuO for the position A, NiO, Cr2O3, Cu and Cr for position B
and Cr2O3 and NiO for position C. Also, the average crystallite size was calculated separately
for the locations A, B, and C using Scherrer formula as 177, 164, and 164 nm, respectively.

2.4.5 X-Ray Fluorescence

X-ray fluorescence (XRF) is extensively used to show the distribution of elements in a solid
nanomaterial as well as imaging. XRF detects the secondary X-ray, which is emitted by the
elements as a result of a primary high-energy beam of radiation. XRF has been applied

200
A B C
Ni
160 Cu Cu Ni
Cr Cr Cr
Counts

120

80

40
10 20 30 40 50 60 70 80
Position-(2θ˚)

Figure 2.15 XRD pattern of Cu/Cr/Ni nanoparticles synthesized using E. campestre leaf extract.
Source: Vaseghi et al. (2019). Copyright © 2019, Elsevier.
 2.4 ­Nanomaterial Characterizatio 49

particularly in biomedicine to detect and measure trace metals such as lead in the organs
near the skin (Ahlgren et al. 1976). Other examples include determination of the level of
gold after chrysotherapy (Scott and Lillicrap 1988) and identification of the maintenance of
platinum and gold in cancer and rheumatology patients (Börjesson and Mattsson 2007).
Also, a quantitative procedure based on XRF was employed to measure the concentration
of gold nanoparticles accumulated in the tumor (Hainfeld et al. 2011; Khlebtsov and
Dykman 2011).
It is worth noting that XRF is the only technique in the elemental analysis that is need-
less of sample pretreatment procedures such as vaporization or digestion (Zhang and
Cresswell 2016). As for nanoparticles, the XRF instrument has two distinct functions:
determining the position of the nanoparticle in the wafer and its elemental analysis. XRF
spectrum of Cd nanoparticles synthesized by Thema et al. (2015) in a green approach using
the leaf extract of Agathosma betulina is depicted in Figure 2.16. The pure chemical com-
position of CdO nanoparticles is confirmed by XRF spectrum as the double Cd L-band is
placed within 3.133–3.528 keV.

2.4.6 X-Ray Photoelectron Spectroscopy

X-ray photoelectron spectroscopy (XPS) is a surface-analyzing technique that provides val-
uable information regarding the chemical composition of the surface of nanomaterials as
well as the binding states of the elements usually under ultra-high vacuum conditions. The
composition of nanomaterials is identified along the direction of the electron beam (Mather
2009). XPS can be used to interpret the interactions during ligand exchange in nanoparticles

Excitation CuKα

Cd L-line
Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10
Energy (keV)

Figure 2.16 Typical X-ray Fluorescence spectrum of the annealed CdO nanoparticles at 500 °C for
2 hours using CuKa radiation as an excitation source. Source: Thema et al. (2015). Copyright © 2015,
Elsevier.
50 2 Nanomaterials

especially for core/shell structures and also as a tool to discriminate between uniform alloy
and core/shell structures. The main advantages of the XPS analytical method are that it
provides depth information usually up to 10 nm depth from the surface and is fast, simple,
and non-destructive for the sample. On the other hand, long sample preparation and inter-
pretation times for the obtained data are considered to be the major obstacles encountered
by researchers (Barth et al. 1988; Carlson 1982; Laajalehto et al. 1997; Tougaard and
Ignatiev 1983).
In this technique, the sample is irradiated with monochromatic X-rays with constant
energy hv leading to the emission of photoelectrons with kinetic energy EK, energies of
which are specific to the elements placed inside the sampling volume (Kohli and Mittal
2011). The basis of XPS function is depicted in Figure 2.17. The balance between hv and EK
is given by (Eq. (2.12)):

hv EK EB (2.12)

where EB is defined as the binding energy of electron to nucleus with respect to Fermi level
and ϕ is a work of function of a solid sample (Konno 2016).
XPS has a wide range of applications in analytical fields among which thin films are of
great importance owing to the short mean free path of electrons through a condensed body
(Bluhm 2011). It has also been used to characterize different metal and metal oxide nano-
particles. For example, ZnO thin films synthesized using spin coating method were charac-
terized by XPS to identify the chemical composition of the synthesized nanomaterial
(Figure 2.18). The XPS spectrum reveals that only C, Zn, and O signals are observed. In
addition, the core level spectrums of Zn2p3/2 and Zn2p1/2 correspond to the binding ener-
gies of 1022.1 and 1044.2 eV that are representative of Zn (Kaviyarasu et al. 2017). Most of
the metal oxides such as CuO, TiO2, and CoO undergo reduction when exposed to XPS. For
instance, Wu et al. (2006) demonstrated that the process of reduction started when CuO
nanoparticles were exposed to photoelectrons from the radiation source and stopped in the
absence of photoradiation or when a bias voltage was applied to the sample. In such cases,
identifying the exact chemical composition of the nanoparticles would be difficult.

Electron energy analyser

X-ray Source

hv
photoelectrons

K.E.=hv-B.E.-Φ

Sample

Figure 2.17 Schematic representation of XPS operation.

 2.4 ­Nanomaterial Characterizatio 51

8 × 105

Zn 2p3/2
Zn 2p1/2
7 × 105

6 × 105
Counts (arb. units)

O 1s
5 × 105

4 × 105 C 1s
3 × 105

2 × 105

1× 105
0
0 200 400 600 800 1000 1200
Binding Energy (eV)

Figure 2.18 XPS spectrum of ZnO nanoparticles. Source: Kaviyarasu et al. (2017). Copyright ©
2017, Elsevier.

2.4.7 Energy Dispersive X-Ray Spectroscopy

Energy dispersive X-ray spectroscopy (EDS) is a quantitative and qualitative X-ray tech-
nique mainly utilized to identify the elemental composition of nanomaterials. It gives
atomic and weight percentage of the containing elements in the nanomaterials (Scimeca
et al. 2018). Typically, SEM apparatus is equipped with an EDS system to chemically ana-
lyze the matter observed in the monitor. When a nanomaterial is bombarded with electrons
in SEM instrumentation, the produced characteristic X-rays can be detected by an EDS
system (i.e. a device which is capable of discriminating among X-ray energies). This cou-
pled system of SEM–EDS is advantageous particularly in cases of failure analysis in which
spot analysis plays a crucial role (Nasrazadani and Hassani 2016). Other advantages of this
analytical system include fast and facile elemental analysis of nanomaterials, two-dimen-
sional elemental mapping of the elements, and high count rates for low-beam currents
(Michalak et al. 2011; Wang and Hou 2007).
As an example of nanomaterial synthesis using EDS equipped by selected area elemental
mapping, consider the trimetallic oxide Cu/Cr/Ni nanoparticles synthesized by Vaseghi
et al. (2018c). Based on data obtained from EDS (Figure 2.19), the existence of all the three
metals along with a relatively high percentage of oxygen is confirmed with the specified
atomic and weight composition. Also, selected area elemental mapping of the nanopowder
in which the target elements are shown with different colors represent the non-homoge-
nous distribution of elements in the trimetallic structure.

2.4.8 Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy provides information about the molecu-
lar structure of molecules on the surface of nanostructures and a means of measuring the
size of nanoparticles in solution as well. NMR spectroscopy can be performed in different
52 2 Nanomaterials

(a)

Atomic percentage Certainty

O 62.4 0.99
Cu 11.1 % 0.98
Cr 9.9 % 0.99
Si 4.4 % 0.98
K 3.1 % 0.98
P 2.3 % 0.97
Al 2.0 % 0.96
S 1.9 % 0.97
Ni 1.9 % 0.93
Ca 1.0 % 0.95
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(b)

Weight percentage Certainty

O 35.7 % 0.99
Cu 25.2 % 0.98
Cr 18.3 % 0.99
Si 4.4 % 0.98
K 4.3 % 0.98
Ni 4.0 % 0.93
P 2.5 % 0.97
S 2.2 % 0.97
Al 1.9 % 0.96
Ca 1.5 % 0.95
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(c)

SEM image O Cr

Ni Cu OCrNiCu

Figure 2.19 EDS spectra of trimetallic oxide Cu/Cr/Ni NPs; (a) Atomic %, (b) Weight %, and (c) selected
area elemental mapping of synthesized trimetallic oxide NPs using the three types of extracts. Source:
Partly taken from Vaseghi et al. (2018c). Copyright © 2018, Elsevier.
 2.4 ­Nanomaterial Characterizatio 53

operational modes such as liquid state, solid state, one dimensional, two dimensional, and
multidimensional in which case nanomaterials’ basic properties including composition,
molecular weight, dynamics, stoichiometry, etc. can be elucidated. It is worth mentioning
that NMR spectroscopy is capable of analyzing nanomaterials in both suspensions and col-
loidal forms with the least sample loss during analysis (Agarwal et al. 2018).
The principle of NMR analysis is devoting magnetic properties to the nuclei of atoms and this
leads to the understanding of the molecular and chemical environments. the spinning motion
of subatomic constituents such as electrons, protons, and neutrons around their own axes can
be imaged. A magnetic dipole is generated along the direction of the spin axis as a consequence
of summation of nucleus spinning. It is believed that a nucleus with the net spin I can align
with an external magnetic field in 2I + 1 ways. a Schematic of the function of NMR spectroscopy
is depicted in Figure 2.20. The term Larmor frequency (ω0) is directly proportional to the gyro-
magnetic ratio (γ) of the nucleus and the external magnetic field (B0) as (Eq. (2.13)):

0 B0 (2.13)

2.4.9 Scanning Electron Microscopy

Electron microscopes have developed due to the incapability of optical microscopes to
observe nanomaterials with high magnification. In contrast to optical microscopes, which
utilize photons, electron microscopes make use of electrons, which possess a much shorter

B0 B0 B0
ω0 = γB0
M0

Larmor precession under Orientation of Net magnetization (M0)

external magnetic field (B0) magnetic moments

90˚— RF pulse

‘H NMR spectrum of ethanol B0

CH3

OH CH2 FT

TMS M0
Time domain signal/
8 6 4 2 0 free induction decay
Chemical shift (δ)—1H (ppm) (FID)
NMR spectrum Transverse
magnetization

Figure 2.20 Schematic representation of the function of NMR spectroscopy. Source: Agarwal et al.
(2018). Copyright © 2018, Elsevier.
54 2 Nanomaterials

wavelength than photons, and this makes objects with atomic resolution observable.
Generally, two types of electron microscopy namely SEM and TEM are used frequently.
Sample preparation in SEM is a rather simple task with no requirement to produce very
thin slices. SEM is mainly applied in analyzing the images of micron or nanometric sizes.
Under good conditions, resolution of the images from SEM can be as small as 3 nm
(Brabazon 2012). When the sample is placed in its proper place in the SEM instrument,
scanning on the sample area using a concentrated beam of electrons leads to the generation
of various output signals. During the course of scanning, the output signals will change
according to the nature of the surface at the area and, consequently, the three-dimensional
structure of the specimen with the fine resolution is imaged (Webb and Holgate 2003; Zhou
and Li 2015). To be more specific on the function of SEM, in the first step, the firing of
primary electrons to be imaged from the sample occurs. Then, secondary electrons (i.e.
those electrons that left the sample and attracted to a positively charged detector) are
recorded, resulting in the displaying of the surface topology of the sample (Brabazon 2012).

2.4.10 Field Emission Scanning Electron Microscopy

Due to some limitations of SEM, especially in imaging nanomaterials with such high reso-
lutions, other methods of imaging such as FESEM were developed. FESEM yields topo-
graphical images with magnifications of the order of approximately 10–300 000 with an
unlimited depth. The resolution of images in FESEM is three to six times enhanced com-
pared to the conventional SEM. FESEM has numerous advantages such as compatibility of
electron voltage with EDS, high quality and low voltage of the images, and no requirement
for coating insulating substances with conducting materials (Mayeen et al. 2018).

2.4.11 Environmental Scanning Electron Microscopy

ESEM is typically a type of high-pressure SEM that has been developed to take morphological
images of wet samples by applying partial water vapor pressure in the sample cavity. The most
striking feature of ESEM is that wet and/or uncoated samples can be analyzed by this instru-
mentation in their native form without the requirement of being dehydrated. Therefore, this
method of analysis is exceptionally appropriate for biological samples where the dehydrating
process may defect the natural characteristics of the moieties. Dehydration of cement is another
example of a chemical reaction that can be imaged by ESEM (Henning and Adhikari 2017;
Mayeen et al. 2018). This issue is of particular importance because controlling the reaction of
the grains with water is imperative for creating a high-quality cement (Liebscher et al. 2018).

2.4.12 Transmission Electron Microscopy

TEM is generally similar to SEM. as its name implies, the only exception is that, unlike
SEM, the electron beam passes through the sample in TEM. Thus, this technique is usually
used to identify the internal structure of the sample, whereas in SEM a picture is taken
from the surface of the sample. In TEM, an extremely focused electron beam with voltage
as high as 80–200 KeV is passed through a solid yet thin sample of typically 100–200 nm
followed by the scattering or diffraction of electrons from lattice planes resulting in phase
 2.4 ­Nanomaterial Characterizatio 55

identification (Ebnesajjad and Ebnesajjad 2013). Figure 2.21 shows the FESEM and TEM
images of Cu/Cr/Ni nanoclusters produced using the reducing ability of E. campestre and
F. subpinnata leaf extracts reported by Vaseghi et al. (2018c, 2019). The images clearly
­display that the nanoclusters produced using E. campestre are in three distinct morpholo-
gies and sizes, while F. subpinnata led to the synthesis of relatively homogeneous plate-like
structures with almost similar sizes. In a TEM image, the area that contains high-density
matrix appears darker as more scattering of the electrons happens within this area. The
lighter area, on the other hand, represents low-density material as more electrons are
­transmitted through (Gouran-Orimi et al. 2018).

(a) (b)

(c) (d)

Figure 2.21 FESEM and TEM images of Cu/Cr/Ni metal oxide nanoclusters synthesized using
(a) and (c) E. campestre and (b) and (d) Froriepia subpinnata, Source: Partly taken from Vaseghi et al.
(2018c, 2019). Copyright © 2019, Springer and Copyright © 2018, Elsevier.
56 2 Nanomaterials

2.4.13 High-Resolution Transmission Electron Microscopy

High-resolution transmission electron microscopy (HRTEM) has been used over the past
four decades for studying the morphology of nanomaterials. The images obtained by HRTEM
are more evident in the nanostructure of nanomaterials than any other microscopic tech-
nique. HRTEM directly takes high-resolution images of the atomic structure of nanomateri-
als. Therefore, direct information is provided about the crystallographic structure of the
materials (Herrera-Ramirez et al. 2019). Unlike the typical TEM, which looks at the absorb-
ance of the beam, HRTEM looks at the way that interference occurs between direct transmis-
sion and diffracted electron beams. Under these conditions, the contrast of the image is
related to the relative phases of the existing beams. Hence, the phrase “phase–contrast imag-
ing” is selected for the imaging mode (Kumar et al. 2016). HRTEM is mainly used in the
detection of crystalline defects in the micro and nanostructures, interfaces, and grain bound-
aries. Other applications of HRTEM include characterizing the morphology, size, and
arrangement of nanoparticles, nanofibers, thin films, and multilayers (Mayeen et al. 2018).

2.4.14 Atomic Force Microscopy

AFM is one of the versatile microscopic techniques used for nanomaterial characteriza-
tion. In 1986, Binnig and colleagues (Ohnesorge et al. 1997) invented AFM enabling them
to produce surface images on conductors and insulators with atomic resolution. This inven-
tion is regarded as a milestone in nanotechnology and has created new opportunities in
filed such as physics, chemistry, biology, medicine, etc. In this technique, a sharp tip of
usually 5–10 nm made from silicon nitride at the end of a cantilever is used to scan over a
surface and measure the forces between the surface of the specimen and the tip in the
course of scanning process, providing topographical images (Hoi et al. 2018). The images
resulting from AFM may be of a few nanometers to hundreds of micrometers in scale
according to the special application of the methodology.
AFM is used for three-dimensional characterization of nanomaterials compared to other ana-
lytical techniques for nanomaterial characterization. Data obtained from AFM is useful in terms
of identifying geometries of nanoparticles, probing into hydrated nanoparticles, determining
some physical properties of nanomaterials, studying the properties of soft and hard nanomateri-
als, and especially measuring the surface roughness (Habibi and Nematollahzadeh 2016).
In order to obtain the desired resolution, AFM apparatus measures the vertical and lat-
eral deflections using the optical lever. In a typical standardized procedure, the optical lever
works by reflecting a laser beam off the cantilever. The reflected beam strikes a four-seg-
ment photodetector, which is position sensitive. The angular deflections of the cantilever
resulting from the location of the laser spot on the detector can be measured by the differ-
ences between the segments of signals of the photodetector. In order to minimize sample
damage, the distance between tip and sample is adjusted so that the deflection of the can-
tilever maintains a constant value. Figure 2.22a depicts the main parts of AFM apparatus.
In practice, two imaging modes are defined for AFM when imaging. In the so-called tap-
ping or non-contact mode, the tip oscillates when scanning the sample. Unlike the contact
mode, this scanning mode is suitable for biological samples to prevent damaging and dis-
torting by the sharp tip (Figure 2.22b) (Hansma 2014; Thomas et al. 2017).
 2.4 ­Nanomaterial Characterizatio 57

(a) (b)
Laser Contact Mode
Segmented
Photodiode

Cantilever Tapping or
with Tip Non-contact Mode

Sample

XYZ Scanner

Figure 2.22 (a) Diagram of a typical AFM apparatus, (b) different imaging modes of AFM. Source:
Hansma (2014). Copyright © 2014, Springer.

2.4.15 Vibrating Sample Magnetometer

VSM is a direct magnetic measurement technique first developed by Foner in the 1950s
(Foner 1956). In the VSM instrument, the microscopic magnetization of the sample is
sensed and measured. To be more specific, a magnetic material is vibrated in a uniform
magnetic field composed of an electromagnet or a superconducting magnet leading to the
generation of an electric current in the sensing coils. The voltage generated in the coils is
proportional to the magnetic moment of the specimen. VSM analysis follows Faraday’s law
of induction pointing out how an electric current produces a magnetic field and, con-
versely, how a changing magnetic field produces an electric field. The measurement of the
produced electric field gives information about the changing magnetic field (Nasrollahzadeh
et al. 2019).
VSM is a facile, versatile, and easy method for studying the magnetic properties of nano-
materials. To expand the temperature limit below and above room temperature, VSM can
use a cryostat or oven, respectively. One of the drawbacks associated with VSM is its low
resolution compared to SQUID magnetometer. From the graph obtained from VSM analy-
sis, magnetization, hysteresis, and coercivity of magnetic nanomaterials (ferromagnetic or
ferrimagnetic) can be elucidated. α-Fe2O3 is one of the important nanomaterials whose
magnetic properties have been studied extensively. For example, in research performed by
Raja et al. (2015), magnetic behavior of α-Fe2O3 nanoparticles was investigated using VSM.
Magnetic hysteresis loop of α-Fe2O3 nanoparticles at room temperature is depicted in
Figure 2.23. The sample exhibited weak ferromagnetic behavior with a noticeably broad-
ened hysteresis loop. Also, magnetic parameters including coercivity, retentivity, and satu-
ration magnetization of α-Fe2O3 nanoparticles were measured to be 3891 G, 0.1617 emu/g,
and 0.4193 emu/g, respectively.
The absence of the hysteresis loop in the VSM graphs can be an indication of the super-
paramagnetic property of the nanoparticles. For instance, γ-Fe2O3 nanosized nanoparticles
with size less than 20 nm shows superparamagnetic property (Mirzayi et al. 2015).
58 2 Nanomaterials

0.03

0.02
Magnetic moment (e mu)

0.01

0.00

–0.01

–0.02

–0.03
–20000 –15000 –10000 –5000 0 5000 10000 15000 20000
Applied Magnetic field (G)

Figure 2.23 Hysteresis loop of α-Fe2O3 nanoparticles. Source: Raja et al. (2015) Copyright © 2015,
Elsevier.

2.4.16 Superconducting Quantum Interference Device

SQUID is a type of magnetometer used to measure the magnetization of tiny samples such
as thin films and those samples that are in the limit of magnetization. Hence, SQUID is
extensively used by scientists throughout the world to determine the magnetization of
materials. Two physical phenomena of SQUID operation include flux quantization and
Josephson tunnel (Grössinger and Sato 2001). Flux quantization in a closed superconduct-
ing loop in units of the flux quantum is given by (Eq. (2.14)):

h 15
0 2.07 10 Wb (2.14)
2e

In superconductors, Cooper pairs with zero spin are responsible for carrying the current.
All of these pairs condense in the same quantum state being described by a collective
superconducting wave function as follows (Eq. (2.15)):

i x ,t
x, t 0e (2.15)

where ϕ(x, t) is the time and phase dependent phase. Also, the Cooper pair density (ns) is
given by (Eq. (2.16)):
2
ns (2.16)
 2.4 ­Nanomaterial Characterizatio 59

In the case where there is a weak connection between two superconductors, Cooper pairs
are capable of exchanging between them. Different arrangements for these weak junctions
exist; the most important one being the so-called superconductor–insulator–superconduc-
tor (SIS) Josephson tunnel junction in which a thin insulating barrier (I) is located between
two superconductors (S) (Schmelz and Stolz 2017).
During recent decades, nano-SQUID (i.e. a SQUID with an extremely small sensitive
area and a sub-micrometric loop diameter being in the range of 100–200 nm) has been
applied to measure the magnetization of nanomaterials (Granata et al. 2008). Nano-SQUID
offers the advantage of direct measurement of magnetic behavior in a small spin system
and detection of single electron spin-flips compared to other magnetization detection
methods. A miniaturized or nano-SQUID is fabricated by placing a SQUID loop on the
apex of hollow quartz pulled into an extremely sharp pipette (Granata and Vettoliere 2016).
Kopanja et al. (2016) have investigated the magnetization of hematite (α-Fe2O3) nanoparti-
cles synthesized using sol-gel combustion method using SQUID magnetometer. Two mag-
netization measurements were performed. The first one was zero-field-cooled magnetization
(ZFC) in which the nanoparticles were first cooled to 5 K, in the absence of a magnetic
field. Then, the temperature of the sample was raised to 300 K by applying a magnetic field.
After that, the magnetic moment was measured. Field-cooled (FC) magnetization meas-
urement was done when the sample was cooled to 5 K in the presence of a magnetic field
and the measurement of magnetization was carried out when the sample was heated to
300 K in the presence of the same magnetic field. The resulting ZFC-FC data are exhibited
in Figure 2.24 as the blocking temperature and the irreversibility temperature were
TB = 27 K Tir = 51 K, respectively. The authors stated that, for temperatures higher than
blocking temperature, magnetization decreases at low speed with enhancement in tem-
perature. For temperatures lower than blocking temperature, rise in temperature of more
than 5 K leads to increase in magnetization owing to unfreezing of the magnetic moments
of nanoparticles along with the orientation of the moments in the direction of the applied
magnetic field. Also, Figure 2.24b shows the ZFC hysteresis loop, which was measured
after cooling the sample from 300 K to 5 K. The value of magnetization parameters such as
coercivity, remanent magnetization, and saturation magnetization at 5 K was found to be
1310 Oe, 1.85 emu/g, and 16.36 emu/g, respectively. The authors have also found that the
FC hysteresis loop is broadened at 5 K (Coercivity value: 1440 Oe) and shifted in the direc-
tion of the negative applied field.

2.4.17 Magnetic Force Microscopy

MFM is a slow-scan, raster-type imaging technique at the nanoscale with the highest reso-
lution. This technique provides information regarding stray field distribution above the
sample, which is in direct relation with the magnetization behavior of the sample. Although
the operation is relatively simple, the data obtained from MFM need to be interpreted very
carefully in order to be reliable (Klapetek 2018). MFM provides images with resolutions
below 20 nm and has no requirement for sample preparation. This imaging technique can
be conducted either at ambient conditions or ultra-high vacuum. The latter is suitable for
studying the clean magnetic surfaces and the relationship between the growth and mag-
netic properties. MFM is an indirect imaging method since it is based on the interaction of
60 2 Nanomaterials

(a)

0.14
FC
0.12
0.10 MZFC
MFC
M [emu/g]

0.08

0.06
H = 50 Oe
0.04

0.02

0.00 ZFC

0 50 100 150 200 250 300

T [K]
(b)

15
T=5 K
10
ZFC
5
M [emu/g]

0 4

2
–5
M [emu/g]

–10 –2

–4
–15 –2 –1 0 1 2
H [kOe]

–45 –30 –15 0 15 30 45

H [kOe]
(c)
0.4
15
M [emu/g]

0.3

10 0.0

–0.3 T=5 K
5 –0.4 FC
M [emu/g]

–1.0 –0.5 0.0 0.5 1.0

0 H [kOe]
4

2
–5
M [emu/g]

–10 –2

–4
–15 –2 –1 0 1 2
H [kOe]

–45 –30 –15 0 15 30 45

H [kOe]

Figure 2.24 (a) Temperature dependence of the zero-field-cooled (ZFC) and the field-cooled (FC)
magnetization in a field of 50 Oe, (b) ZFC hysteresis loop at 5 K, and (c) FC hysteresis loop at 5 K.
Source: Kopanja et al. (2016). Copyright © 2016, Elsevier.
 2.4 ­Nanomaterial Characterizatio 61

two magnetic bodies, the probe and the sample (Rave et al. 1994). MFM is applied in the
magnetic studies of nanomaterials in order to clarify the effect of structural characteristics
on magnetic properties. Besides various applications of MFM such as solid state physics
research, magnetic recording projects, and superconducting phenomena, special effort has
been devoted to using this technique in chemical and nano-biological areas mainly to study
the magnetic properties of nanoparticles (Nasrollahzadeh et al. 2019).

2.4.18 Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) is a thermoanalytical technique in which heat flux
to a sample or the power-compensation is monitored based on time or temperature.
Practically, the difference in heat flux to a chamber containing the sample and to an empty
pan is monitored and compared to each other. DSC apparatus involves two identical (based
on heat losses) measuring cells. The sample is placed in one of the cells, while the other cell
is the reference holder. The cells are separately heated up to a defined temperature and
separate sensors are used for the measurement of temperature in the cells. The tempera-
ture of both cells, which is controlled by an average-temperature control loop, can be varied
linearly. As soon as some exothermic or endothermic processes result in a temperature
gradient, the second differential control loop starts its function to adjust the heat or power
supply. Finally, the differential heat or power supply is recorded as a function of the precise
temperature of the sample (Groenewoud 2001). As an example of the application of DSC
analysis, Wang et al. (2017) have performed DSC characterization on the graphite nanopar-
ticles dispersed into a phase change emulsion containing 30 wt.% of OP10E (a commercial
organic phase change material). The DSC thermograms of the phase change emulsions
with different weight fractions of graphite nanoparticles are depicted in Figure 2.25. The
figure clearly shows that unlike the freezing points, which vary depending on the mass
fraction of the samples, the melting points remain approximately constant.

2.4.19 Thermogravimetric Analysis

Thermogravimetric analysis (TGA) is an analytical technique mainly utilized to measure
the thermal stability of nanomaterials and volatile or combustible fraction. Other parame-
ters being studied using TGA include life expectancy, activation energy, thermal stability,
and decomposition profile. TGA functions by monitoring the change in the weight of the
sample when heated at a constant rate (Polini and Yang 2017; Rajisha et al. 2011).
Thermogravimetry was developed for the first time in the 1900s. TGA can be used to deter-
mine the weight loss of material components as a result of decomposition, oxidation, and
volatile loss. Thermogravimetric experiment results in a plot of mass as a function of time
or temperature. Electromagnetic weight compensation enables repeated mass recording
and compensations (Buschow et al. 2001).
As for nanomaterials, the heating results in decomposition and vaporization of compo-
nents with different degradation temperature and mass change. Considering the initial
mass of a smaller sample, organic ligands of the nanomaterial sample can be identified
both quantitatively and qualitatively. TGA was used to study the thermal stability of a
wide range of nanomaterials. For example, the purity and thermal stability of CNTs were
determined using TGA by Mansfield et al. (2010). They have concluded that TGA is
62 2 Nanomaterials

(a)
Exo
4.0 wt% graphite
2.0 wt% graphite
1.0 wt% graphite
Heat flow (a.u.)

0.5 wt% graphite

0.25 wt% graphite
0.0 wt% graphite

Endo
–10 –5 0 5 10 15
Temperature (˚C)

(b)
Exo

4.0 wt% graphite

Heat flow (a.u.)

2.0 wt% graphite

1.0 wt% graphite

0.5 wt% graphite

0.25 wt% graphite

0.0 wt% graphite

Endo
–10 –5 0 5 10 15 20
Temperature (˚C)

Figure 2.25 DSC curves of the graphite nanoparticles-dispersed phase change emulsions with
different mass fractions of graphite nanoparticle: (a) melting curves; (b) freezing curves. Source:
Wang et al. (2017). Copyright © 2017, Elsevier.

extremely sensitive to allow quality control in both macroscale (carbon-to-metal ratio)

and nanoscale (single-walled to multi-walled ratio). Recently, a technique called micro-
thermogravimetric analysis (μ-TGA) with the same principles as conventional TGA except
for the small mass of the sample (i.e. 1 μg) has been applied, which is able to detect mass
change of less than 1 ng. Thus, μ-TGA is more promising than conventional TGA since it
uses far less sample and also improves the detection limit of conventional TGA (Mourdikoudis
et al. 2018; Sebby and Mansfield 2015). Sebby and Mansfield (2015) used the technique to
determine the amount of PEG bound to the gold nanoparticles to avoid the high consump-
tion of nanomaterials in conventional TGA. μ-TGA assumes that, except gold metal, every
component will decompose. Also, the results obtained from μ-TGA fluorescence were
used to evaluate the size of ligand footprint (i.e. average area occupied by each ligand on
the particle’s surface).
 2.4 ­Nanomaterial Characterizatio 63

2.4.20 Brunauer–Emmett–Teller Physisorption Method

The Brunauer–Emmett–Teller (BET) method generally provides information regarding the
specific surface area in terms of probing gas adsorption isotherm measurements. BET is
named after its developers, Brunauer, Emmett, and Teller. BET is recognized as a simple,
precise, and rapid method for pore size distribution, pore volume, and the surface area
measurement of materials. The most commonly used adsorbate probing gas is known to be
nitrogen. Thus, the measurements are carried out mainly at the boiling temperature of
nitrogen (i.e. 77 K). In order to make measurements at different temperatures practically
feasible, other probing adsorbates including argon, carbon dioxide, and water are also uti-
lized. The main assumption in the BET analysis is the multilayer adsorption of a gas on the
surface of the adsorbent. The probing gases used in this analytical technique are such that
do not chemically react with the surfaces of the adsorbate (Hanaor et al. 2014; Jaroniec
et al. 1998). Nematollahzadeh et al. (2012) calculated the surface area of Verapamil molec-
ularly imprinted nanoparticles using BET analysis. The calculated values were 186 m2/g,
while the value for a non-imprinted polymer was found to be 168.2 m2/g. The average pore
size was determined using a Wheeler equation as follows (Eq. (2.17)):

Pore volume cm3 / g

d p nm 4000 (2.17)
Surfacearea m 2 / g

2.4.21 Dynamic Light Scattering

Dynamic light scattering (DLS) determines the hydrodynamic radius of molecules by
measuring the intensity of laser light when scattered by molecules in a solution (Bauer and
Schnapp 2007). It is worth noting that, while other characterization methods are mostly
utilized to confirm the existence of crystals, their morphology, and crystalline structure,
DLS gives valuable information on the particle size and its distribution at various time
intervals. In this way, the calculation of particle size and size distribution would be feasible
using the Stokes–Einstein equation. In fact, stochastic Brownian motion of particles caused
by interference of particles with the surrounding medium leads to a time-dependent scat-
tering intensity. In Eq. (2.18), the first-order autocorrelation function in terms of the scat-
tering radius q and delay time of τ is given. The diffusion coefficient Dt obtained from
Eq. (2.17) can be utilized to calculate the hydrodynamic radius with assuming spherical
geometry for particles with known viscosity of η at a temperature of T (Eq. (2.19)). Size
distribution can be elucidated by computational Fourier decomposition followed by the
identification of multiple signals in the raw data (Coe et al. 2015).
q 2 Dt
g q, e (2.18)

K BT
Dt (2.19)
6 r

The hydrodynamic diameter of trimetallic Cu/Cr/Ni nanoparticles was investigated

using DLS by Vaseghi et al. (2019). They concluded that for the two-particle size ­distribution
64 2 Nanomaterials

a­ verage nanoparticle size is 104.59 nm (76.7%, PDI = 0.12) and 5.2 μm (23.3%, PDI = 0.37)
(Figure 2.26).

2.4.22 Zeta-Potential
Theoretically, zeta-potential (ξ-potential) is the electric potential within the interfacial double
layer of a dispersed particle in regards to a point in the continuous phase located far away
from the interface. The most important parameters influencing zeta-potential are the pH of
the medium, followed by ionic strength, additive concentration, and temperature (Lu and
Gao 2010). In fact, the zeta-potential of a sample is representative of the stability of its col-
loidal dispersions. Particles with intense positive or negative charges have the tendency to
repel each other, leading to the formation of stable colloidal solutions with almost no ten-
dency for agglomeration. When a colloidal nanoparticle suspension is analyzed by zeta-
potential, obtaining low values for zeta-potential indicates flocculation of the colloids, which
corresponds to values near to the isoelectric point of the system. Thus, in order to have stable
solutions without aggregation and flocculation, the values of zeta-potential should be high,
either positive or negative. Typically, solutions with zeta-potential values in the range of ±
(20–30) mV or higher are regarded as stable solutions. Interestingly, DLS also gives informa-
tion on the aggregation of a solution. Therefore, the combination of DLS and zeta-potential
can lead to a more comprehensive characterization (Mourdikoudis et al. 2018).
Zeta-potential analysis of Ag nanoparticles synthesized using an acidophilic actinobacte-
rium strain—namely Streptacidiphilus durhamensis HGG 16n—is shown in Figure 2.27.
From the figure, it is clear that maximum zeta-potential value of −32 mV was obtained,
which is representative of the stability of the particles owing to electrostatic repulsion
between the solvated particles. Instability of the system was observed at the pH range of
2–4 corresponding to the zeta-potential value of 0 ± 0.5 mV. At pH 5–10 oscillation of zeta-
potential occurred and for pH values greater than 10 a plateau of the model curve was
registered (Buszewski et al. 2018).

30
76.7%

25
Density distribution

20

15
23.3%

10

0
1 10 100 1000 10000
Particle size (nm)

Figure 2.26 Histogram showing particle size distribution of ternary Cu/Cr/Ni. Source: Vaseghi et al.
(2019). Copyright © 2019, Elsevier.
 References 65

–5

–10
Zeta (mV)

–15

–20

–25
Sigmoidal model
–30
Experimental data

–35
2 4 6 8 10 12
pH

Figure 2.27 Zeta-potential of bio (AgNPs) according to pH value. Error limits (grey shadow).
Source: Buszewski et al. (2018). Copyright © 2018, Elsevier.

2.5 ­Conclusion

The field of nanotechnology is expanding rapidly, leading to a growing amount of research

throughout the world on nanoscience and nano-based products (i.e. nanomaterials). Based
on this chapter and other cited literature, nanomaterials are classified into various catego-
ries, which we have illustrated in this chapter. Also, various methods applied for the synthe-
sis of different types of nanomaterials are selected according to the existing facilities, primary
materials, potential application of the nanomaterial, and other environmental and economi-
cal limitations. Characterization of nanomaterials is another important issue that we have
illustrated comprehensively by introducing the existing techniques yet utilized for the char-
acterization of nanomaterials. Thus, the choice of suitable synthesis methods and charac-
terization tools is necessary to attain the desired features of a specific nano-product.

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 83

Recent Advances on Classification, Properties, Synthesis,

and Characterization of Nanomaterials
Veer Singh, Priyanka Yadav, and Vishal Mishra
School of Biochemical Engineering, IIT (BHU), Varanasi, Uttar Pradesh, India

3.1 ­Introduction

Nanotechnology is an emerging application in various fields such as medicine, biofuel pro-

duction, wastewater engineering, drug delivery, etc. (Figure 3.1). The nanoparticle is con-
sidered to be a fundamental unit of nanotechnology. Nanoparticles are varied in size from
1 to 100 nm. The smaller size of nanoparticles provides some unique properties to the
nanomaterials (Hasan 2015).
Different organizations have their own opinions about nanomaterials. According to the
US Environmental Protection Agency (USEPA), nanomaterials exhibit various properties
that are different from their non-nano forms (Boverhof et al. 2015). According to the US
Food and Drug Administration (USFDA) and the International Organization for
Standardization (IOS), nanomaterials must have at least one dimension, and be 1–100 nm
in size (USFDA 20112).
Nanomaterials are widely used in the field of electronics, optical communications
technology, cancer therapy, and biofuel production. The application of nanomaterials
depends on many factors including their physical properties, larger surface area to vol-
ume ratio, which provides possibilities for improvement of functionality of nanomateri-
als. Nowadays, a new and emerging field of nanotechnology has emerging applications
in the fields of bioenergy, enzymology, and pharmaceuticals. Nanotechnology plays a
major role in the enhancement of productivity and long-term stability, as well as being
low cost, which enables the production of inexpensive products. Recent research has
reported that the addition of nanomaterials along with algal culture medium enhances
both cell growth and lipid accumulation. Hence, nanotechnology improves the lipid con-
tent in microbial cells that minimize the production cost and enhance the productivity of
biodiesel. This chapter aims to review nanoparticle synthesis, its properties, and the
characterization methods.

Green Synthesis of Nanomaterials for Bioenergy Applications, First Edition. Edited by Neha Srivastava,
Manish Srivastava, P. K. Mishra and Vijai Kumar Gupta.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
84 3 Recent Advances

Biofuel
production

Wastewater
Health care
treatment

Nanotechnology

Food
Biomedical
products

Electronics

Figure 3.1 Figure shows the application of nanotechnology in various fields.

3.2 ­Classification and Types of Nanomaterials

The nanomaterials are classified based on materials used in the synthesis process, the ori-
gin of materials and based on the structure or dimension of nanomaterials (Figure 3.2).

3.2.1 Classification of Nanomaterials Based on Materials

3.2.1.1 Carbon-Based Nanomaterials
These types of nanomaterials are made up of carbon content and have various morpholo-
gies. The carbon-based nanomaterials can be hollow tubes or spheres, carbon nanofibers,
Fullerenes, and graphene. The different methods used for the synthesis of carbon-based
nanomaterials include chemical vapor deposition (CVD), arc discharge, and laser ablation
(Kumar and Kumbhat 2016).
(a) Fullerenes: Fullerenes (C60) are spherical carbon molecules made up of carbon atoms
arranged in sp2hybridization. Fullerenes contain 28–1500 carbon atoms arranged in
spherical structures with a diameter of up to 8.2 nm for a single-layered fullerene and
4–36 nm for a multi-layered fullerene.
(b) Graphene: This is the carbon-containing network. The arrangement of carbon atoms
forms a hexagonal pattern in the graphene network and makes two-dimensional pla-
nar surfaces. The thickness of a two-dimensional sheet is about 1–2 nm.
(c) Carbon nano tubes (CNTs): These are nanofoils made up of carbon-containing gra-
phene. The carbon atoms are arranged in honeycomblattice in carbon nanotubes and
 3.2 ­Classification and Types of Nanomaterial 85

Classification of nanomaterials

Classification of Classification of
nanomaterials based on nanomaterials based on
materials the dimension

Carbon-based Zero dimension

nanomaterials nanomaterials

Inorganic-based one dimension

nanomaterials nanomaterials

Organic-based Two dimension

nanomaterials nanomaterials

Composite based Three dimension

nanomaterials nanomaterials

Figure 3.2 Schematic diagram showed the basic classification of nanomaterials.

form hollow cylinders of 0.7 nm diameter for a single-layered CNT and about 100 nm
for a multi-layered CNT. The length of CNT varies from a few μm to a few mm.
(d) Carbon nanofibers: The graphene nanofoils used in the production of carbon
nanofibers are the same as CNTs but the structure is different. The graphene mol-
ecules are arranged in cone or cup form rather than the regular cylindrical tubes
of CNTs.
(e) Carbon black: This is an amorphous nanomaterial made up of carbon atoms that
are arranged in a spherical shape with diameters from 20 to 70 nm. The carbon black
­particle–particle interaction is so high that they bind with each other and form
­aggregates around 500 nm.

3.2.1.2 Inorganic-Based Nanomaterials

It is these nanomaterials that make up metals and metal oxides. They can be synthesized
from metals such as Ag, Au, and Fe: and the metal oxides are TiO2, ZnO, and MnO2.
Semiconductor nanomaterials are also synthesized from silicon and ceramic materials
(Jeevanandam et al. 2018).

3.2.1.3 Organic-Based Nanomaterials

The organic-based nanomaterials are made up of organic matter other than carbon and
inorganic material. The synthesis of these nanomaterials is through self-assembly or trans-
formation from organic matter into the desired structure. The noncovalent (weak) interac-
tion applies in these types of materials (Kumar and Kumbhat 2016).
86 3 Recent Advances

3.2.1.4 Composite-Based Nanomaterials

Composite nanomaterials are made up of one more layer of nanoparticles. These nanoma-
terials are combined with other nanoparticles, bulk materials, or more complex materials
like metal frameworks. The composites may be made up of many types of materials such as
metal, ceramic, organic, inorganic, carbon-based, or bulk polymers. These materials have
different morphologies depending on the synthesis and required properties for the desired
applications (Badrossamay et al. 2010; Gokarna et al. 2014; Li et al. 2012; Zhang et al. 2011).

3.2.2 Classification of Nanomaterials on the Basis of Dimension

The small nanomaterials have various dimensions: zero dimensions, one dimension, two
dimensions, and three dimensions. The dimensions of nanomaterials and some of their
important characteristics are explained in Figure 3.3. The nanomaterials have at least one
dimension of 1–100 nm. The nanomaterials exist in single, aggregated or fused forms with
several shapes such as tubular, spherical, and irregular. The most common types of nano-
materials are nanofibers, nanotubes, quantum dots, and nanosheets. Siegel also classified
nanomaterials into four types: zero-dimensional, one-dimensional, two-dimensional, and
three-dimensional nanostructures (Jeevanandam et al. 2018).

3.2.2.1 Zero-Dimensional Nanomaterials

Zero-dimensional nanoparticles are the most common type of nanomaterials, with dimen-
sions within nanoscales (the dimension of no nanoparticle is larger than 100 nm). These
nanoparticles are point like particles i.e. as small as a point). The most common examples
of these particles are quantum dots (uniform particle arrays), hollow spheres, nano lenses,
etc. (Jeevanandam et al. 2018).

3.2.2.2 One-Dimensional Nanomaterials

These types of nanoparticles are at least one dimension larger than nanoscales and other
dimensions are within nano range. The most common examples of one-dimensional nano-
particles are nanofibers, nanotubes, and nanorods (Jeevanandam et al. 2018).

Nanomaterials

Zero dimension one dimension Two dimension Three dimension

•Fullerenes •Nanotubes •Nanodiscs •Embedded clusters

•Quantum dots •Fibres and filaments •Nanolayers •Equiaxed crystallites
•Rings •Whiskers and belts
•Atomic clusters •Spirals and springs

Figure 3.3 Classification of nanomaterials on the basis of dimensions.

 3.3 ­Properties of Nanomaterial 87

3.2.2.3 Two-Dimension Nanomaterials

These types of nanomaterials are two dimensions larger than nanoscle (100 nm). The most
common examples of this class are nanofilms, nanolayers, and nanocoating. This class of
nanomaterials have plate-like structures (Jeevanandam et al. 2018).

3.2.2.4 Three-Dimensional Nanomaterials

Three-dimensional nanomaterials have all three dimensions larger than 100 nm but their
components are below 100 nm in size. Nano range particles come together to form three-
dimension nanomaterials. These materials are generally nonporous in nature and have
many applications. The most common examples of three-dimensional nanomaterials are
nanocomposites, bundles of nanofibers, multinanolayer-type structures (Jeevanandam
et al. 2018).

3.3 ­Properties of Nanomaterials

The nanoparticles have more surface area and some unique properties compared with bulk
materials. The properties of nanomaterials are described in terms of physical and chemical
properties.

3.3.1 Physical Properties

The physical properties of nanomaterials are described in terms of optical, mechanical s,
hydrophilicity, hydrophobicity, suspension, settling, thermal properties, magnetic, and
electrical properties.

3.3.1.1 Optical Properties

The optical properties of nanoparticles include color, light penetration, reflection, absorp-
tion, and ultra violet (UV) absorption properties. The optical properties of nanoparticles
are size dependent and also represent a strong UV–visible extinction band; these properties
are not present in the bulk materials.

3.3.1.2 Mechanical Properties

The mechanical properties of nanoparticles include elasticity, tensile strengths, hardness,
and flexibility. These mechanical properties play a key role in the nanomaterials in differ-
ent fields.

3.3.1.3 Magnetic and Electrical Properties

The magnetic and electrical properties are defined in terms of conductivity, semiconductiv-
ity, and resistivity. Based on these properties, nanoparticles can be used in modern
electronics.

3.3.1.4 Other Properties

Nanomaterials have been shown to have some other important properties such as hydro-
philicity, hydrophobicity, suspension, diffusion, and thermal conductivity.
88 3 Recent Advances

3.3.1.5 Chemical Properties

Chemical properties are important factors in nanoparticles. The chemical properties of
nanomaterials include stability and sensitivity, toxicity, corrosive and anti-corrosive, oxida-
tion, reduction, antifungal, antibacterial, and disinfection. These chemical properties
determine the role of nanomaterials in chemical and biomedical engineering.
The chemical properties of nanomaterials also depend on the size of nanomaterials and
change with respect to size. The small nanomaterials have larger numbers of atoms on
their surface compared with bulk materials. Hence, nanomaterials show more reactivity
than bulk materials. The following are some of the chemical properties:
1. The larger number of atoms on the surface of nanomaterials is a major reason for the
change in behavior nanomaterials. As up to half of atoms in nanomaterials are present
on the surface, properties of materials such as electrical transport are enhanced in
nanomaterials compared with bulk materials.
2. The large numbers of atoms on the surface of nanomaterials results in higher average
energy than longer structures. For example, nanomaterials have more catalytic activity
than bulk materials. The nanomaterials show more chemical activity per atom of
exposed surface. The catalytic activity decreases in bulk materials compared with nano-
materials. The greater chemical activity is due to the large number of atoms exposed on
the surface.
3. Impurities may be attracted to the surfaces of nanomaterials and the interactions
between nanoparticles and those small dimension impurities can depend on the struc-
ture of nanomaterials and type of chemical bonding between those impurities and
nanoparticles.

3.4 ­Synthesis of Nanomaterials

Two methods, bottom–up and top–down, are generally used to synthesize the nanoparti-
cles. The bulk materials are converted into small particles by top–down approaches. Small
particles are aggregates and form nanoscale range crystals through bottom–up methods.
The bottom–up and top–down processes are simplified in Figure 3.4.

3.4.1 Bottom-Up Method

This method is also considered useful because it is based on nanocrystal formation from
atomic range particles. Various types of synthesis processes are used in a bottom–up pro-
cess, such as sol-gel, chemical, spinning, and CVD methods.

3.4.1.1 Sol-gel
This is the colloidal solution of solid particles present in the liquid medium. This gel is a
small solid molecule suspended in a solvent solution. Sol-gel method, a wet process in
which chemical regents are used as precursor molecules for nanoparticle synthesis, is
widely used for nanoparticle synthesis due to its simplicity. Metal oxide and chlorides
are widely used in this method (Ramesh 2013). These metal oxides, as well as chlorides, are
dispersed in liquid medium by heating, followed by stirring, shaking, or sonication.
 3.4 ­Synthesis of Nanomaterial 89

Bottom-up Top-down
Method Method

Atoms Bulk
(small size) Materials

Clusters Powder

Nanoparticles

Figure 3.4 Nanoparticle synthesis methods.

The resultant consists of solid as well as liquid phase. The nanoparticles are separated by
various processes such as centrifugation, filtration, and sedimentation (Mann et al. 1997).

3.4.1.2 Spinning
The nanoparticle synthesis through spinning followed by the use of a spinning disc
reactor (SDR). An SDR consists of a rotatory disc inside a reactor under controlled phys-
ical parameters such as temperature and humidity. The inert environment is also ­created
by using nitrogen or other inert gases, which are responsible for removal of oxygen from
the reaction chamber. It is necessary to remove oxygen from the reaction chamber
because it can initiate various chemical reactions during nanoparticle formation and
decrease the efficiency nanoparticle synthesis (Tai et al. 2007). The small molecules are
fused together due to spinning and precipitation occurs. These precipitated materials
are collected and washed several times before being dried in a hot-air oven (Mohammadi
et al. 2014). The characteristics of nanomaterials are dependent on various parameters
of the SDR such as rotation speed, temperature, precursor to liquid ratio, surface area of
the disc, etc.

3.4.1.3 Chemical Vapor Deposition

This method depends on the deposition of gaseous molecules on the solid substrate, which
is performed at optimum temperature in a reaction compartment. The reaction initiates
when gaseous molecules come into contact with the heated substrate (Bhaviripudi et al.
2007). This method has many advantages such as formation of very pure, hard, and uni-
form nanoparticles. The nanoparticle synthesis reaction is highly dependent on the tem-
perature. However, this method also has some disadvantages such as formation of toxic
gaseous byproducts and the requirement for specialized equipment for the synthesis pro-
cess (Adachi et al. 2004).
90 3 Recent Advances

3.4.1.4 Pyrolysis
This is the most important method for nanomaterial synthesis and is generally used for
nanoparticle synthesis at industrial level. Pyrolysis is based on the burning of precursor
materials at high temperature and pressure. The precursor materials used in this process
are in liquid or gaseous form and these precursors are passed through a small hole into the
furnace (Kammler et al. 2001). This process has many advantages in comparison with other
processes such as cost effectiveness, simplicity, and being a continuous process with high
nanoparticle synthesis yield (Amato et al. 2013).

3.4.1.5 Biosynthesis
This is both a cost-effective and ecofriendly method of nanoparticle synthesis. The synthe-
sized nanomaterials generated by biosynthesis are non-toxic and biodegradable in nature
(Kuppusamy et al. 2014). Various biological materials such as bacteria biomass, plant
extracts, and fungal biomass, along with precursors, have been used for biosynthesis of
nanomaterials. This method has unique and enhanced properties that have many applica-
tions in bio-medication engineering (Hasan et al., 2015).

3.4.2 Top-Down Method

The top-down method is a destructive method of nanoparticle synthesis. It is based on the
decrement of bulk materials into nanometric-scale materials. Mechanical ball milling,
thermal decomposition, and laser ablation are the most widely used top–down methods for
nanomaterial synthesis.

3.4.2.1 Mechanical Milling

Compared to other top–down methods, the mechanical ball milling technique is generally
beneficial for the synthesis of various types of nanoparticles. Some materials are milled in
the inert atmosphere during the nanoparticles production process. Some important factors
that influence the production process and help to maintain quality of nanomaterials are
plastic deformation, particle fracture, and cold welding. Plastic deformation is responsible
for maintaining the shape of nanoparticles, fracture is responsible for decrement in nano-
particle size, and cold welding plays an important role in minimizing particle size (Yadav
et al. 2012).

3.4.2.2 Nanolithography
The study of constructing nanometric-scale particles from bulk materials is termed nano-
lithography. The synthesized nanoparticles generated through this method vary from 1 to
100 nm in size. Various nanolithography processes have been used for synthesis of nanoma-
terials such as electron beam, optical process, nanoimprint, and high energy proton beams.
Generally, lithography is the procedure of production of nanoparticles of a required shape
and size from light-sensitive bulk materials, which selectively removes the portion of bulk
materials to produce the required shape. The synthesis of single-size nanoparticle clusters
of a desired shape and size is the main advantage of this method. There are a few disadvan-
tages to nanolithography such as the requirement for expensive and complex equipment
(Ding et al. 2005; Duan et al. 2003; Hulteen et al. 1999; Pimpin and Srituravanich 2012).
 3.5 ­Characterization of Nanomaterial 91

3.4.2.3 Laser Ablation

This is one of the basic methods for nanoparticle synthesis from different precursor sol-
vents. The metallic solution is mixed in the liquid medium under exposure of a laser
beam and the condensed metallic solution produces nanoparticles of 1–100 nm in size.
This method provides an alternative solution to conventional chemical reduction meth-
ods for synthesis of nanoparticles. The chemical methods generate various types of toxic
substances during the synthesis process. Therefore, this method is ecofriendly, cost effec-
tive, and does not produce toxic byproducts. Laser ablation created stable nanoparticles
from organic solvents as well as from water. This method does not require any toxic
chemicals or stabilizing agents. Hence, it is a green process of nanoparticle synthesis
(Amendola and Meneghetti 2009; Delmée et al. 2017; Fong et al. 2013; Saitow et al. 2012;
Zhang et al. 2017).

3.4.2.4 Sputtering
This method is based on the deposition of nanoparticles on the surface through collision
between the ejecting materials and ions (Ealias and Saravanakumar 2017). The deposition
of nanoparticles on the surface is usually in the form of a thin layer. The thickness of the
deposited layer depends on the temperature as well as the duration of annealing time, the
materials used for synthesis, etc. These parameters also determine the size as well as the
shape of nanoparticles (Lugscheider et al. 1998).

3.4.2.5 Thermal Decomposition

This method is endothermic in nature. The major reaction in this method occurs in the
presence of heat. The synthesis of nanoparticles depends on deposition and the breaking of
chemical bonds within compounds in the presence of heat. The temperature at which the
reactant metal compounds decompose is called the specific decomposing temperature. The
decomposition reaction also requires various parameters such as pressure and reaction
time. The decomposing reactant produces stable and small-size nanoparticles (Salavati-
niasari et al. 2008).

3.5 ­Characterization of Nanomaterials

Nanomaterials have several applications in the field of medical science as well as in a num-
ber of engineering fields. The characterization of nanomaterials before their use is very
important in many fields. The characteristics of nanomaterials determine their properties.
Surface characterization of nanomaterials is also determined by various techniques. The
characteristics of nanomaterials are explained in Table 3.1.
Figure 3.5 depicts the common methods used for the characterization of nanoparticles
like scanning electron microscopy (SEM), transmission electron microscopy (TEM), low
energy electron diffraction (LEED), extended X-ray absorption fine structure (EXAFS)
used for the detection of surface topography, and secondary ion mass spectroscopy (SIMS),
auger electron spectra (AES), electron probe microanalysis (EPMA) etc. used for the detec-
tion of the surface complexes etc.
92 3 Recent Advances

Table 3.1 Characteristics of nanomaterials in solid, liquid, and gaseous phases.

Characteristics of
nanoparticle Solid phase Liquid phase Gaseous phase Reference

Size of Electron Centrifugation Optimal particles Xuanxuan et al.

nanoparticles microscope and photon counter equipment (2017)
and laser correlation
diffraction spectroscopy
Surface area of BET NMR equipment Differential mobility Baer et al.
nanoparticles and chemical analyzer and scanning (2013), Szekeres
titration mobility et al. (2002)
Particle sizer
Composition X-ray Analyzed by This technique is based Asmi et al.
Photoelectron mass on collection and (2010), McMurry
Spectroscopy spectrometry collected particles et al. (2009)
and chemical and ion analyzed by
digestion chromatography spectrometric or wet
method chemical methods
Surface Imaging Particles Particles collected by Hodoroaba et al.
Morphology of performed by deposition and filtration and (2014), Wu et al.
nanoparticles SEM and TEM analysis through electrostatically The (2013)
SEM and TEM collected particles
imaged by SEM and
TEM
Surface charge Point zero Zeta potential Differential Marsalek (2014),
on nanoparticles charge mobility analyzer Kumal et al.
(2015), Schleh
et al. (2012)

3.5.1 Size
The size of the particles is considered to be a beneficial characteristic of nanomaterials. For
the measurement of nanoparticle characterization, the particle is considered to be a basic,
as well as the most important, measurement. The size of nanoparticles varies from 1 to
100 nm. During the synthesis process, various sizes of nanoparticles are formed. The
desired size nanoparticles are selected by using electron microscopy. SEM and TEM are
generally used to determine the size of nanoparticles. Laser diffraction technique is useful
for determining the bulk sample in the solid phase. Photon correlation spectroscopy is used
for measurement of particle size in the liquid phase. When nanoparticles present in the
gaseous phase then size measurement is very difficult. This problem can be solved by scan-
ning mobility particle size (SMPS). SMPS used for the determination of particle size in the
gaseous phase. This method provides the fast and effective measurement of nanoparticle
(Dukhin et al. 2010; Liao et al. 2006; Marsalek 2014; Tso et al. 2010; Xuanxuan et al. 2017).

3.5.2 Surface Area

It is also an important factor of nanoparticles. The surface area of a nanoparticle affects its
properties. Surface area is generally measured in terms of surface to volume ratio. The
measurement of surface area of nanoparticles is commonly performed by BET analysis.
 3.5 ­Characterization of Nanomaterial 93

COMMON METHODS FOR CHARACTERIZATION OF NANOPARTICLES

Surface state

Particle size Surface area Surface Complexes Surface topography Surface composition

•SIMS •SEM
•AES •TEM
•Electron Microscopy •XPS •LEED
•Magnetic measurements •EPMA •EXAFS
•X-Ray diffraction •EXAFS

•Raman
•NMR
•IR
•UV-Vis

Figure 3.5 Common methods for the characterization of nanoparticles.

Simple titration methods are generally used for determination of surface area nanoparti-
cles in the liquid phase but these titration methods are laborious. So, nuclear magnetic
resonance spectroscopy (NMR) is used for surface area determination. It is an effective and
time saving process. Differential mobility analyzer (DMA) is a most effective tool for deter-
mination of surface area in the gaseous phase (Baer et al. 2013; Szekeres et al. 2002; Zhou
et al. 2009).

3.5.3 Composition
The composition is also a major component of nanoparticles. Nanoparticles are made up of
a desired composition of chemicals and elements, which are responsible for their activity
and efficiency. Undesired components reduce the activity and efficiency of nanoparticles
and may cause secondary harmful or useless reactions. The composition also determines the
purity of nanoparticles. The nanoparticle components are generally determined by the help
of X-ray photoelectron spectroscopy (XPS) (Sharma and Rao 2014). Some other techniques
also have useful applications in the liquid phase for analysis of nanoparticles such as mass
spectrometry ion chromatography. Gaseous phase particles are also analyzed by some mass
spectrometric techniques (Asmi et al. 2010; Bzdek et al. 2011; McMurry et al. 2009).

3.5.4 Surface Morphology

Nanoparticles have several shapes and sizes, which play an important role in their proper-
ties. Some examples of shapes are tubular, cylindrical, flat, spherical, and conical, as well
as irregular shapes. Nanoparticles have two types of surface morphology: crystalline as well
as amorphous structures with both uniform and irregularities on the surface. The surface
94 3 Recent Advances

morphology of nanoparticles is also determined by SEM and TEM imaging ­methods. The
nanoparticles in liquid medium form a layer on the surface and analyzed by imaging meth-
ods. Gaseous phase particles also capture and analyze surface morphology (Hodoroaba
et al. 2014; Rades et al. 2014; Wu et al. 2013).

3.5.5 Surface Charge

Nanoparticles are made up of various metallic and non-metallic materials that have a fixed
charge on their surface. The surface charge plays an important role in properties of nano-
particles. The positively/negatively charged nanoparticles can interact with their opposite
charges on the target site to form a complex. The surface charge of nanoparticles is deter-
mined by using zeta potentiometer. DMA determines the surface charge of nanomaterials
in the gaseous phase (Kumal et al. 2015; Marsalek 2014; Schleh et al. 2012).

3.5.6 Crystallography
This method is based on the study of materials at atomic and molecular level. Atoms are
arranged in a unique form that determines the structure and properties of nanomaterials.
The crystallography method is widely used for determination of the arrangement of atoms
in the nanomaterials. This analysis is carried out by X-ray and high-energy electron beams
(Dorofeev et al. 2012; Ingham 2015; Sharma et al. 2012; Yano et al. 1996).

3.5.7 Concentrations
The nanoparticles concentration has an emerging application in the gaseous phase reac-
tion medium. It determines the amount of gas in terms of volume required for reaction.
The various parameters like size, concentration, and the distribution of nanoparticles play
a key role in determining performance and efficiency of nanomaterials. Measurement of
the concentration of the nanoparticles is done with the help of the condensation particle
counter (CPC).

3.6 ­Conclusion

In this chapter, we have focused on nanoparticles and their properties, classification,

­synthesis, and the characterization technique. Nanomaterials are classified on the basis of
their dimensions and the materials used for synthesis. The nanomaterials are synthesized
using various methods such as the top–down and bottom–up methods. Among these meth-
ods, biosynthesis is widely used as a non-toxic and cost-effective method. Biosynthesis of
nanomaterials is based on biological dead or living materials such as plant biomass, algal
biomass, fungal biomass, and agricultural waste materials. Various types of techniques are
used for characterization of nanomaterials such as surface morphology determined by
SEM, TEM, and XRD. The size of nanoparticles is measure by BET analysis. A nanoparticle
shows various types of properties such as optical, magnetic, and electric properties and also
varies in size from 1 to 100 nm. The nanoparticles have different applications in the field of
 References 95

wastewater engineering, health science, and biofuel production. This chapter has given an
overview of nanoparticles and provides up-to-date information about nanoscience.

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 99

Synthesis of Metallic and Metal Oxide Nanomaterials

Ayse Demirbas1, Tuna Karaytuğ2, Nihan Arabacі3, Ebru Sebnem Yilmaz4,
and Ismail Ocsoy5
1
Faculty of Fisheries and Aquatic Sciences, Recep Tayyip Erdogan University, Rize, Turkey
2
Department of Biology, Institute of Natural and Applied Sciences, Cukurova University, Adana, Turkey
3
Department of Biology, Faculty of Arts and Sciences, Cukurova University, Adana, Turkey
4
Department of Biology, Faculty of Arts and Science, Hatay Mustafa Kemal University, Antakya, Hatay, Turkey
5
Department of Analytical Chemistry, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey

4.1 ­Nanomaterials

Today, the metallic nanomaterials defined as having at least one dimension in the range
1–100 nm, including metal oxides, quantum dots, and plasmonic, have introduced advances
into science. Nanomaterials have been actively used in several diverse industrial and scien-
tific fields, ranging from electronics, medicine, and agriculture to catalysis owing to their
optical, luminescent, and magnetic properties (Lee et al. 2004; Veiseh et al. 2010; Dreaden
et al. 2012; Peng et al. 2014; Qiu et al. 2015; Prasad et al. 2017). Nanomaterials were initially
synthesized as single components, however, researchers have shifted to the development of
synthesis of hybrid nanomaterials (including at least two components) for these potential
reasons: (i) suppression of drawback of single component nanomaterials, (ii) improving
corresponding intrinsic properties, and (iii) introducing new functionalities (Kim et al.
2006; Sanvicens and Marco 2008; Lee et al. 2012; Ocsoy et al. 2013a).
In recent decades, instead of focusing on the production or design of new nanomaterials,
the most attractive and exciting research has been to create novel nanomaterial synthesis
methods. In general, the two most common approaches to synthesis of nanomaterials are
the “top–down” and “bottom–up” methods(Herzer 1989) (Figure 4.1). The principle of the
top–down approach uses physical methods to convert bulk materials into nano size.
Although the top–down approach enables us to produce large-scale nanomaterials in a
short time, a lack of control over morphology, size, and size distribution are considered to
be major drawbacks of that approach. In contrast, the bottom–up approach enables the
production of monodispersed nanomaterials in very narrow and homogeneous size distri-
bution with certain morphology. However, nanomaterials production yield with this
method is quite low when compared to the top–down approach.

Green Synthesis of Nanomaterials for Bioenergy Applications, First Edition. Edited by Neha Srivastava,
Manish Srivastava, P. K. Mishra and Vijai Kumar Gupta.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
100 4 Synthesis of Metallic and Metal Oxide Nanomaterials

Nanomaterials

Bulk

Bottom-up
Top-down

Seeds
Powder

Nanomaterials
Atoms

Figure 4.1 Illustration of the most common approaches, top–down and bottom–up, in
nanomaterials preparation.

The most common nanomaterial synthesis methods in the bottom–up approach are
chemical and biological methods. The chemical method generally uses solvothermal/
hydrothermal and thermal decomposition synthesis strategies in which hydrophobic nano-
materials have been produced in organic solvents and surfactant molecules. The surfactant
molecules usually consist of long hydrophobic hydrocarbon chains (oleic acid, oleylamine,
dodecanethiol, and trioctylphosphine oxide) with various functional end groups to reduce
metal ions, stabilizing and protecting nanomaterials from aggregation and oxidation,
respectively. Although hydrophobic nanomaterial synthesized in those abovementioned
strategies show long-term stability in nonpolar solvents, effective control over morphology,
size and size distribution, and very high crystallinity, using toxic materials including
organic solvents, reducing, stabilizing agents, and hydrophobic surfactants can strictly
limit their production and use especially in nanomedicine. In order to use hydrophobic
nanomaterials in bioanalytical and biomedical applications, the ligand exchange proce-
dures are needed to transfer hydrophobic nanomaterials into the aqueous solutions. The
hydrophobic nanomaterials are not water soluble and deposition of those nanomaterials
into aqueous solutions may cause rapid and inevitable aggregation. This means that if
directly implemented during in vivo experiments, blood circulation in living organisms is
blocked and may cause a threat to human or animal lives. Therefore, it is imperative that
the nanomaterials are made water soluble by exchanging hydrophobic ligands with hydro-
philic ones or creating a hydrophilic coating on them.
The potential mechanism for the formation of monodisperse materials documented for
the first time in the 1940s by LaMer and colleagues (Figure 4.2). They explained that the
rapid and short nucleation occurs in the first step, followed by slow growth without further
nucleation, which results in the production of colloidal materials with a narrow size distri-
bution (LaMer and Dinegar 1950). However, this proposed theory was initially developed
to elucidate the formation mechanism of sulfur hydrosols and oil aerosols. Although it
 4.1 ­Nanomaterial 101

I II III
Nucleation Growth
Monomer conc.

Sc

Reaction time

Figure 4.2 Illustration of proposed LaMer mechanism for formation of nanomaterials with
nucleation and growth steps. Supersaturation (S) and critical supersaturation (Sc) are given on the
graph. Source: Schladt et al. (2011).

works for micro size materials, researchers applied and transferred the theory to materials
produced in nano size. The LaMer theory divides the nucleation and growth process into
three steps for the formation of materials (Schladt et al. 2011).
In the first step, the monomer concentrations of materials should reach the point of
supersaturation with gradual increase for the initiation of nucleation, however, no seeds
formation is observed, material formation happens under heterogeneous nucleation. For
the creation of homogeneous nucleation, the monomer concentrations can be determined
based on the intrinsic properties including Fermi level energy or energy barrier defined as
the Gibbs free energy as shown in phase II in Figure 4.2.
It is worth mentioning that the LaMer theory has been offered and successfully imple-
mented to microparticles, however, it can be easily applied to formation of nanomaterials
with some considerations. For instance, surface free energy is quite high in nanomaterials
compared to micromaterials, which causes uncontrolled growth and particle agglomera-
tion during the formation of nanomaterials (Alivisatos 1996). In order to block the agglom-
eration of the nanomaterials, the various types of surfactant including hydrophobic,
hydrophilic, and amphiphilic capping or reducing surfactant or agents that have phos-
phines, amines, thiols, and carboxylates functional groups have been employed (Hyeon
2002; Cheon et al. 2004; Sun et al. 2004; Jun et al. 2007; Shukoor et al. 2012; Ocsoy et al.
2013b, 2016, 2017d; Yasun et al. 2012).
Briefly, various amphiphilic molecules including amphiphilic polyethylene glycol (PEG)
block copolymer, amphiphilic PEG-phospholipids, cetyltrimethylammonium bromide
(CTAB), and lipid deoxyribonucleic acid (DNA) have been used intensively for phase transfer
of the hydrophobic nanomaterials. In principle, amphiphilic molecules can intercalate
between the tail and head of hydrophobic capping surfactant molecules through hydropho-
bic–hydrophobic and van-der-Waals interactions, which turn the nanomaterials into aqueous
solutions (Palma et al. 2007; Qin et al. 2007; Chen et al. 2012). Additionally, heterobifunc-
tional polymeric ligands such as dopamine-PEG-COOH, poly(analine), poly(methylidene
102 4 Synthesis of Metallic and Metal Oxide Nanomaterials

malonate), poly(pyrrole), poly(lactic acid), poly(glycolic acid), and poly(ethylene imine) may
show cross-linking between the ligands or chelation between ligand and metal oxides to pro-
duce stable water-soluble nanomaterials (Gawalt et al. 2003; Yu et al. 2007; Ocsoy et al. 2013b;
Peng et al. 2014). In addition to ligand exchange, the hydrophobic nanomaterials can be
transfered to aqueous solutions by forming silica coatings on the nanomaterials (Figure 4.3).
In typical silica coating procedure, hydrophobic nanomaterials and tetraethoxysilane (TEOS)
are dispersed in cyclohexane, then condensation of TEOS is initiated by the addition of
­aqueous NH4OH to form a silica coating on the nanomaterial surface (Warner et al. 2000;
Santra et al. 2001; Santra et al. 2004; Sen et al. 2006; Deng et al. 2008; He et al. 2009).
The stability of the nanomaterials under physiological conditions is important for their
use in nanomedicine. As mentioned, several strategies have been developed for efficient
phase transfer. However, almost all phase transfer procedures require intensive labor, com-
plicated ligand design or synthesis, expertise, and expensive tools. To address those issues,
the biogenic methods have been developed for synthesis of biocompatible and water-solu-
ble nanomaterials using a variety of biomolecules.

4.2 ­Biogenic Methods for Synthesis of Biocompatible

and Hydrophilic Nanomaterials

In recent years, different biomolecules including DNAs, proteins, enzymes, peptides,

amino acids, plant extracts, and standard molecules have been used as reducing and/or
stabilizing agents for formation of metallic and metal oxide nanomaterials. For instance,
Watson–Crick base pairing and the design features of DNAs make them act as templates
for nucleation, growth, and positioning of metallic nanoparticles (Ma et al. 2006;

hilic
Hydrophobic Amphip
Nanomaterials Lig ds
a n Hydrophilic
ate Nanomaterials
i - d ent
l t rs
Mu lyme
P o

al
t i on
nc s
-fu nd
Phase Transfer Bi Liga

ell
Silica Sh
Hydrophobic Hydrophilic
Nanomaterials in Nanomaterials in
Apolar Solvents Aqueous Solutions

Figure 4.3 The most common strategies for the phase transfer of hydrophobic nanomaterials into
the aqueous solutions.
 4.2 ­Biogenic Methods for Synthesis of Biocompatible and Hydrophilic Nanomaterial 103

Rothemund 2006; Pal et al. 2011a, b; Ocsoy et al. 2013c; Schreiber et al. 2014; Strayer et al.
2016). Research related to DNA-guided metal-nanoparticle formation on graphene oxide
surface is shown in a graphical scheme in Scheme 4.1 and TEM images in Figure 4.4 (Ocsoy
et al. 2013b).
Previous reports have proven that the metal ions preferentially bind to nucleotides and
phosphates, and both nucleotides and phosphates are parts of nucleic acids (Gugliotti et al.
2004; Berti and Burley 2008). In addition to DNA, proteins and enzymes act as templates or
nanoreactors for synthesis of biocompatible nanomaterials. The metal ions may interact
with functional groups’ (amine, thiol, or carboxyl) protein to form primary nanocrystals or
seed, then the introduction or presence of reducing agents results in the formation of nano-
materials. Up to now, several types of nanomaterials such as metallic nanomaterials, metal
oxide, and quantum dots have been successfully produced. It is worth mentioning that while
proteins are considered as template or stabilizing agents for synthesis of nanomaterials with
the presence of reducing agent, enzymes act as stabilizing agents owing to their protein
structure and also cause the production of hydrogen peroxide (H2O2) and the superoxide
anion (O2−) through enzymatic reactions, which are used as reducing agents for synthesis of
biogenic nanomaterials (He et al. 2013; Shi et al. 2014; Leng et al. 2016; Ocsoy et al. 2018a).
Similar to proteins and enzymes, amino acids and peptides also act as reducing agents for
the reduction of metal ions and stabilizing agents for synthesis of nanomaterials (Hwang
et al. 2011; Zare et al. 2013; Maruyama et al. 2015; Lee et al. 2018; Kulkarni et al. 2018).
Although synthesis of various biocompatible and stable nanomaterials is accomplished
with DNAs, proteins, enzymes, peptides, and amino acids, those biomolecules also have a
series of drawbacks:
●● they are quite expensive,
●● they have low stability in experimental conditions,
●● they can be easily contaminated, and
●● they provide low production yield in nanomaterial synthesis.
Recently, plant extracts or standard molecules have captured researchers’ attention and are
being considered as alternative biomolecules for synthesis of nanomaterials with high yield.
There are a number of potential advantages of plant extracts in nanomaterial synthesis:
●● they are freely available to obtain,
●● there are simple and easy procedures for preparation of extract solution,
●● they are free or very affordable,
●● they are quite stable in harsh environmental or experimental conditions, and
●● there are no contamination risks.
Biomolecules directed synthesis of nanomaterials are shown in Figure 4.5.

4.2.1 Biological Resources-Directed Plasmonic Nanoparticles

Plasmonic nanoparticles (PNPs) are a very attractive class of nanomaterials. Depending on
their material, size, shape, stabilizing agent, and the roughness of their surface single metal
NPs have a specific plasmon band (Yu et al. 2013a; Altinsoy et al. 2018). PNPs are being
investigated continuously for their application for enhancing catalytic conversions, owing
 COOH COOH COOH OH COOH COOH COOH OH

COOH COOH
O O
O O
O O O O O

HO HO
HO HO

COOH COOH

O O
O O
FITC-labeled O O
O O
O dsDNA O
OH OH

COOH COOH COOH COOH

Graphene
oxide COOH COOH COOH OH

COOH
O
O
O O

HO
HO
Metal ions
COOH

O O
Reducing
O O
agent O
OH

COOH COOH

Double stranded FITC dye Partially quenched Completely quenched

Metal nanoparticle
DNA FITC dye FITC dye

Scheme 4.1 Graphical illustration of the synthesis of MNP@dsDNA–GO composites. Source: Ocsoy et al. (2013b).
 4.2 ­Biogenic Methods for Synthesis of Biocompatible and Hydrophilic Nanomaterial 105

(a) (b)

(c) (d)

Au
Cu

Au
Cu Pt Cu
Pt
Au
Pt

0 1 2 3 4 5 6 7 8 9 10 1112 13 14 15keV

Figure 4.4 TEM images of nano composites (a) ∼ 13 nm Cu NPs, (b) ∼ 10 nm Pt NPs grown on
dsDNA–GO, (c) ∼ 20 nm Au/Cu/Pt alloy NPs grown on dsDNA–GO using sodium ascorbate, and (d)
Pt, Au, and Cu elemental analysis of the Au/Cu/Pt alloy–GO composite. Source: Ocsoy et al. (2013b).

sDNA/dDNA

Protein/Enzyme With/Without
Reducing agent
Mn+

Peptide/Amino acid

Plant extract/molecule Nanomaterials

Figure 4.5 Biomolecules-directed synthesis of nanomaterials.

106 4 Synthesis of Metallic and Metal Oxide Nanomaterials

to their attractive property of robust interaction with light. Copper (Cu), gold (Au), and
silver (Ag) nanoparticles in the solution display vivid colors by localized surface plasmon
resonance (LSPR). PNPs are quite remarkable because of their unique optical properties,
which are now widely utilized in solar cells, nanomedicine, biosensing, and spectroscopy.
Plasmonic nanomaterials are more of an aforethought these days as up-and-coming sys-
tems for catalysis applications (Gellé and Moores 2019).
Within the last 20 years, bio-synthesized nanoparticles prepared with plant extracts, DNA,
protein, and enzymes have attracted more attention than chemically synthesized nanoparti-
cles because of features such as biocompatibility, low toxicity, cleanliness, ecofriendliness,
and their use in the process of material synthesis (Cheng et al. 2007; Thakkar et al. 2010;
Akhtar et al. 2013; Demirbas et al. 2017; Sood and Chopra 2018). Hitherto, several methods
for developing and producing nanoparticles have used organisms like plants, yeast, fungi,
bacteria, and viruses (Demirbas et al. 2017; Ildiz et al. 2017). Of these diverse methods, the
usage of all plant extracts tissues and living plants for synthesizing nanoparticles has proved
attractive as a confident and promising method because of its ecofriendly characteristics
(Cheng et al. 2007; Akhtar et al. 2013; Demirbas et al. 2016; Sood and Chopra 2018).
Plant extracts have been generally used for nanomaterial synthesis, medicine, biosensing,
and food (Ocsoy et al. 2017b). The morphological components of plants like roots, leaves,
seeds, and latex are used in order to the synthesize metal-based nanoparticles (Delamare
et al. 2007; Grzegorczyk et al. 2007; Maksimović et al. 2007; Marshall et al. 2007; Bar et al.
2009; Sathishkumar et al. 2009; Taarit et al. 2009; Krishnaraj et al. 2010; Castro et al. 2011;
Singhal et al. 2011; Duman et al. 2016; Shamaila et al. 2016; Dogru et al. 2017; Ramkumar
et al. 2017; Seker Karatoprak et al. 2017; Ocsoy et al. 2018a; Pugazhendhi et al. 2018; Some
et al. 2019). Plant extracts contain plenty of molecules—including, bioactive polyphenols,
terpenoids, flavonoids, proteins, sugars, and enzymes—that can be used as stabilizing and
reducing agents in order to biosynthesize the metallic nanoparticles in green synthesis
(Haverkamp and Marshall 2009; Mittal et al. 2014; Shamaila et al. 2016; Seker Karatoprak
et al. 2017; Ocsoy et al. 2017b). Therefore, the addition of hazardous agents is not necessary
for synthesis (Beg et al. 2018; Some et al. 2019) and these kinds of metabolites play a signifi-
cant role in the bioreduction of gold (Au) and silver (Ag+) (Some et al. 2019).
Plant extracts have been of interest to researchers (Some et al. 2019) due to their advan-
tages over other biomolecules in metallic nanoparticle synthesis:
●● they are inexpensive, quite easy, and stable in production,
●● there is low risk of contamination,
●● they are stable in harsh conditions (temperature, pH, salt concentrations, etc.),
●● they are easily prepared in a short time,
●● there is no need for special storage conditions, and
●● they provide large-scale and environmentally harmless nano molecules that are applica-
ble in medical processes (Schröfel et al. 2014; Duman et al. 2016; Ocsoy et al. 2017a, c).
In metal nanoparticle synthesis, biogenic nanoparticle synthesis is regarded as a non-
toxic, one-step, speedy, safe, ecofriendly, and economic procedure (Some et al. 2019). The
various parts of the plant have functionalities as potential stabilizing and reducing agents
to create Ag nanoparticles (AgNPs) of different structures. For example, researchers syn-
thesized spherical, square, hexagonal, and triangular AgNPs (10–90 nm) using extracts
 4.2 ­Biogenic Methods for Synthesis of Biocompatible and Hydrophilic Nanomaterial 107

from leaves (Elavazhagan and Arunachalam 2011; Phillip 2011; Ocsoy et al. 2017b). Besides
AgNPs, the plant extracts reacted with Au ions to form Au nanoparticles (AuNPs) and to
reduce them (Ocsoy et al. 2017b). AuNPs are very important because of their nontoxicity
and optical features, which increase the preferability for them in a large variety of scientific
fields such as nanomedicine, catalysis, nano biosensing, and nanoelectronics (Aromal and
Philip 2012; McLamore et al. 2016; Ocsoy et al. 2017b).
In green synthesis methods, using plant extracts to mediate nanoparticle synthesis
(Table 4.1), the mixture of the extract and metal salts is prepared and then nanoparticle
formation is achieved at a suitable temperature (from room temperature to 100 °C) and
time interval (min to h) (Schröfel et al. 2014; Duman et al. 2016).

4.2.2 Plant Extract-Directed Metal Oxides

Metal oxide nanoparticles (MO-NPs) have been used for bioremediation, agriculture, catal-
ysis, molecular imprinted modified surface detection, and drug release systems. When con-
ventional chemical methods are used in MO-NP synthesis, it contains toxic substances and
out of equilibrium reaction conditions. However, economic and ecofriendly green synthe-
sis methods are used as an alternative to this situation.
Nanoparticles can be divided into several subsections: metal oxides, metallic, carbon
based, ceramics, lipid based, polymeric, and semiconductors (Khan et al. 2017). Metal
oxides are prominent in various processes such as biomedical and water treatment applica-
tions (Koca et al. 2018). They have been synthesized largely by utilizing physical meth-
ods—including spray pyrolysis (Nazari et al. 2017), ultrasonication (Kumar et al. 2000),
chemical evaporation, etc.—and chemical methods. These nanoparticles can also be chem-
ically synthesized due to the collapse of the metal ion precursor required in a redox or
aqueous solution phase (Duan et al. 2015; Arabi et al. 2017). During chemical processes,
they often release toxic substances that pose a threat to the environment and human health.
Biological-derived green chemistry methods are cheaper and ecofriendly. It is very impor-
tant to protect human health and nature: green synthesis, in other words, biosynthesis
techniques, does so by reducing or eliminating the usage of toxic reaction precursors
(Zikalala et al. 2018).
It is known that various organisms are used in biological-controlled nanoparticle
synthesis. In such a particle synthesis, by using biological systems, the particle size,
surface area, and composition of the synthesized particles can be controlled. Although
it is a controllable synthesis, only a limited number of nanoparticles are synthesized in
some organisms. It has been shown that metal oxide nanomaterials can emerge as a
result of biodegradation of the toxicity of the metal ions in green synthesis (Jayaseelan
et al. 2012).
In the synthesis of various MO-NPs, some bacterial strains or biomass are used.
Magnetotactic bacteria and cell wall S-layer containing bacteria are some of these microor-
ganisms. Generally, metal ions are harmful to bacteria, and bacteria turn these metal ions
into nanoparticles to cope with this toxicity. In physiological stress conditions, bacteria
synthesize specific proteins for stabilizing the formed nanoparticles. The formation of nan-
oparticle equivalents from metal ions by microorganisms is associated with cellular bio-
molecules (Iravani 2014; Zikalala et al. 2018).
108 4 Synthesis of Metallic and Metal Oxide Nanomaterials

Table 4.1 Plants used in green synthesis of plasmonic nanoparticles.

Plants name(s) Nanoparticle(s) Size (nm) Shape(s) Reference(s)

Achillea biebersteinii Ag 12 ± 2 – Some et al. (2019)

(flowers)
Camellia sinensis Cu2+ – Hybrid Baldemir et al.
(leaves) nanoflower (2017)
Citrus limon (fruits) CuO 10 Spherical Zikalala et al. (2018)
Nephelium ZnO 25–40 Spherical Zikalala et al. (2018)
lappaceum L. (peels)
Polygala tenuifolia ZnO 33–73.5 Spherical Zikalala et al. (2018)
(roots)
Rhizophora Ag 60–95 – Asmathunisha and
mucronata Kathiresan (2013)
Trifolium Ag 17 Spherical Some et al. (2019)
resupinatum (seeds)
Viburnum opulus L. Cu2+ – Hybrid Ildiz et al. (2017)
(fruits) nanoflower
Zingiber officinale Ag 10–20 – Some et al. (2019)
(rhizome)
Zea mays Ag 249.12 Spherical Patra and Baek
(2016)
Pisonia grandis R. Br. Zn, Mn – – Joghee et al. (2019)
leaf
Eucalyptus globulus Cu 44–145 Nanowires Pinto et al. (2019)
bark
Sansevieria Gold – – Kumar et al. (2019)
raxburghiana leaf
Tropaeolum majus Ag – Cubic Valsalam et al.
(2019)
Croton caudatus Gold 20 Spherical Kumar et al. (2019)
Geisel
Hevea brasiliensis Fe – –
Muell. Arg. Bark
Persea americana Pd-Ag 9–13–16 – Meva et al. (2019)
bark
Leucasaspera extract Ag 25–80 Clustere, Kumaran and
irregular Vijayaraj (2017)
Dodonaeviscosa, Ag 60–90 Spherical Giridharan et al.
Capparisdeciduas (2014)
leaf
Viburnum opulus Ag 10–50 Spherical Moldovan et al.
fruit (2017)
Pteristriparita Sw leaf Ag 32 Hexagonal, Baskaran et al.
spherical abd (2016)
rod-shaped
 4.2 ­Biogenic Methods for Synthesis of Biocompatible and Hydrophilic Nanomaterial 109

Table 4.1 (Continued)

Plants name(s) Nanoparticle(s) Size (nm) Shape(s) Reference(s)

Terminalia catappa, Ag 7–10–11 Spherical Mohamed El-Rafie

Terminalia mellueri, and Hamed (2014)
Terminalia bentazoe,
Terminalia bellerica
leaf
European black Ag 20–80 Spherical
elderberry fruit
Calophyllum Ag – Spherical Govindappa et al.
tomentosum leaf (2018)
Piper nigrum fruit Ag 40–100 Spherical, Seethalakshmi
cuboidal (2015)
Salvia officinalis leaf Ag 16 Spherical Baharara et al.
(2017)
Trianthema ZnO 10–20 Spherical Yadav et al. (2018)
portulacastrum Linn.
Leaf
Heriteira fomes, Zn, Ag 65–66 Spherical
Sonneratiaa petala (Ag),
hexagonal
(Au)
Litchi chinensis Au, Ag – – Murad et al. (2018)
Prunus domestica gum Au, Ag 7–30 Spherical Islam et al. (2017)
Andrographis ZnO 96–115 Spherical, Rajakumar et al.
paniculata leaf hexagonal (2018)

Some of the advantages of bacteria-mediated MO-NP synthesis are that the method is
scalable, cheaper, limits the use of toxic chemicals, and produces particles that are free of
additional impurities. This technique also has its disadvantages such as the selection of the
correct microorganisms being time consuming, the risk of contamination, and difficulties
in providing some control in particle synthesis (Agarwal et al. 2017).
Bacterial-induced synthesis is also known as biomineralization and occurs in iron-reduc-
ing bacterial species such as magnetotactic bacteria. Magnetotactic bacteria are a heteroge-
neous prokaryotic group with various cellular morphologies (Faivre and Schüler 2008).
This group’s members synthesize magnetite nanocrystals in a linear vesicle surrounded by
magnetosome. Biological synthesis of Fe2O3 nanoparticles (magnetite) is a multistage
molecular mechanism including the formation of magnetosomes, transport of iron to the
magnetosome vesicles, and crystallization of iron oxide nanoparticles (Zikalala et al. 2018).
Reductases and iron chelating siderophores make possible the transportation of iron into
the magnetosome. When Fe+3 is reduced on the cell surface, it is transported to the cell
membrane and oxidized, dehydrated, and precipitated to produce magnetite (Banzylinski
and Schubbe 2007). When the products (magnetite iron oxide nanocrystals) are examined,
it has been shown that their chemical purity has narrow size distribution and a special
arrangement in crystal morphology (Zikalala et al. 2018).
110 4 Synthesis of Metallic and Metal Oxide Nanomaterials

Microorganisms are mediated and convenient conditions for the extracellular synthesis
of inorganic nanoparticles by this synthesis. They produce detoxified nanoparticles, one of
the protective mechanisms against high concentrations of metal ions. The mechanism
comprises the exchange of redox potentials of metal ions, aggregation, and precipitation
(Agarwal et al. 2017; Zikalala et al. 2018).
Fungi is a group of eukaryotic organisms that contains yeast, mold, and mushrooms.
Fungi cell walls contain several biological molecules that ensure some advantages of nano-
particular synthesis compared to other microorganisms (Zikalala et al. 2018). In the synthe-
sis of different MO-NPs, reducing agents such as various enzymes and proteins contained in
fungal biomass are used (Khandel and Shahi 2018).
In green synthesis, by using fungi, secreted enzymes in various parts of the cell convert
metal ions into MO-NPs. Cationic metal ions attached by microorganisms cause the bio-
synthesis of MO-NPs (Suzuki et al. 2002). Similarly, metal ions stimulate specific biomol-
ecules such as proteins in fungi, which in turn hydrolyze the metal ion complexes bringing
about the genesis of nanoparticles. The fungal biomolecules also play a role in capping
agents and rendering nanoparticles ecofriendly and more stable (Raliya and Tarafdar 2013).
In general, the MO-NPs’ synthesis by using fungi is seen to be more favorable because
they are more suitable for bioaccumulation, more resistant to metals, and more economical
compared to the production processes of other prokaryotic particles (Zikalala et al. 2018).
Fungi surrounded by mycelia show more interaction with metal salts surface (Siddiqi and
Husen 2016). Furthermore, by using fungi in the formation of nanoparticles, larger amounts of
nanoparticles are formed compared to the use of bacteria and proteins (Taherzadeh et al. 2003).
In a general synthesis, the fungal biomass is added to the metal ion solutions and shaken
during incubation. Optimal temperature is needed to produce corresponding MO-NPs.
Some examples of MO-NP syntheses that have been performed by fungi can be given: pro-
duction of ZnO3 from ZnO by Aspergillus fumigatus TFR-8; synthesis of silica (SiO2), titania
(TiO2) and magnetite (Fe2O3), bismuth oxide (Bi2O3) and barium titanate (BaTiO3) by
Fusarium oxysporum (Zikalala et al. 2018).
Different yeast biomass is also used efficiently in nanoparticle synthesis. For example,
titanium dioxide (TiO2) nanoparticles have been prepared to utilize Saccharomyces cerevi-
siae (Bharde et al. 2006).
Algae ranging from unicellular (microalgae-Chlorella vulgaris) to multicellular (macroal-
gae-gigantic kelps) form a group of the eukaryotic and photosynthetic organism of the king-
dom Protista. As they are primary oxygen producers, such as photosynthetic prokaryotes,
they have an important role in the ecosystem. Macroalgae have the ability to convert toxic
metal forms to less toxic forms. Their ability to stabilize nanoparticles results from the pres-
ence of bioactive complexes (polysaccharides such as fucoidan, laminarin, alginate, etc.) that
contain hydroxide, carboxyl, and sulfhydryl groups in the cell walls. Moreover, their cell walls
also include secondary metabolites, like plants and seaweeds (Thema et al. 2016). Since the
metabolites present in algae are similar to those that exist in higher plants (spermatophytes),
the stages involved in the synthesis are obliged to be identical (Zikalala et al. 2018).
Plant extracts, used for a variety of purposes in the food industry, have also been used for
a long time in the production of nanoparticles. The ions penetrate into specific cavities of
the plant organelles where different metabolites exist in that organelles transform the
metal ions into nanoparticles (Makarov et al. 2014). The cavity works as a template and
identifies the morphology and size of the resultant nanoparticles. This technique is slow
 4.2 ­Biogenic Methods for Synthesis of Biocompatible and Hydrophilic Nanomaterial 111

compared to other biological systems for nanoparticle formation (Gardea-Torresdey et al.

2002; Zikalala et al. 2018).
Moreover, recovery of produced metals is inconvenient. Because the concentration, cavity
size, and types of stabilizing agents available in plants affect the process, the resultant metal
oxides’ size and morphologies are very miscellaneous. In vitro plant-based MO-NP synthesis
is gaining popularity in research. In this synthesis, all plant biomass, extracts, or certain parts
(leaves, roots, flowers, fruit peels, stem) are selected (Imran Din and Rani 2015; Zikalala et al.
2018). The extracts contain stabilizing agents such as terpenoids, flavonoids, phenolic acids,
proteins, polysaccharides, and organic acid for nanoparticles (Ahmed et al. 2017).
Generally, the target region is separated from the plant, washed, dried, and divided into
small pieces. The alkaline pH supports the generation of MO-NPs, which is stabilized by the
phytochemicals in the extract (Singh et al. 2016). Optimal reaction conditions and amount
of metal precursors play a critical role in nucleation and, either isotropically or anisotropi-
cally, the growth. At the end of the physical applications such as precipitation and washing
MO-NPs are obtained. Among biogenically synthesized nanoparticles are zinc oxide and
iron oxide. In addition, CuO and MgO2, NiO2, CeO2, SiO2 are nanoparticles synthesized by
plants, which are used in many applications (Arumugam et al. 2015; Ahmed et al. 2017).
Promising results have been reported in the literature on AgNPs’ antimicrobial activity
against several pathogenic microorganisms. Using AgNPs could inhibit the growth of mul-
tidrug-resistant bacteria shown in Figure 4.6.

4.2.3 Metallic Hybrid Nanoparticles

Among a range of hybrid nanoparticles, core-shell nanoparticles consist of two or more
materials, such as biomolecules and metals, one of which forms the center core, while the
other material/materials around the center core develop a shell (Khatami et al. 2018).
Organic–inorganic hybrid particles with diameters varying from ten to several hundred

Membrane damage

ROS
Mediated
damage
Protein
Damage

e–
DNA
Damage

Interruption in
electron transport chain

e– AgNPs

Figure 4.6 Antimicrobial mechanism of silver nanoparticles.

112 4 Synthesis of Metallic and Metal Oxide Nanomaterials

nanometers are important classes of hybrid materials with potential applications in a variety
of fields ranging from encapsulation and managed release of active ingredients to use as
additives for the paint and coating industries. Compared to individual nanoparticles, the
hybridization of noble metals (gold, silver, palladium, and platinum) with MO-NPs has
superior features. In several cases, metal oxides operate as semiconductors, such as nano
zinc oxide or titanium oxide nanoparticles, where their hybridization with silver nanoparti-
cles increases their photocatalytic efficacy significantly. Biomolecule–nanoparticle—or
quantum-dot (QD)—hybrid systems combine biomolecule validation and biocatalytic struc-
tures with the unique optical, catalytic, and electronic characteristics of nanoparticles to
produce composite materials with new functionality (Willner et al. 2007). Biomolecule–
nanoparticle hybrid systems enable the development of the synthesis of metallic nanowires,
new biosensors, and the manufacture of metallic or magnetic nanoparticle nanostructured
patterns on surfaces. These advances in nanobiotechnology are illustrated by the develop-
ment of amperometric glucose sensors through the electrical contact of redox enzymes
through AuNPs, and the design of an optical glucose sensor through the biocatalytic growth
of AuNPs (Willner et al. 2007). Metallic nanoparticles’ biocatalytic growth is used to manu-
facture Ag and Au nanowires on surfaces. Semiconductor quantum-dots’ fluorescence prop-
erties are used to develop competitive maltose biosensors and to test protease biocatalytic
functions. Similarly, when generating photocurrents, semiconductor nanoparticles associ-
ated with electrodes are used to photoactivate bio electrocatalytic cascades. Indeed, signifi-
cant progress has been made in the use of biomolecule–nanoparticle hybrid systems as
functional units for nanobiotechnology in recent years, and several articles have summa-
rized the various nanobiomolecular constructions and their potential applications (Niemeyer
2001; Willner 2002; Katz and Willner 2004).
In a recent work, new magnetic hybrid materials were developed by encapsulating iron
oxide nanoparticles into a chitosan matrix using eucalyptus extract as a reduction agent: a
green synthesis method was performed to obtain metallic nanoparticles (Martínez-Cabanas
et al. 2016).

4.2.4 Magnetic Nanoparticles in Biofuel Production

Biofuels are advancing rapidly as alternative renewable energy sources because of their
nonpolluting characteristics and cost-competitiveness compared to fossil fuels. However,
the focus is shifting to the use of technologies to maximize their yields in order to quickly
track their development. Due to their exquisite properties, nanoparticles are gaining
increasing interest among researchers, enabling them to be applied in diverse fields such as
the food industry, electronics, pharmaceuticals, and agriculture. To improve the perfor-
mance of these bioprocesses, they are also being explored in biofuels. This also elucidates
the different types of nanomaterials used in these bioprocesses (metallic, nanofibers, and
nanotubes) (Sekoai et al. 2019). The consequences of immobilized nanoparticles on biofu-
els like biodiesel are also assessed alongside the opportunity for nanoparticles under cer-
tain conditions to effectively suppress inhibitory chemicals. In addition, due to strong
paramagnetic properties and high coercion during the methanogenesis process, magnetic
nanoparticles (MNPs) can also be used for biogas production (Antunes et al. 2017). For the
production of biodiesel from waste cooking oil, one study used Pseudomonas cepacia lipase
 References 113

enzyme immobilized on MNPs (Yu et al. 2013b). For the synthesis of fatty acid methyl
esters (FAME) with soybean oil as a feedstock, some researchers had also previously immo-
bilized lipase from Pseudomonas cepacia onto MNPs (Mak 2008; Mak et al. 2009; Andrade
et al. 2016; Kim et al. 2018).

4.3 ­Conclusion
This review has systematically discussed major synthesis approaches, methods, and sur-
face properties of nanomaterials. It has been explained that although uniform and mono-
dispersed nanomaterials with great properties have been produced in the chemical
synthesis approach, use of toxic materials is the main disadvantage of those nanomaterials
especially when used in bioanalytical and biomedical applications. The researchers have
offered surface engineering strategies for phase transferring nanomaterials into aqueous
solutions or synthesis of biomolecules-directed biocompatible nanomaterials into aqueous
solutions to safely use them in nanomedicine.

­Acknowledgments

We thank authors Ayse Demirbas, Ismail Ocsoy, Nihan Arabacı, and Tuna Karaytuğ for
writing sections of 4.1, 4.2, 4.2.1, 4.2.2, 4.2.3, and 4.3. We also appreciate the assistance of
Nihan Arabacı, Tuna Karaytuğ, and Ebru Sebnem Yilmaz with the writing of sections 4.2
and 4.3. We also thank Recep Tayyip Erdogan University Scientific Research Office for the
grant (FBA-2018-903) that has supported the project on which this review is partially based.

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 125

Analysis of Green Methods to Synthesize Nanomaterials

Pavlos Nikolaidis
Department of Electrical Engineering, Cyprus University of Technology, Limassol, Cyprus

5.1 ­Introduction

The increasing presence of material goods of different types and forms in daily human life
makes them almost invisible to people. However, potential environmental concerns relat-
ing to their production and disposal are of vital importance. Indeed, lifecycle analyses
focused on demand and environmental impacts have revealed that material production,
utilization, maintenance, disposal, and replacement constitute energy‐intensive and high‐
cost procedures have a considerable influence on availability of world metals and overall
efficiency (Ra 2001; Beaudin et al. 2010). Our entry into the fourth industrial revolution is
set to modernize our daily life and requires us to integrate sustainable development goals
and actions to address the critical damage caused by the previous industrial revolutions
(Poizot et al. 2018).
With the fast‐paced changing technologies in both light and heavy industry, new chem-
istry references addressing new technologies are coming to the market. Nanoengineering
does show promise for combining the electrochemical advantages of different materials,
yielding composite designs with satisfactory performance in terms of safety and sustaina-
bility (Xin et al. 2017). Aiming to continuously increase the reactive density, the developing
nanoscale and nanostructured materials based on metals and metal oxides are among the
sought‐after candidates for next‐generation automobile and stationary systems.
Dealing with materials possessing at least one dimension smaller than 100 nm, green
nanotechnology is used to develop clean manufacturing processes in order to minimize
human health risk, encourage substitution of existing products, and be friendlier to the
environment. This combination of nanotechnology and the principles of green chemistry
may hold the key to building an environmentally sustainable society. The term is applica-
ble to any sort of systems and devices that require engineering to prepare nanoproducts
and nanomaterials without the use of toxic ingredients, operating at very low tempera-
ture, using less energy, exploiting renewable inputs, and using lifecycle concepts in all
design stages.

Green Synthesis of Nanomaterials for Bioenergy Applications, First Edition. Edited by Neha Srivastava,
Manish Srivastava, P. K. Mishra and Vijai Kumar Gupta.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
126 5 Analysis of Green Methods to Synthesize Nanomaterials

Innovations achieved in numerous applications involve medicine, energy, transportation,

metering and computing science, robotics, advanced manufacturing, flexible electronics,
intelligent sensing, and other biomedical and agricultural sectors. Based on the extensive
literature, this chapter provides a comprehensive classification, discussion, and analysis of
the operating principles along with the potential applications relating to green nanomaterial
synthesis methods. An overview of the major nanotechnology processes is carried out. The
remarkable properties and the main technical characteristics of the systems that make up
each nanotechnology process are analyzed. Also, the natural resources used and the chemi-
cal substances relating to each method are reviewed. Finally, the associated applications in
various fields are provided in order to evaluate the feasibility of such systems and their
future contribution to the development of a sustainable nanomaterial economy.
In section 5.2, emphasis is placed on the classification of nanoparticles and different
methodologies for their characterization. In section 5.3, a variety of natural resources that
are utilized for the synthesis of nanomaterials are discussed, with special focus on biosyn-
thesis by viable organisms. Section 5.4 emphasizes the synthesis methods and applications
of nanoparticles in different fields along with the current green nanotechnology achieve-
ments. Finally, the conclusions are drawn in section 5.5.

5.2 ­Classification of Nanomaterials

Inspired by the mechanisms of nature, the scientific community involved with nanotech-
nology is always turning to the natural world to study and imitate the artifacts of nature
that have endured in time and provide functional solutions to the survival of the organisms
and the construction of their surrounding space. In evolution, nature has chosen and
maintained those structures that are functional and viable for organisms by applying the
thermodynamic laws and principles of conservation of energy, matter, and load, obeying
both macro and quantum phenomena.
Nanotechnology mainly involves the synthesis of nanoparticles distinguished by their
size, shape, chemical composition, controlled dispersity, or potential use for human bene-
fits. They can be separated by size into nanoclusters, nanopowders, and nanocrystals or
distinguished by shape into nanorods, nanocups, nanospheres, nanodiamonds, nanostars,
or quantum dots. More recently, general and multifunctional classes were reported to define
the chemical composition of nanoparticles introducing liposomes, superparamagnetic nan-
oparticles, fullerenes (carbon‐based nanoparticles), dendrimers, and liquid crystals.
Liposomes or lipid vesicles find ready application as polymer nanocontainers in several
fields such as biology, biophysics, chemistry, pharmaceutical industry, cosmetics, etc.
They can serve as an excellent example of catalysis, energy conversion, and photosynthe-
sis, while in biochemistry they can help to better understand the biological function of
proteins specifically in secretion, trafficking and signaling, gene delivery, and other func-
tions in cell biology. Owning an iron oxide (Fe2O3) core and coated by either inorganic
(e.g. silica (SiO2), gold (Au)) or organic (like phospholipids, fatty acids, polysaccharides,
peptides, surfactants, polymers) materials, the second category includes the super-
paramagnetic nanoparticles. Due to their unique properties of superparamagnetism,
greater surface‐to‐volume ratio, and easy separation methodology, superparamagnetic
 5.2 ­Classification of Nanomaterial 127

­ anoparticles have been the focus of much consideration recently (Abbasi et al. 2014).
n
They are capable of attracting to a magnetic field without retaining residual magnetism
after the removal of the field. This constitutes a property that makes them appealing to
various biomedical applications including selective bio‐separations and contrast enhanc-
ing agents for diagnostics (e.g. MRI) in drug delivery systems, therapeutics (like magnetic
hyperthermia), and other ailments treatment (such as magnetically assisted transfection
of cells) (Nath and Banerjee 2013; Mahmoudi et al. 2011).
Fullerenes are molecules made entirely of carbon and they can take on distinct forms,
namely spheres, ellipsoids, tubes, etc. Inside their molecule, a hollow core or empty region
of space exists. As a result, the spherical forms of fullerenes are also known as “buckyballs”
whereas the tubular forms are referred to as “buckytubes” or carbon nanotubes. Structurally,
fullerenes resemble graphite, which is composed of stacked sheets often linked by pentago-
nal, hexagonal, or, rarely, heptagonal rings. Material sciences, optics, and electronics are
among the interested scientific fields that spend a great amount of time on research to
exploit their unique properties (Fahmy and Mobarak 2011). Dendrimers are nanosized,
radially symmetric molecules with well‐defined homogeneous and monodisperse struc-
ture. They are composed of a central core, an interior dendritic structure (the branches),
and an exterior surface with functional surface groups (Abbasi et al. 2014). They are built
layer‐by‐layer from core to circumference by repetitive covalent bond‐forming reactions,
generating molecules with uniform size and narrow weight distribution. In nanomedicine,
dendrimers have been found to be an invaluable tool in attaching fluorescent dyes, enzymes
cell recognition labels, and other molecules because of the many molecular “hooks” pre-
sented on their surface (Fahmy and Mobarak 2011; Nath and Banerjee 2013).
The last category refers to liquid crystals, which are typically pharmaceuticals composed
of liquid organic crystalline materials. They are designed to mimic naturally occurring bio-
molecules like proteins or lipids. Being able to target a particular area of the body where
tissues are inflamed and capable of detecting tumors, liquid crystals are interpreted as a
very safe and specific carrier of drug delivery (Yuan and Ozcan 2015). Besides, various
kinds of metal nanoparticles are available, like silver (Ag), gold (Au), platinum (Pt), cad-
mium (Cd) and metal oxide such as zinc oxide (ZnO), cerium dioxide (CeO2), titanium
dioxide (TiO2), and zirconium dioxide (ZrO2). A nanometer scale comparison for different
nanoparticle categories is provided in Figure 5.1.
Among the various nanostructured oxide materials, special attention has been directed
to TiO2 nanotubes due to its enhanced properties, cost‐effective construction, and higher
surface‐to‐volume ratio (Meissner and Shepherd 2001). The TiO2 with high‐specific surface
area, ion‐exchangeable ability, and photocatalytic property has been considered for various
potential applications as catalyst in pool boiling (Chen et al. 2009), photocatalysis (Costa
and Prado 2009), electrochromic device (Macak et al. 2007), hydrogen generation
(Nikolaidis and Poullikkas 2017), corrosion resistance (Narayanan and Seshadri 2006),
solar cells (Mor et al. 2006), sensors (Varghese et al. 2003), storage device (Nikolaidis and
Poullikkas 2017), catalyst support (Liang et al. 2007), waste water treatment (Liu et al.
2009), wettability (Balaur et al. 2005), and especially in the biomedical field (Crawford et al.
2009). In addition to the aforementioned applications, nowadays the TiO2 is used to remove
the environmental pollutants (Zhang et al. 2011) and as a volatile organic content sensor
(Kilinç et al. 2011; Indira et al. 2015).
128 5 Analysis of Green Methods to Synthesize Nanomaterials

Water Glucose Antibody Virus Bacteria Cancer cell A period Tennis ball

(nm)
10–1 1 10 102 103 104 105 106 107 108

Liposome Dendrimer Gold nanoshell Quantum dot Fullerene

Figure 5.1 Nanotechnology chart ruler. Source: Pradhan et al. (2015).

Ag nanoparticles (AgNPs) have conquered extreme interest, owing to their beneficial

characteristics of antimicrobial, antioxidant, and antitumor activities, which aid in the
development of pharmaceutical drugs. The applications of AgNPs have also been success-
fully applied to agriculture and other textile and food industries (Shanmuganathan et al.
2018). Pt‐nanoparticles (PtNPs)possess an excellent oxidation property. A representative
example is the oxidization and reduction in the process of coenzyme Q (Kim et al. 2008).
Of great interest are also the polymer‐doped Pt‐NPs and template‐directed synthesis of
cadmium sulfide nanoparticles (CdSNPs). Specifically, PtNPs are capable of exhibiting
various functions in the form of electrode nanomaterials, optical and biological nanosen-
sors as well as catalysis and photocatalysis (Ramkumar et al. 2017), while the distinct
method used for CdSNPs has spurred the research interest due to its size‐dependent tune-
able spectroscopic properties (Das et al. 2012).
ZnO and CeO2 nanoparticles can be used in cancer diagnosis and treatment because of
some reliable physiological attributes. In respect to their nanosize, they acquired immense
curiosity in the earlier decade due to their outstanding optical, electronic and chemical
properties, which are not exhibited by the bulk counter parts. More recently, these kinds of
nanoparticles have been widely utilized in alternative sectors including biomedicine, indus-
tries, electronics, environmental remediation, etc. (Hameed et al. 2017). Metal nanoparticles
such as Ag and Au possess wider biomedical applications in the field of medical nanoengi-
neering, pharmaceuticals, and theragnostic, especially for the treatment of cancer, arthritis,
and chronic diseases. Within the metal nanoparticles category, AuNPs have been considered
as promising contributors to a wide variety of applications in different sectors. Due to their
relative ease in the synthesis, controlled size, shape, optical characteristics and biocompati-
bility, AuNPs become advantageous in drug and gene delivery, nucleic acid labeling and
 5.3 ­Natural Sources for Green Nanomaterial 129

tissue engineering, biosensors and bioimaging process, photodynamic‐based therapy against

human pathogens, protein detection, etc. (Suganthy et al. 2018).
Finally, ZrO2 may be the one for which there is the largest debate among scientists,
industrials, or clinicians. It constitutes a robust transition metal that resembles titanium
due to its strong resistance to corrosion and biomedical grade zirconia. As an implant bio-
material, dental crown, or femoral heads, ZrO2 is widely used for total hip replacement,
solid oxide fuel cell electrolytes, catalytic activity, high chemical stability, and ionic conduc-
tivity. Its wide range of applications lies in its outstanding biocompatibility, increasing
strength, and low wear cost. However, in biomedical industries there is no clear informa-
tion about its respective metabolic interactions and toxicity (Fathima et al. 2017).

5.3 ­Natural Sources for Green Nanomaterials

Numerous chemical substances can serve as suitable reducing agents for nanoparticle syn-
thesis; plant extracts, biopolymers, vitamins, proteins, peptides (such as glutathione), and
sugars (like glucose and fructose) are some representative examples of resources provided
by nature (Virkutyte and Varma 2011). Given their abundance, plant extracts (plant leaf,
root, latex, seed, and stem) are considered among the most promising natural reducing
agents with particular success in synthesizing metal nanoparticles used in both medical
applications and electronics (Kumar and Yadav 2009). Nontoxic, green reduction agents,
including plants, algae, bacteria, and fungi, are commonly used to improve the biocompat-
ibility in the synthesis processes.
Biopolymers form a further family of natural resources exploited as reducing and stabiliz-
ing agents for metal nanoparticle synthesis. These polymeric carbohydrate molecules have
already been used in various industries and thus are readily available for large‐scale nano-
particle production. Examples of biopolymers for nanoparticle synthesis include chitosan,
cellulose, and dextran, which are isolated from the exoskeleton of crustaceans, plants, and
sugar cane, respectively (Yuan and Ozcan 2015). Vitamin C is a well‐known natural reduc-
ing agent or antioxidant. Similar to other natural reducing agents, vitamin C can reduce
metal ions (Mn+) in an aqueous solution to produce metal nanoparticles (Nadagouda et al.
2007). Interestingly, it can also be used to synthesize iron (II, III) oxide (Fe3O4) nanoparticles
by reducing colloidal iron hydroxide (or colloidal ferric hydroxide—Fe(OH)3) under hydro-
thermal conditions (Li et al. 2011). Other vitamins utilized for metal nanoparticle synthesis
include vitamin B and vitamin E. Despite the widespread commercial availability of vita-
mins, their relative cost‐effectiveness may be an obstacle for commercial application. Other
natural sources, such as proteins and peptides, also suffer from cost‐related challenges and
thus are ill‐suited for the large‐scale production of nanoparticles (Scott et al. 2008).
Vegetable oil has been proven to be a viable precursor for high‐quality carbon nanotube
formation. Using different catalysts, both single‐walled and multiwalled carbon nanotubes
can be produced whereas other examples make reference to use of sugars and biopolymers
as precursors for carbon quantum dots (Titirici et al. 2015). Microorganisms are also
regarded as potential resource alternatives for producing nanoparticles via biological pro-
cesses. Such methods appear to be more advantageous due to certain features, namely the
selective adsorption of Mn+, operation under a wide variety of environmental conditions
130 5 Analysis of Green Methods to Synthesize Nanomaterials

(pH, ionic strength, and temperature), and their undemanding, renewable, and economic
availability, great biosorption capability, and the potential to contribute by making use of
feed-stocks that can be obtained in large quantities (Shankar et al. 2003). Bacteria, fungi,
and yeast, which fall into the microorganism family, play an essential role in ecology for
mineral reprocessing. In addition, they have been developed and proven as novel materials
for the fabrication of semiconductors, quantum dots, and metal nanoparticles of different
sizes and shapes (Saratale et al. 2018).
Bacteria and actinomycetes are also suitable for the reduction of Mn+ into metallic (M0)
nanoparticles. Two main categories exist distinguishing the biological and microbial sys-
tems for nanosynthesis as intracellular and extracellular. With respect to the category into
which they are grouped, the nanoparticles are formed or synthesized based on their loca-
tion in the bio‐reducing system. Due to several advantages, bacterial systems represent a
useful basis for the extracellular synthesis of metal nanoparticles. Among them, their eco-
nomic and increased growth rate in combination with their ease of genetic manipulation
are the most valuable benefits. On the other hand, actinomycetes possess proteins, which
significantly raise the productivity through biosynthesis. The actinomycete‐mediated syn-
thesis of nanoparticles offers the advantage of easy scaling‐up processes. In contrast to
bacterial fermentation processes, which require systematic instruments to extract clearer
filtrates from colloidal broths, actinomycetes are easily filterable due to a filter press, thus
avoiding the cost associated with investments in heavy equipment (Saratale et al. 2018).
Yeasts are mainly eukaryotic single‐celled microorganisms classified as members of the
fungus kingdom. Owing to their higher tolerance to toxic metals they can be successfully
employed for metallic nanoparticle syntheses. Yeast‐based biosynthesis processes are
mainly the result of enzymatic reduction and chelation with extracellular peptides/poly-
saccharides ending at different sizes, morphologies, and nanoparticle synthesis sites.
Similar properties are also provided by fungi. Some of the most important characteristics
are the resistance to toxicity, the ease of manipulation and scaling up their process, the
large surface areas leading to increased rates of production, the simple and convenient
downstream processing, and economic viability. Over the bacterial systems, their unique
features include the larger spectrum of nanoparticle morphologies and intracellular metal
uptake capabilities (Gericke and Pinches 2006).
Revolutionized by nanoscale engineering, viruses have played a special role in the way we
prevent, detect, and treat diseases. Since they can function as prefabricated nanoscaffolds
providing distinct characteristics and easy modifications, virus‐based nanocomposites are
considered very important biomaterials for producing smart nanoobjects. The interiors of
virus particles can encapsulate and protect sensitive compounds, while the exteriors can be
altered to display large and small molecules in precisely defined arrays (Koudelka et al.
2016). Viruses provide unique properties in nano‐conjugates synthesis with noble metal
nanoparticles, relying on their monodispersal capability, stable and robust nanoparticle
formation. Hence, monodispersed nanoparticles of different morphologies and sizes can be
created in a controlled manner and contribute as actively targeted drug delivery systems to
expand on and improve current pharmaceutical options (Saratale et al. 2018).
Finally, algae are equally important organisms in the synthesis of nanoparticles. Algae is an
informal term for a large, diverse group of eukaryotic oxygenic photoautotrophs that are not
necessarily closely related and is thus polyphyletic (Castro et al. 2013). Some of them are able
 5.3 ­Natural Sources for Green Nanomaterial 131

to accumulate various heavy metals. Nowadays, their culture is gaining tremendous attention
as a response to the rapid industrialization, economic development, and ­overpopulation; the
main drivers for the release of organic and inorganic substances into the environment, which
further leads to environmental pollution and water contamination (Agarwal et al. 2019).
They display a wide variety of shapes and sizes and can be found in different habitats, either
aquatic or semiaquatic, such as seas, lakes, rivers, swamps, polar areas, and deserts
(Dahoumane et al. 2016; Saratale et al. 2017). Algae‐mediated synthesis becomes advanta-
geous in terms of production scale and cost as well as in the nucleation and crystal growth
rate (Sharma et al. 2015). However, it is influenced by operational parameters, namely pH,
temperature, substrate concentration, and surface‐bound proteins along with their amino
acid ingredients involved in the capping and stabilization of nanoparticles (LewisOscar et al.
2016). A wide variety of nanoparticles synthesized using different natural resources are listed
in Table 5.1 along with their nanosized dimension (Saratale et al. 2018).

Table 5.1 Nanoparticles synthesized using different natural resources.

Reducing/stabilizing agent nanoparticles Size (nm)

Aspergillus flavus silver – Ag 7.3–10.5

Aspergillus fumigatus silver – Ag 5–25
Bacillus cereus silver – Ag 4–5
Bacillus licheniformis silver – Ag 50
Brevibacterium casei gold – Au, silver – Ag 10–50
Candida utilis gold – Au N/A
Corynebacterium glutamicum silver – Ag 5–50
Desulfovibrio desulfuricans lead – Pb 50
Enterobacter sp. mercury – Hg 2–5
Escherichia coli gold – Au 20–30
Escherichia coli silver – Ag 50
Escherichia coli cadmium – Cd/Te – tellurium 2–3.2
Fusarium oxysporum Au–Ag alloy 8–14
Fusarium oxysporum silver – Ag 5–50
Neurospora crassa silver – Ag, Au/Ag 20–50
Phaenerochaete chrysosporium silver – Ag 50–200
Plectonema boryanum UTEX 485 gold – Au 10–6000
Plectonemaboryanum gold – Au <10–25
Pseudomonas aeruginosa gold – Au 15–30
Pyrobaculum Islandicum U(VI), Tc(VII), Cr(VI), Co(III), Mn(IV) N/A
Rhodococcus sp. gold – Au 5–15
Rhodopseudomonas capsulate gold – Au 10–20
Sargassum wightii gold – Au 8–12

(Continued)
132 5 Analysis of Green Methods to Synthesize Nanomaterials

Table 5.1 (Continued)

Reducing/stabilizing agent nanoparticles Size (nm)

Shewanella algae gold – Au 10–20

Shewanella algae platinum – Pt 5
Shewanella oneidensis gold – Au 7–17
Shewanella sp. selenium – Se 141–221
Trichoderma viride silver – Ag 5–40
Trichoderma viride silver – Ag 2–4
Ureibacillus thermosphaericus gold – Au 50–70
V. luteoalbum gold – Au N/A
Verticillium sp. silver – Ag 17–33
Yarrowia lipolytica gold – Au 15
Yeast Au/Ag 9–25

Source: Nath and Banerjee (2013).

5.4 ­Green Methods to Synthesize Nanomaterials

Generally, the synthesis of nanoparticles can be achieved by a variety of physical and

chemical methods possessing several drawbacks such as: high cost of production; require-
ment of high energy input; and generation of potentially hazardous to the environment,
toxic, and perilous byproducts that are responsible for various biological risks. Attrition
and pyrolysis are the most commonly used physical methods. In attrition, the main steps
involve a size‐reducing mechanism for the particles grinding, their air‐classification and
the final recovery of the oxidized nanoparticles. Pyrolysis involves an intermediate burning
of the precursor by passing the particles through an orifice at high pressure. The rest of the
process is similar to attrition; the obtained ash is then air‐classified to recover the oxidized
nanoparticles (Chen et al. 2012).
On the other hand, the chemical methods are represented by chemical reduction, elec-
trochemical techniques, and photochemical reactions in reverse micelles. Chemical reduc-
tion is the simplest and most commonly used route for the growth of stable metal
nanoparticles, colloidal dispersions in liquid solvents containing various reactants.
Particularly, sodium borohydride (NaBH4), potassium bitartrate (KC4H5O6), methoxy‐poly-
ethylene glycol (C34H40O20), or hydrazine (N2H4) are typically used as reducing agents.
Sodium dodecyl benzyl sulfate (C18H29NaO3S) or polyvinyl pyrrolidone (C6H13NOP2) are
added to the reaction mixture as stabilizing agents to prevent the agglomeration of metallic
nanoparticles.
Chemical methods are low cost for high volume, while their demerits are the contamina-
tion from precursor chemicals, the use of toxic solvents, and the generation of hazardous
byproducts. The main drawbacks of physical methods include the low production rate,
high production cost, and high energy consumption. Therefore, there is a requirement for
new, environmentally safe methods for synthesizing nanoparticles in order to replace the
 5.4 ­Green Methods to Synthesize Nanomaterial 133

toxic ingredients evolved while improving the overall performance. To overcome this,
research is focused on biological techniques, since they are generally regarded as more
cost‐effective, nontoxic, and ecofriendly processes.
Figure 5.2 presents such a concept, i.e. the mechanism of metal nanoparticles ­synthesis.
According to the process, the Mn+ are reduced to M0 nanoparticles by heading with the
aqueous form of plant extracts. Different sizes and shapes of metallic nanoparticles are
successfully synthesized using bio‐organisms by varying the process’ synthesizing param-
eters of pH, temperature, incubation time, and/or oxygenation (Bai and Zhang 2009; Keat
et al. 2015). Figure 5.2 shows the various steps employed in the synthesis of nanoparticles
using microorganisms. Bioresources containing enzyme, protein, and bioactive molecules
will bind to Mn+ due to the stress by the metal salts. Then, they can be reduced to nuclei
followed by the growth of nanoparticles (Saratale et al. 2018).
The development of biologically inspired experimental processes is evolving into an
important branch of nanotechnology. Two approaches exist: top–down and bottom–up, as
illustrated in Figure 5.3 (Llop et al. 2017). In bottom–up approaches, nanoparticles can be
synthesized using chemical and biological methods by self‐assembly of atoms into new
nuclei, which grow into a particle of nanoscale. In top–down approaches, suitable bulk
material breaks down into fine particles by size reduction through various lithographic
techniques such as grinding, milling, casting, sputtering, or thermal/laser ablation. In
other words, the first pathway makes use of synthesizing the appropriate materials from
the atomic or molecular level of matter through chemical reactions, allowing the elemen-
tary particles to grow in size. On the other hand, the opposite route starts with large‐sized
materials that are gradually reduced to a smaller scale through different energy conversion
steps. Both approaches play an important role in modern industry and of course in nano-
technology processes (Letsinger et al. 2002; S. Information 2010).

Metal salt Biomolecules (cellulose, lignin Nanoparticles

pectin, chitosan & proteins) synthesis

Mn+ Mn+ M0 M0
Mn+ M0
Mn+ Mn+
M0 M0

Nucleation

Capped and stable Capping of nanoparticles Growth of

nanoparticles with biomolecules nanoparticles

Figure 5.2 Schematic representation of a proposed mechanism for plant-mediated synthesis of

metal nanoparticles. Source: Saratale et al. (2018).
134 5 Analysis of Green Methods to Synthesize Nanomaterials

M+ Metal ions

M+
M+ M0
O
Reduction Nucleation
M0 Metal
OH O Nanoparticle
Stabilization

M0
O
OH
Reduction metal atoms
OH
Plant reducing agent

Figure 5.3 Synthesis of metallic nanoparticles through reduction and stabilization. Source: Keat
et al. (2015).

Based on their different physical and chemical properties, the associated green chemistry
principles and the corresponding green nanotechnology responses are listed in Table 5.2.
So far, the emerging trend of synthesizing nanoparticles has been developed over various
biogenic methods relying on bacteria, fungi, yeast, algae, viruses, and on using biomole-
cules. Green nanosynthesis refers to the employment of reducing and stabilizing agents
from plants and other natural resources to fabricate nanomaterials. It does not engage the
use of extremely poisonous chemicals or elevated energy inputs during the synthesis.
Instead, various kinds of phototrophic eukaryotes mentioned in section 5.3 (algae,
microbes, plants, etc.) are reflected as an efficient and environmentally benign green nano-
factories, that have been notably designed for the synthesis of different inorganic nanopar-
ticles. Highly stabilized organisms are capable of reducing the Mn+ to metal nanoparticles,
reaching the end goal of producing high‐purity products. Hence, the novelty of green syn-
thesis is progressing as a key branch of nanotechnology where the use of biological entities,
ranging from microorganisms to plant extracts, could be a potential alternative for produc-
ing nanoparticles in an ecofriendly manner (Kim 2010).
In the top–down approach, nanoparticle synthesis is generally realized at atmospheric
pressure through evaporation–condensation. A tube furnace assists the center‐placed
material to vaporize into a carrier gas. Silver, gold, lead‐sulfide, and fullerene nanoparticles
can be synthesized using this technique. Despite the large space occupied by tube furnaces,
utilizing them as the main source of heat constitutes an energy and time intensive proce-
dure in order to adequately raise the temperature and achieve thermal stability (Prathna
et al. 2011; Sintubin et al. 2011). Referred to as surface chemistry, another critical limitation
of the method are the surface–structure imperfections that violate the physical attributes of
nanoparticles, which are strongly dependent on it (Ahmed et al. 2016).
Other conventional approaches for generating organic or inorganic nanoparticles are
referred to as chemical vapor deposition (CVD) (Sladek and Herron 1972), pyrolysis (Han
et al. 2013), hydrothermal synthesis (Lindh 2000), and sol‐gel (Gonzalez‐Oliver et al. 1982).
Exploiting different aspects of chemistry, these methods aim to create nanostructured mate-
rials, the geometrical features of which can be controlled by various anodization (potential,
 5.4 ­Green Methods to Synthesize Nanomaterial 135

Table 5.2 The principles of green chemistry and the corresponding green nanotechnology
response.

Green Chemistry Principles Green Nanotechnology Response

1) Prevent waste Design chemical syntheses to prevent waste, leaving no waste to

treat or clean up.
2) Design safer chemicals Design chemical products to be fully effective yet have little or
and products no toxicity.
3) Design less hazardous Design syntheses to use and generate substances with little or no
chemical syntheses toxicity to humans and the environment.
4) Use renewable feedstock Use raw materials and feedstocks that are renewable rather than
depleting (e.g. made from agricultural products or wastes of
other processes rather than from fossil or mined fuels).
5) Use catalysts, not Minimize waste by using catalytic reactions since catalysts are
stoichiometric reagents used in small amounts and can carry out a single reaction many
times in contrast to stoichiometric reagents, which are used in
excess and work only once.
6) Avoid chemical derivatives Avoid using blocking or protecting groups or any temporary
modifications if possible. Derivatives use additional reagents and
generate waste.
7) Maximize atom economy Design syntheses so that the final product contains the
maximum proportion of the starting materials. There should be
few, if any, wasted atoms.
8) Use safer solvents and Use innocuous chemicals instead of solvents, separation agents,
reaction conditions or other auxiliary chemicals.
9) Increase energy efficiency Run chemical reactions at ambient temperature and pressure
whenever possible.
10) Design chemicals and Design chemical products to break down to innocuous
products to degrade after substances after use so that they do not accumulate in the
use environment.
11) Analyze in real time to Include in‐process, real‐time monitoring, and control during
prevent pollution syntheses to minimize or eliminate the formation of byproducts.
12) Minimize the potential for Design chemicals and other forms (solid, liquid, or gas) to
accidents minimize the potential for chemical accidents, including
explosions, fires, and releases to the environment.

Source: Schmidt Pen (2007).

time, and temperature) and electrolyte parameters (composition, pH, viscosity, and conduc-
tivity) (Kilinç et al. 2011). For example, the CVD method involves the transport of gaseous
reactants in an activated (heat, light, plasma) environment. This transport is performed
above a heated substrate upon which the stable solid product can deposit. As a relatively
mature technique, CVD offers the distinct advantages of producing highly dense and pure
materials at a reasonable cost as well as being able to coat complex shaped components
while controlling the crystal structure, surface morphology, and orientation (Choy 2003).
Spray pyrolysis constitutes an alternative to the CVD method. It is a versatile technique
for ceramic materials production on industrial scales, with a wide variety of particle sizes,
136 5 Analysis of Green Methods to Synthesize Nanomaterials

c­ ompositions, and morphologies (Li et al. 2005). In general, a thin film can be observed by
spraying a solution on a heated surface. A typical process may evolve as follows: (i) an initial
solution with precursors (e.g. TiCl4 for titanium or NH4F for nitrogen/fluorine) is first ­atomized
by a nebulizer; (ii) the formed droplets pass through a high‐temperature tube undergoing
pyrolysis; (iii) the final collection of the generated powder can be realized on a ceramic filter at
the end of the tube. Another technique falling into the high‐temperature approaches is known
as hydrothermal synthesis. Hydrothermal synthesis is achieved by chemical reactions taking
place at elevated temperature and pressure conditions in a sealed heated solution. This mecha-
nism follows a liquid nucleation model the understanding of which is necessary to best fit a
synthesis method to its synthesized material’s properties (Feng and Xu 2001).
The final conventional approach is known as sol‐gel method and provides great custom-
izability in the final product. It sits between the well‐established synthesis approaches
for the preparation of innovative metal oxide nanoparticles. According to (Hench and
West 1990), “sol” is used to define the dispersed colloids and nanoparticles contamination
into a liquid, whereas “gel” denotes the rigid network formed by interconnected pores
(size < 1000 nm). Following sequentially the processes of hydrolysis, condensation, and
drying, the initial metal precursor is processed into metal hydroxide solution, dimensional
gel, and Xerogel or Aerogel, respectively. However, the utilized solvent can be either water
or organic solution, classifying the process performed in aqueous or non‐aqueous, respec-
tively. Then, a calcination process takes place in order to remove the volatile species and
provide the desired product with high purity. A further advantage regards the homogenous
multicomponent systems that can be easily obtained (Gonzalez‐Oliver et al. 1982).
Following are a number of alternative approaches able to enhance some critical parame-
ters and overall performance in the synthesis of metal nanoparticles. Ranging from the
non‐conventional to the more advanced, the various processes include laser ablation, micro-
wave‐assisted, photocatalytic degradation, ultrasonic irradiation, sonochemical decomposi-
tion, solvothermal, electrochemical synthesis, etc. Laser ablation of metallic bulk materials
in solution has been successfully employed for the synthesis of Ag‐nanospheroids (20–
50 nm). The formation efficiency and final product characteristics depend on the laser flu-
ence, wavelength and pulses duration, the effective liquid medium (in the presence or
absence of surfactants), and the ablation duration time. Compared to other approaches for
production of metal colloids, this technique ensures preparing pure and uncontaminated
metal colloids due to the absence of chemical reagents in solutions (Iravani et al. 2016).
The introduction of microwave‐assisted techniques in nanoparticle synthesis methods
has seen considerable improvements in terms of yields, selectivity, and conversions
achievement that would otherwise be impossible. It is becoming a widespread method for
preparing organic and inorganic nanostructured materials, offering distinct advantages
compared to a synthesis assisted by a traditional chamber furnace. Apart from the faster
response and kinetics, no further annealing is required for developing crystalline materials.
(Han et al. 2013). It is known that most preparation methods (including sol‐gel) require
high temperatures (450–1400 °C) and longer processing time (6–8 hours) to obtain crystal-
line particles, whereas by using the microwave in the preparation, temperatures below
250 °C can be seen for quality crystalline production (Kuo et al. 2015).
Similar results have been observed in the synthesis of other doped and undoped metal
oxides such as TiO2 (Wilson et al. 2002), CuO (Wang et al. 2002), and copper‐sulfate (CuS)
 5.4 ­Green Methods to Synthesize Nanomaterial 137

nanorods (Gorai et al. 2005). Consequently, the microwave‐assisted technique is ­considered

to be an industrially viable approach that offers an energy efficient, ecofriendly, rapid, and
convenient process (Periyat et al. 2010). A microwave process for the synthesis of nanocrys-
talline titania showed a surface area up to 240 m2 g−1, which was almost six times higher
than that of a traditional sol‐gel approach (40 m2 g−1). The final product was rapidly
­produced at comparatively low microwave power intensity (300 W) and irradiation for
two minutes (Han et al. 2013).
Photocatalysis constitutes the reaction by which a photocatalyst can be activated.
Activating photocatalysts can modify a chemical reaction rate. This way, microwave irradia-
tion can take place in the preparation of visible‐light (>400 nm) active materials, which can
be used to break down contaminants of interest. A microwave‐assisted (at 200 °C for 30 min-
utes) hydrothermal treatment has been employed for the formation of photocatalytic anatase
TiO2 using a mixed solution of tetra butyl titanate (C16H40O4Ti) – hydrogen fluoride (H2F2)
solution and additional water (Li et al. 2012). Ag‐nanoparticles have been successfully pre-
pared through the photoreduction of silver nitrate (AgNO3) in a stabilizing agent layered by
inorganic laponite clay suspensions. The properties of generated nanoparticles varied by UV
irradiation time. Specifically, bimodal size distribution and relatively large nanoparticles
were observed after three hours of UV irradiation, while further irradiation was needed in
order for a relatively stable, smaller size to be obtained in a single distribution mode
(Suprabha et al. 2009). Silica‐coated ZnO‐nanoparticles have also been obtained via a simple
microwave‐irradiation method. These particles possess certain photocatalytic attributes,
where the silica effectively inhibits the photocatalytic activity of the ZnO‐nanoparticles
(Siddiquey et al. 2008). Generally, the use of microwave irradiation has been found to be a
proven approach in reducing the time needed as well as the energy used in the fabrication
of various nanomaterials in the field of nanoscience (Teimouri et al. 2018). Photochemical
synthesis is considered to be a clean alternative with convenience of use and high versatility.
In addition, it enables the fabrication of nanomaterials in a wide variety of mediums (such
as cells, emulsion, polymer films, surfactant micelles, glasses, etc.) (Iravani et al. 2016).
Ultrasound radiation is a further research area that has been experiencing great interest
for studying different chemical effects in the last decades. This notable example targets the
production of hot spots, which are able to initiate and transmit energy‐intensive chemical
reactions. Such spots are observed after intense local heating and high pressure by generat-
ing ultrasound irradiated in a liquid phase. In this regard, nanoparticle synthesis can be
realized at considerably lower temperatures. Other potential benefits are the simplicity of
ultrasonic technology, its improved efficiency, and one‐step green synthesis method. A
Materials group reported the successful production of superparamagnetic γ‐Fe2O3 nanopar-
ticles by using ultrasonic irradiation of an Fe(II) alkoxide derivative (Gun’ko et al. 2001;
Afsheen et al. 2018). In relation to nanoparticles, there have been many other investigations
carried out in the field of sonochemistry. For example, Ag‐nanoparticles have been prepared
using the sonoelectrochemistry technique, which utilizes ultrasonic power to mechanically
manipulate the material by alternating sonic and electric pulses (Iravani et al. 2016).
Sonochemical decomposition of Fe(CO)5 was performed in the presence of various
surfactants, retrieving stable colloids of undecenoate (C13H24O2), dodecyl sulfonate
(C12H25NaO4S), and octyl phosphonate (C8H17Na2O3P) coated Fe2O3 nanoparticles of
5–16 nm diameter (Shafi et al. 2001). Relating to the production of nanosized materials, the
138 5 Analysis of Green Methods to Synthesize Nanomaterials

main objective of sonochemistry is to the develop facile, rapid, green, convenient, and
­additive free synthesis. One such protocol was achieved using a simple synthesis method to
prepare ZnO‐nanoparticles (Bhatte et al. 2012; Khanjani et al. 2013). Moreover, many
­different morphologies achieved by similar processes, such as PbWO4 nanostructures (Geng
et al. 2006), Zn(II)bis(1‐(2‐pyridylazo)‐2‐naphthol) or Zn(PAN)2 complex nanorods, porous
Cu2O nanospheres, and single‐crystalline PbF2 nanorods (Han et al. 2013). Finally, a novel
sonochemical process was carried out under high‐intensity ultrasonic irradiation (20 kHz,
100 W cm−2) at 90 °C for three hours. Anatase nanocrystalline TiO2 was directly synthesized
after the hydrolysis of tetraisopropyl titanate (C12H28O4Ti) (Guo et al. 2003). As a result,
sonochemical routes possess a potential role in functional materials synthesis at decreased
temperatures and in substantially shorter times compared to conventional methods.

5.5 ­Conclusion

Nanotechnology is mainly associated with the synthesis of nanoparticles of variable sizes,

shapes, chemical compositions, controlled dispersity, and potential uses. A wide variety of
green nanomaterial synthesis methods and concepts are available while others will emerge
in the future. Advanced approaches in the field are playing an increasingly important role
in modern society, including both automobile and stationary systems. Therefore, the first
aspect of green nanotechnology aims at using more environmentally friendly or more
energy‐efficient manufacturing processes. The second aspect involves developing products
that benefit the environment.
Nanomaterials or products are able to directly clean hazardous waste sites, desalinate
water, or sense and monitor pollutants in the environment. Indirectly, lightweight nano-
composites for automobiles and aircraft can save fuel and reduce material use.
Nanoengineering‐enabled technologies such as fuel cells, electrochemical storage systems,
and light emitting diodes (LEDs) can assist in pollution mitigation in all of the energy pro-
duction sectors and help conserve fossil‐fuel reserves. Moreover, nanoscale self‐cleaning
surface coatings could even eliminate the need for cleaning chemicals of different kinds.
Finally, extended battery life spans can be achieved, leading to decreased material use and
waste. Figure 5.4 offers a wide overview of the applications of surface‐modified nanoparti-
cles and more broadly nanostructures. The term surfactant‐decorated nanoparticles is typi-
cally associated with manmade nanostructures for applications including imaging, drug
delivery, composites, catalysis, energy conversion devices, purification systems, and a vari-
ety of other technologies shown in Figure 5.5.
Ways forward may include: (i) advancements relating to the recent interest in semi‐con-
ducting layered materials; (ii) the emergence of new nanomaterials with advanced optical,
mechanical, electrical, and other functional attributes; and (iii) the extensive use of self‐
assembled chemical substances on nanoparticles. Research and development efforts should
not only be concentrated toward small‐scale nanomaterial synthesis processes, but also at
different points and levels of use where their suitability assessment is of critical impor-
tance. Finally, there is still a plenty of scope, since a biological nanosynthesis system is
always less controllable compared to a chemical process.
 5.5 ­Conclusio 139

Bulk

AuNPs
Atomic/molecular
Condensation
Vapour Deposition Sputtering

P U
Nanopartice Synthesis

Bottom
Sol-gel Process Chemical etching
Spray Pyrolysis Thermal/Laser ablation

Chemical/Electrochemical Mechanical/Ball missling

Nuclei Powder
Deposition

Top
Explosion Process
Aerosol Process

own D
Bioreduction

Atoms/Molecules
AuNPs

Figure 5.4 Protocols employed for different synthesis methods of nanoparticles. Source: Llop et al.
(2017).

Silica-based NPs Metal-based NPs

Liposomes
SiO2 Metal oxide NPs
Au

Ag
QDots
Magnetic NPs

Carbon-based NPs

Drug loading & delivery, anti-

bacterial, antifungal and antiviral

Biosensors and contrast agents for Polymer-based NPs

medical diagnostics and cell study
Bioseparation & purification or
Photoacoustic, photothermal or bioactive substances immobilization
photodynamic imaging & therapy
Magnetically()-assisted Immunoassay & biomedical diagnosis
imaging & hyperthermia

Figure 5.5 Applications of surface-modified nanoparticles. Source: Zhao et al. (2015).

140 5 Analysis of Green Methods to Synthesize Nanomaterials

Biosensors Detergents,
and brighteners, Removal of
Diagnostics Implants for cosmetics metal ions,
Therapeutics bone and
(organic/ toxins
teeth
inorganic)
Medical Cleaning and Column
Applications Purification materials in
chromato-
Functional graphy
hydrogers

Nanoparticle
Decoration with
Surfactants

Fillers for
nanocompo- Solar
sites cells
Structural Energy and
Materials Catalysis
Electrodes
Building
in batteries
materials Coatings, Catalysts and fuel
paints, for organic cells
latexes reactions

Figure 5.6 Nanotechnology application range in modern society. Source: Llop et al. (2017).

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 145

Biosynthesis of Silver Nanoparticles from Acacia nilotica

(L.) Wild. Ex. Delile Leaf Extract
Karishma I. Sheikh and Kalpesh B. Ishnava
Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences (ARIBAS), Anand,
Gujarat, India

6.1 ­Introduction

Nanotechnology is growing rapidly and has potential in a wide range of commercial

­products. By definition, “Any particle whose size ranges between 1 nm to 100 nm is called a
nanomaterial.” A blend of nanoparticles from plants is known as green amalgamation of
nanoparticles and consolidates the information of nanotechnology and plant biotechnol-
ogy. Other than plants, nanoparticles can likewise be blended from microscopic organisms,
parasites, green growth, and actinomycetes (Kelly et al. 2014).
Nanotechnology includes the integration of smaller structures into larger materials, keep-
ing the construction of new material at nano level. Nanoparticles are also present in differ-
ent morphologies such as rods, spheres, prisms, and tubes (Rout et al. 2014). Nanoparticles
made of zinc, iron, titanium, alginate, copper, gold, and silver are known to exhibit antimi-
crobial properties (Azam et al. 2012). Due to their smaller size, nanoparticles offer advan-
tage by improving biocompatibility. It appears that microscopic organisms are more averse
to creating opposition against nanoparticles on the grounds that nanoparticles have an
expansive range of antibacterial movement and for microbes to be safe numerous changes
must happen. The present examination manages the green amalgamation of nanoparticles.
Regardless of current advancements like UV-radiation, laser removal, ­photochemical
decrease, and lithography, advances have been effectively used to create nanoparticles. Their
more noteworthy expense and utilization of destructive synthetic compounds denotes their
impediments (Iravani et al. 2014).
In this examination, which centers around silver nanoparticles (AgNPs), the antimicrobial
property of silver is credited to the measure of silver discharged. Silver is latent in its metallic
state yet upon presentation to skin dampness it gets ionized and the ionized silver is pro-
foundly responsive. It ties to proteins present in the tissue and generates basic changes in the
cell mass of bacterial-prompting apoptosis. The impact of silver particles on microscopic
organisms can be seen by changes in silver’s structure. Silver is accepted to collaborate with

Green Synthesis of Nanomaterials for Bioenergy Applications, First Edition. Edited by Neha Srivastava,
Manish Srivastava, P. K. Mishra and Vijai Kumar Gupta.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
146 6 Biosynthesis of Silver Nanoparticles from Acacia nilotica

three primary segments of bacterial cells. The cell divider is made of peptidoglycan, plasma
film, bacterial DNA, and bacterial proteins and meddles with different cell forms. It oncen-
trates on activity of AgNPs as it permits electrostatic communication between contrarily
charged bacterial cell films and emphatically charged nanoparticles (Hamouda 2012).
There are different hypotheses on the activity of AgNPs on microorganisms that causes
the microbicides impact. The arrangement of free radicals by the AgNPs might be viewed
as another instrument by which the cells pass on. Electron turn reverberation spectroscopy
considers that there is development of free radicals by the AgNPs when in contact with the
microscopic organisms, and that these free radicals can harm the cell layer and make it
permeable, which can at last lead to cell passing (Kim et al. 2007).
It has likewise been recommended that generation of silver particles can be caused by the
nanoparticles, and these particles can associate with the thiol gatherings of numerous fun-
damental catalysts and inactivate them (Matsumura et al. 2003; Feng et al. 2008). The bacte-
rial cells in contact with the silver take in silver particles, which hinder some capacities in
the cell and harm them. At that point, there is the age of the receptive oxygen species to
consider, which are created potentially through the restraint of a respiratory chemical by
silver particles and assault the cell itself. The activity of these nanoparticles on the cell can
make the response happen, hence leading to cell demise. Another reality is that the DNA
has sulfur and phosphorus as its significant segments; the nanoparticles can follow up on
these delicate bases and demolish the DNA, which would prompt cell demise (Hatchett and
Henry 1996). The connection of the AgNPs with the sulfur and phosphorus of the DNA can
prompt issues in the DNA replication of the microscopic organisms, terminating them. It
has additionally been discovered that the nanoparticles can tweak the flag transduction in
microbes. Phosphorylation of protein substrates in microscopic organisms impacts bacterial
flag transduction. Dephosphorylation is noted just in the tyrosine deposits of gram-negative
microscopic organisms. The phosphotyrosine profile of bacterial peptides is changed by the
nanoparticles. It was discovered that the nanoparticles dephosphorylate the peptide sub-
strates on tyrosine buildups, which prompts flag transduction restraint and consequently
stops development. It is anyway important to comprehend that further research is required
on the theme to completely set up the cases (Shrivastava et al. 2007).
Green synthesis of nanoparticles includes three methods:
1) Green synthesis using bacteria as a medium: Bacteria are known to produce an inorganic
material which makes them capable of producing nanoparticles. It is flexible and suitable
method for large scale production however slow synthesis ability marks their limitations.
2) Green synthesis using fungi as a medium: As like bacteria, fungi too have tolerance
against silver and high binding capacity which makes it an efficient substrate for syn-
thesis of nanoparticle. It is easy to handle and is suitable for large scale production.
3) Green synthesis using plant extract: The first report on formation of nanoparticles from
plants was obtained with alfalfa sprouts. The main advantage of using plant extract is
that it is easily available, environment friendly, and it consist of variety of biomolecules
which help reduce silver ions (Rauwel et al. 2014).
The advantages of green synthesis of nanoparticles are:
●● cost effectiveness,
●● safety,
 6.1 ­Introductio 147

●● no use of chemicals,
●● shorter production time, and
●● no input of high energy or pressure
There are two elective methodologies for amalgamation of metallic nanoparticles: the
top–down approach and the bottom–up approach. In the top–down methodology, a reason-
able beginning material is diminished in size utilizing physical (mechanical) or concoction
implies. Numerous procedures of blending AgNPs, e.g. synthetic decrease of silver parti-
cles by inorganic and natural lessening specialists with or without balancing out operators,
warm disintegration in natural solvents, substance decrease and photo reduction in turn
around micelles, laser removal, gamma illumination, and electron light have been
accounted for in the literature (Lee et al. 2001; Bogle et al. 2006; Long et al. 2007).
The greater part of these strategies is incredibly costly, uses a great amount of vitality,
and, furthermore, includes the utilization of poisonous and perilous synthetic substances,
which may present potential ecological and organic dangers. A noteworthy disadvantage of
the top–down methodology is the defect of the surface structure that can have a critical
effect on physical properties and surface science of the metallic nanoparticles because of
the high viewpoint proportion (Vigneshwaran et al. 2007; Xie et al. 2007a, b). Interestingly,
there is an expanding need to create a green combination strategy comprising of naturally
adequate dissolvable framework, ecofriendly lessening and topping operators, high return,
ease, and nonharmful and earth amiable techniques, for union of metallic AgNPs.
The natural way to deal with materials combination could be utilizing microorganism
(green growth, organisms, yeast, microbes, and infections) (Klaus et al. 1999; Willner et al.
2006; Nayak et al. 2011; Pradhan et al. 2011), chemical, plant or plant concentrate, and
flower concentrate for union of nanoparticles (Haverkamp and Marshall 2009; Nethradevi
et al. 2012; Saha et al. 2012; Vijayaraj et al. 2012). The natural techniques for combination
of nanoparticles have turned out to be superior to the synthetic strategies due to slower
energy, which offers a superior power over precious stone development and decreased cap-
ital engaged with creation. Both unicellular and multicellular creatures have been known
to deliver intracellular or extracellular inorganic materials. Among the green growth,
microalgae are considered to be cell industrial facilities for nanoscale molecules, because
of the high development rate and high biomass efficiency with shorter development time.
Plus, they have been demonstrated as a potential life form for overwhelming metal detoxi-
fication and creation of monetarily vital metabolites, including biodiesel (Mata et al. 2010;
Ahmad et al. 2011). Algal biomass can be effectively reaped and the intracellular nanopar-
ticles can be resealed by upsetting the cell by financially accessible preparing gear.
However, there are several factors affecting formation of nanoparticles, including com-
position of biomolecules in plants, pH value, incubation time, temperature, and presence
of proteins in extract (Makarov et al. 2013). Synthesis of nanoparticles is attractive to many
researchers nowadays because of their characteristic features: owing to their smaller size
they have application in various fields including biofuel energy. They have remarkable
physicochemical and biological properties. Solutions of colloidal silver have application in
biofuel energy due to their nano catalyst properties. The interaction of nanoparticles with
organic catalysts is the current topic of interest in the research (Salem et al. 2011).
Conservative, natural, and vitality security issues caused by excessive dependence on oil
are driving nations all through the world to look at plants and biodiesel. The advancement
148 6 Biosynthesis of Silver Nanoparticles from Acacia nilotica

of different and sustainable fills has become an extremely extraordinary investigation space
during the most recent decade (Farrell et al. 2006). The utilization of biodiesel, generated
from the transesterification of vegetable oils, animal fats, and waste cooking oils, has
increased fundamentally in some nations around the globe. A particular method that
stands out among the most well-known strategies used to down size oil consistency is
transesterification. Biodiesel, a fuel that includes mono-alkyl esters of long chain unsatu-
rated fats, is the most generally acknowledged elective fuel for diesel motors in view of its
specialized, ecological, and key focal points. Besides, biodiesel is regular, oil-determined
diesel fuel and needs pretty much no adjustments in the fuel circulation foundation. When
contrasted with petro-diesel, with biodiesel we see a decrease of general fumes emanations.
Its attractions include biodegradability, higher glimmer point, natural lubricity, and the
fact that its source is household waste (Haas et al. 2001; Enweremadu et al. 2011). With the
growing request for biodiesel,it is imperative to scale back burdens on edible oil and to
synthesize biodiesel from waste non-edible fatty acids to stop pollution and reduce the cost
of biodiesel production. Biomass is being increasingly considered as a sustainable power
source supply, on account of world fossil oil demand and issues over the ecological effect of
carbonic corrosive gas creation. In contrast to non-renewable energy sources, biomass
incorporates a scope of advantages such as having low discharges of sulfur oxide (SOx),
carbon dioxide (CO2), being unbiased, sustainable, and locally made. Warm transformation
procedures of biomass epitomize coordinate ignition, pyrolysis, compound change, and
state change. Snappy change of biomass is considered to be a promising thermo-concoction
technique during which biomass is reborn to shift oil, char, and gas (Zhang et al. 2011).
For hundreds of years, running out of power has been a chief challenge that has affected
financial improvement in nearly all international locations around the world. As a result,
in order to meet the severity of this requirement daily, extraordinary nations have shifted
their attention to day-to-day energy saving in different forms that comprise diverse ener-
getic ability, and are capable of meeting the economic requirements of international loca-
tions. Bioenergy is one of the principal non-traditional resources of energy, created from
energy. There is a high dependence on traditional assets of power, which have been imme-
diately, or maybe in a round about way, acquired from fossil fuels. Petroleum and coal have
proven detrimental to the global environment. Bioresources are promising because they
are no longer created, are self-regulated. A number of the major bioresources include bio-
mass, animal carcasses, useless plant life, and cow dung. But there is limited biomass usage
in every day because of insufficient biochemical amendment strategies to facilitate profit-
ability. Nanotechnology refers to a new dimension of fabric sciences. It is a measurement
that is multidisciplinary in nature since it originates from diverse, overlapping herbal sci-
ences fields. It has supplied faster and dependable methods of optimizing electricity gen-
eration from the bioassets.
Sustainable power sources have the potential to substitute biofuels for petroleum products
for a healthy future scenario. Production of biofuels is probably going to be progressed and we
will see novel research territories, which offer elective wellsprings of vitality (Pandey et al.
2012; Srivastava and Jaiswal 2016). A number of countries are taking an interest in the gen-
eration of renewable energy, explicitly in the area of biofuels. Among these, Brazil and USA
are contributing 80% of global production of biofuels to the creation of bioethanol (Hussein
2015). Hence, creation of biofuels enables us to meet transport fuel demand. Biofuels creation
requires a greater number of processes, but does not have a ­detrimental effect on the environ-
 6.1 ­Introductio 149

ment. However, widespread use of non-renewable energy sources is still a long way off due
the high generation costs (Lund 2007; Sahaym and Norton 2008; Srivastava et al. 2014).
Therefore, continuous endeavors are being made for the cost-effective production of biofuels
worldwide for economic benefit. In this specific situation, lignocellulosic biomass can reduce
the expense of the biofuels generation procedure when it is utilized as the substrate.
Lignocellulosic biomass is organic waste; it is cheap, potential carbon source, sustainable in
nature, and easily accessible on Earth in enormous amounts (Chandra et al. 2010). These
lignocellulosic biomasses can be successfully utilized as the substrate for the creation of bio-
fuels. Cellulases chemicals are required for the bioconversion of lignocellulosic biomass into
fermentable sugars, which are further changed into biofuels (Bhalla et al. 2013). In spite of
the fact that cellulose enzyme assumes a key role in the creation of biofuels, production of the
cellulase compound itself is considered to be the most cost-concentrated advance in the
whole of biofuels production (Sun and Cheng 2002). Therefore, high generation cost of cel-
lulase enzyme raises the expense of biofuels, contributing 40% of the total cost. Then again,
nanomaterials are gaining huge interest in the area of proteins and compounds due to their
potential and adaptable physicochemical properties (Han et al. 2011; Singh et al. 2016).
Nanomaterials can be used to enhance the creation procedure of biofuels by participating as
the impetus in the response. For example, it has been accounted for that cellulase chemical
production, stability, pretreatment of lignocellulosic biomass, and enzymatic hydrolysis pro-
ficiency can be significantly enhanced by utilizing nanomaterials.
The use of engineering and nanomaterials within the biofuel business has emerged as a
promising tool for providing efficient and process-efficient strategies to boost biofuel pro-
duction (Serrano et al. 2009; Sekhon 2014). Nanomaterials can enhance the creation proce-
dure of biofuels by participating as the impetus if the response in the biofuel business is to
use each the scientific (green and chemical process chemistry) and engineering solutions
along within the quest of ecofriendly energy sources (Ramsurn and Gupta 2013). A great
deal of analysis has been conducted to style and fabricate nanomaterials together with
advanced purposeful catalysts for biofuel and energy-related applications (Trindade 2011).
What makes nanomaterials exceptional candidates in varied biofuel systems are their high
surface areas and distinctive characteristics such as a high degree of crystallinity, chemical
process activity, stability, surface assimilation capability, durability, and economical storage
that might jointly optimize the general system. They even have high potential for recovery,
reusability, and usage (García-Martínez 2010). Some examples of use of engineering in
biofuel technologies embrace transesterification, anaerobic digestion, pyrolysis, chemical
process, and chemical process for the assembly of fatty esters, biogas, renewable hydrocar-
bons, etc. The combinations of those technologies with engineering are verified to be eco-
nomical and mature, despite being still largely only used at laboratory and pilot stages, they
are expected to be a promising way forward for replacement of standard systems once
developed on a business scale (Zhang et al. 2010).
The oil business is facing increasing difficulties, in light of the fact that the call for power
administrations is growing, which with the present innovation setup is converted into
greater interest in fluid petroleum derivatives. Oil makers and refiners confront issues such
as finding enough suitable, attractive, unrefined petroleum at reasonable costs. They must try
to meet an increasing demand while satisfying consumer pressure for natural resources and
better air quality. Moreover, issues around the impacts of industry on the atmosphere, brought
about by the outflow of ozone-depleting substance from the assembly and utilization of fluid
150 6 Biosynthesis of Silver Nanoparticles from Acacia nilotica

hydrocarbons. This offers biofuels a commercial greater middle infiltration opportunity

within the change, like a yet unclear new energy fate (Trindade 2011).
Hence, the present study was carried out to synthesize the characterize silver nanopar-
ticles using leaf extract.

Plant Information
Taxonomical Classification
Kingdom: Plantae
Division: Magnolophyta
Class: Magnoliopsida
Family: Mimosaceae
Genus: Acacia
Species: Nilotica
Morphology: This is a perennial tree, usually but not always, 1–2.5 m in height. The bark
is a dark brown to black-gray color when mature. Plants are spiny with about 10–50 mm of
spine present in a pair at the base of the leaf. Leaves are small and dark green colored.
Leaflets are usually found in 15–25 pairs per pinnae. The stems tend to exude gum when
damaged. Flowers are bright yellow, round headed, nectar-less, and have a sweet smell;
and most of them are functionally male. Pods may vary in color from brown to gray; some-
times they have a velvety appearance and are thick. Seeds number 10–12 per pod, are oval
in shape, and have a darkish brown color. Plants have woody taproot.
Phytochemical studies show the presence of phenols, alkaloids, saponinss, glycosides,
and tannins. Various types of polyphenols are present in pods, most of which are gallic
acid, catechin, cholorogenic acid, and robidandiol. Gum consists of Larabinose, galactose,
and L-rhamnose. Root contains lapachol, sitosterol, and botulin. Flowers provide isoquer-
cetin, leucocyanidin, and stearic acid (Rasool et al. 2014).
Acacia is widely known for its multipurpose use. Solar energy conversion efficiency, bio-
mass production, growth rates, mineral content of different components, and mineral use
efficiency in energy plantations of Acacia nilotica (5264 plants/ha) raised under semi-arid
non-irrigated conditions. The absolute biomass created (counting root biomass) at 12 and
multi-months old was 16.0 and 154.0 stove dry t/ha, individually. The relating yield of utiliz-
able biomass (stem, wood, bark, and branches) was 10.9 and 110.1 stove dry t/ha. The sun-
light-based vitality change effectiveness ran from 0.79% at 12 years old to >1.53% at
multi-months old; the occasional vitality transformation proficiency at the pinnacle time of
development was 2.5%. The results indicated that A. nilotica has a high mineral use efficiency
compared with other species grown under energy plantation conditions, suggesting a low
nutrient requirement and efficient internal cycling of nutrients (Gurumurti et al. 1986).

6.2 ­Materials and Methods

6.2.1 Collection of Plant Material

Mature A. nilotica (Mimosaceae) (Figure 6.1) plant leaves were collected during June 2015
from the Ashok & Rita Patel Institute of Integrated Study and Research in Biotechnology
and Allied Sciences (ARIBAS), ADIT Campus of New Vallabh Vidyanagar. The plant was
 6.3 ­Green Synthesis of Silver Nanoparticles from Acacia nilotica Leaf Powde 151

Figure 6.1 Acacia nilotica plant leaves.

identified by plant taxonomist Dr. Kalpesh Ishnava at ARIBAS; and the plant leaves were
collected from six-month old plants.

6.2.2 Preparation of Leaves Extract of Acacia nilotica

The leaves were first washed thoroughly in running tap water and then with distilled
water. After being allowed to dry in shade for five to seven days, the dried leaves were
crushed into powder form by a Maharaja mixer grinder and the powder was passed
through 60 mm mesh. For the extraction process, the cold extraction method was used.
Four solvents, —, methanol, ethyl acetate, chloroform, and hexane were used. Twenty-five
grams of leaf powder was added to 125 ml of each solvent in a 250 ml flask, and that flask
was kept on a shaker for 24 hours. After 24 hours, the mixture was filtered through Whatman
filter paper into a beaker. The contents of the beaker were emptied into a petriplate in order
to evaporate the solvent. The extract was then scrapped off from the petriplate, weighed, and
placed in centrifuge tubes for further use: the percentage yield of extract was calculated.

6.3 ­Green Synthesis of Silver Nanoparticles from Acacia

nilotica Leaf Powder

6.3.1 Preparation of Plant Materials

The A. nilotica leaves were dried for five to six days in shade and ground to a fine powder
using a mixture grinder and a sieve. One gram of leaf powder was added to 10 ml D/W in a
50 ml flask, boiled for eight to ten minutes, and cooled to room temperature. The cooled
solution was filtered by Whatman filter paper no. 1 and the filtrate collected.
Preparation of 1 mM AgNo3: 16 m : g AgNo3 in 100 ml D/W. This gives 1 mM AgNo3.

6.3.2 Green Synthesis of Silver Nanoparticles

One millimeter of filtrate was added into 99 ml of AgNo3 solution in a 250 ml flask. OD of
this starting solution was taken by an ultraviolet–visible (UV-VIS) spectrophotometer
from 300 to 700 nm. Afterwards, this reaction mixture was kept at room temperature on a
152 6 Biosynthesis of Silver Nanoparticles from Acacia nilotica

magnetic stirrer. After about 18–24 hours, the color change was noted, and nanoparticle
formation was monitored using UV-VIS spectroscopy from 300 to 700 nm.

6.4 ­Characterization of Silver Nanoparticles

6.4.1 UV–VIS Spectroscopy

UV–VIS spectroscopy was performed for the detection of AgNPs synthesized by leaf extract of
A. nilotica. Λmax between 400 and 450 nm indicates the formation of AgNPs (Dhas et al. 2013).

6.4.2 Fouriter Transform Infrared Spectroscopy

To distinguish the bio-atoms related to the union of nanoparticles by plant intervention
was performed by utilizing fouriter transform infrared (FT-IR) spectroscopy (Baharara
et al. 2014).

6.4.3 Energy Dispersive X-Ray Spectroscopy

The presence of elemental silver was carried out by using scanning electron microscopy
(SEM) equipped with an energy dispersive X-ray (EDX) system (Usha and Rachel 2014).

6.4.4 Scanning Electron Microscope

SEM was used to find out the surface topography of AgNPs using high objectives SEM
(Salem et al. 2011).

6.4.5 Transmission Electron Microscopy

Transmission electron microscopy (TEM) was carried out to discover the morphology of
AgNPs using high objectives indicative TEM (Rauwel et al. 2015).

6.5 ­Result and Discussion

Nanotechnology is a fast-growing field that has potential in a wide range of commercial

products. A nanomaterial is defined as being an particle that is between 1 and 100 nm in
size. Union of nanoparticles from plants is known as green blend of nanoparticles. It offers
the favorable characteristics of being savvy, safe, and free of destructive synthetics. Silver
tends to tie the proteins present in the tissue and get essential change in the bacterial mass.
Combining AgNPs through this procedure was prudent and quick. A. nilotica is a perennial
tree, commonly known as babul, which has can be used for multipurposes. Phytochemical
studies show the presence of phenols, alkaloids, saponins, glycosides, and tannins. A. nilot-
ica are rich sources of biologically active compounds and have been used for a wide variety
of purposes for many years. This examination was carried out to choose the depictions of
plant leaf concentrate and AgNPs.
 6.5 ­Result and Discussio 153

6.5.1 Yield of Extract in Different Organic Solvents

Out of the four solvents used, methanol gave maximum yield (7.6%) followed by ethyl ace-
tate (7.12%), chloroform (1.6%), and hexane (1.2%) (Figure 6.2). Three of these organic sol-
vents were polar in nature, methanol in which maximum yield was obtained being the most
polar of all. Hexane in which minimum yield was obtained is a nonpolar ­solvent. Thus, we
can conclude that yield of the extract obtained depends on the polarity of the solvent.

6.5.2 Color Change of the Solutions

The color of the solution in the flask containing the 1 mm AgNO3 and plant extract mixture
was initially light yellow. This gradually changed to dark brown after 24 hours of incuba-
tion in a magnetic stirrer. The reduction of AgNO3 into nanoparticles was obviously appar-
ent from the shading change. Leaf extricate was incorporated into the AgNO3. Inside
24 hours, the presence of darker shading was seen, which demonstrated the arrangement
of AgNPs. The shading was changed from yellow to a dull darker yellow after 30–35 min-
utes. After18–24 hours, shading was changed into darker yellow. Anuj and Ishnava (2013)
studied Tinospora cordifolia plant form AgNPs, in which they observed, after a few hours,
the appearance of a brown color that indicated the formation of AgNPs (Inbathamizh et al.
2013). A similar type of result was observed in our plant, A. nilotica.

6.5.3 UV–VIS Spectral Analysis

The decrease of silver particles by plant removal was observed by estimating the UV-obvious
ingestion range in a 24-hour observation period. The example was weakened into refined
water and was checked at the 400 and 700 nm wavelength on a thermo logical spectropho-
tometer. A solitary pinnacle was seen at 424 nm due to surface plasmon reverberation (SPR)

Figure 6.2 Graph showing % yield of extract 8 7.6

in different solvents. 7.12
7
6
5
% yield

4
3
2 1.6
1.2
1
0
E
L

TE

RM

N
O

A
N

TA

EX
FO
A

CE
H

RO

H
ET

LO
M

L
Y

CH
H
ET

SOLVENTS
154 6 Biosynthesis of Silver Nanoparticles from Acacia nilotica

of free electrons present in the AgNPs (Figure 6.3). By UV-VIS spectroscopy we got the
λmax at 424 nm, which provided solid proof for the arrangement of AgNPs. Silver nanopar-
ticles were affirmed by the ingestion maxima filtered by UV–VIS spectroscopy at the 300–
700 nm wavelength. Earlier we discussed adsorption spectra of the blended AgNPs utilizing
the leaf concentrate of A. nilotica. The peak reduction of AgNO3 into AgNPs was observed
at around at 420–450 nm. Metal nanoparticles have free electrons, which gives SPR an
understanding of the process due to the joined vibration of electrons of metal NPs in rever-
beration with light wave. Inbathamizh et al. (2013) revealed the in vitro assessment of can-
cer prevention agents and antibacterial capability of Morinda pubescens-blended AgNPs
(Inbathamizh et al. 2013). Yogeswari et al. (2012) announced the UV-VIS spectra at 430 nm
of the AgNPs incorporated from leaf concentrate of Ocimum santcum.

6.5.4 FT-IR Spectroscopy Analysis

FT-IR range was examined for identification of various biomolecules that were adsorbed on
the outside of AgNps and likewise their conceivable job associated with reduction and
adjustment of nanoparticles. The spectrums of both leaf extract and AgNPs were compared.
In the spectrum of leaf extract, a broad peak at 3418.06 cm−1 was observed, which was due
to stretching vibrations of the hydroxyl group and intermolecular H-bonding among them.
The peaks at 1609.40cm−1 and 1445.72 cm−1 correspond to 1° amines and the presence of
aromatic rings in the extract, respectively, whereas a peak at 1707.01 cm−1 corresponds to
the carbonyl group (Figure 6.4). In FT-IR spectra of AgNPs the stretching vibration of the
hydroxyl group became narrow and the peak was shifted to 3417.92 cm−1 (Figure 6.5). These
results indicated that the carbonyl group of proteins are strongly absorbed on the surface of
AgNPs resulting in their reduction and stabilization. The ingestion peak at1631.77 cm−1 is
reported for local protein, which proposes that proteins are cooperating with biosynthesized
nanoparticles and amide plays no role in the production of AgNPs (Fayaz et al. 2010).

6.5.5 Energy Dispersive X-Ray Spectroscopy

EDX analysis was done to obtain the information about the elemental profiles of both
extract and AgNPs in terms of percentage. EDX analysis gives a full element profile of the

Figure 6.3 Absorption spectra of AgNPs.

 6.5 ­Result and Discussio 155

100.0
95
90
838.38
85
80 779.76
75 2856.55 672.39

70
1317.49 927.50
65
2926.73 600.59
60
997.16
55
50 1108.45
%T

45 1631.77
1053.60
40
35
30
25
20 3417.92
15
1384.52
10
5
0.0
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
Sample Name-AgNP cm–1 ID No-1866/2015–16

Figure 6.4 FT-IR spectra of plant extract.

100.0
1082.50
95 1445.72

90
85
80
75 866.12
673.02
70
815.07 619.09
65 943.55
60
758.92
55
2850.34 1520.04
50 1127.09
%T

45
1707.01 1032.88
40 2919.5
1353.40
35 3418.06 1206.65
30
1317.34
25
20 1609.40
15
10
5
0.0
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
cm–1

Figure 6.5 FT-IR spectra of AgNPs.

sample and indicates the amount of any element present in terms of percentage. In
Figures 6.6 and 6.7, the vertical axis shows X-ray count and the horizontal axis shows
energy in keV. A comparison of the EDX analysis of both extract and AgNPs showed the
presence of carbon and oxygen.
A peak at 3.0 keV relates to restricting vitality of silver. Independent of alternate compo-
nents present in the example, silver holds the most astounding specific binding site. This is
156 6 Biosynthesis of Silver Nanoparticles from Acacia nilotica

unmistakably portrayed in the pinnacles of the diagram in Figure 6.6, which shows bio
decrease of silver particles into essential silver. Investigation through EDX spectroscopy
affirmed the specific natural silver signs of AgNPs (Tables 6.1 and 6.2). The EDX examina-
tion uncovered solid flags in the silver locale and affirms the development of AgNPs.
Metallic silver nano precious stones, for the most part, show common optical assimilation
peak at around 3 keV because of SPR (Thangaraju et al. 2012). EDX study reveals that the
elemental silver is present in concentration of 25.94%. There were other EDX peaks for Mg,
and Na (Figure 6.7), suggesting that they are blended with another substance present in the
plant extricate. It likewise demonstrates the nearness of bio-natural and bio-inorganic
­interference, which is naturally occurring in the leaf extract of A. nilotica. Thus, the analysis
confirms the bio-reduction of silver from ionic silver to elemental silver, i.e. Ag+ to Ag0.

117k O

104k
0.91k
Ag
0.78k
0.65k
0.52k Na
Ag
0.39k Na
Mg
0.26k Mg
0.13k
0.00k
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
Lsec:100.0 0 Cnts 0.000 keV Det Octane Plus Det

Figure 6.6 EDX graph of plant extract.

Table 6.1 Presence of different elements in EDX pattern of extract.

Element Weight % Atomic % K ratio Z A F

CK 29.75 36.06 0.15 1.03 0.5 1

OK 70.25 63.94 0.23 0.99 0.33 1

Table 6.2 Presence of different elements in EDX pattern of AgNPs.

Element Weight % Atomic % K ratio Z A F

OK 60.84 82.77 0.18 1.09 0.27 1

NaK 8.99 8.51 0.02 1 0.27 1
MgK 1.62 1.45 0.01 1.02 0.36 1
AgL 25.94 5.23 0.21 0.75 1.09 1
 6.5 ­Result and Discussio 157

2.50k
O
2.25k
2.00k
1.75k
1.50k
1.25k
1.00k
0.75k
C
0.50k
0.25k
0.00k
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
Lsec:100.0 0Cnts 0.000 keV Det Octane Plus Det

Figure 6.7 EDX graph of AgNPs.

6.5.6 Scanning Electron Microscopy

SEM helps to investigate the surface morphologies of the sample. Figure 6.8. shows a SEM
micrograph of plant extract that is seen to have a rough surface and larger particles. The
morphological characterization of AgNPs was studied by SEM. SEM with EDX analysis
was performed by using Phillips, Netherland Model: XL 30 ESEM.
Looking at the picture of AgNPs extracted from A. nilotica leaf in Figure 6.8, we can see
that the suspended AgNPs in sterile refined water were used for SEM. The large AgNPs
might be the result of a collection of the smaller ones. The nanoparticles were seen to be
round with 60–95 nm diameter and a couple of particles are agglomerated shapes (Figure 6.9).
The electron microscopy has been used to portray the size, shape, and morphologies of
framed AgNPs. The results of SEM analysis support the results of Govindaraju et al. (2008).

6.5.7 Transmission Electron Microscope

The use of AgNPs is profoundly subject to compound organization, shape, size, and disper-
sion of the particles, so to acquire information about potential applications, nanoparticles
were portrayed utilizing TEM. TEM analysis was performed by using Phillips, Netherland
Model: Tecnai20.
A TEM image of plant extract is shown in Figure 6.10, which shows larger particle size
AgNPs derived from A. nilotica leaf powder. Through this analysis we got the spherical
shape AgNPs produced by our plant. A TEM image recorded from the silver was coated on
a carbon-coated copper TEM grid. The morphology of the nanoparticles was circular in
nature. The large number of non-uniform AgNPs in the TEM image revealed that the
AgNPs were produced by reaction of Ag+ with the leaf extract of A. nilotica.
Under careful observation, agglomeration of AgNPs may be seen due destabilization of
electric double layer of silver ions. Synthesized AgNPs were highly uniform in size: 12–15 nm
(Figure 6.11). Usha and Rachel (2014) reported the synthesis of AgNPs using leaf extract of
the same plant. Their microscopic observation agreed with the UV–VIS spectroscopic stud-
ies. In other research, Anuj and Ishnava (2013) detailed extracellular manufacture of AgNPs
utilizing Pseudomonas aeruginosa (Anuj and Ishnava 2013).
158 6 Biosynthesis of Silver Nanoparticles from Acacia nilotica

Figure 6.8 SEM micrograph of plant

extract.

Figure 6.9 SEM micrograph of AgNPs.

6.6 ­Conclusion

AgNps were effectively orchestrated utilizing A. nilotica leaf extract. Synthesis of

AgNPs through this process was economical and rapid. The FT-IR, SEM, EDX, and
TEM further characterized AgNPs and pointed toward the fact that AgNPs have
emerged as a promising bioenergy agent to use in the production of biodiesel or
bioethanol. It is probably going to be helpful that the biosynthesized AgNPs can be
productively utilized for enhancing the properties of biodiesel or as an added sub-
stance for biodiesel.
 References 159

Figure 6.10 TEM micrograph of plant extract.

Figure 6.11 TEM micrograph of AgNPs.

­Acknowledgments

The authors are grateful to and recognize the Sophisticated Instrument Center for Applied
Research and Testing (SICART), DST, India for IR and NMR investigations. Likewise we
are grateful to Charutar Vidya Mandal (CVM), VallabhVidyanagar, Gujarat, India, and
Director of Ashok and RitaPatel Institute of Integrated Studies and Research in
Biotechnology and Allied Sciences (ARIBAS), New Vallabh Vidyanagar – 388121, Gujarat,
India, for giving important help with research and laboratory facility.

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 165

Nanomaterials for Enzyme Immobilization

Nihan Arabacı1, Tuna Karaytuğ2, Ayse Demirbas3, Ismail Ocsoy4,
and Ahmet Katı5
1
Department of Biology, Faculty of Arts and Sciences Çukurova University, Adana, Turkey
2
Department of Biology, Institute of Natural and Applied Sciences, Çukurova University, Adana, Turkey
3
Faculty of Fisheries and Aquatic Sciences, Recep Tayyip Erdogan University, Rize, Turkey
4
Department of Analytical Chemistry, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey
5
Department of Detergent and Chemical Technologies, Hayat Kimya Research and Development Center, Kocaeli, Turkey

7.1 ­Enzymes

Enzymes are macromolecules (generally proteins, occasionally ribonucleic acids) that

­catalyze specific biochemical reactions. That is, each enzyme catalyzes a uniform chemical
reaction. Some enzymes also catalyze a similar reaction group. This specificity is due to the
enzyme’s three-dimensional structure. In a catalysis reaction, enzyme and substrate are
temporarily combined to constitute the enzyme–substrate complex (Yeşilada 2010). The
stability of the enzyme–substrate complex determines the ability of the substrate to
be transformed into the product by the enzyme (Arsalan and Younus 2018).
Enzymes are generally much larger than the substrates. Weak bonds such as van der
Waals forces, hydrogen bonds, and hydrophobic interactions are necessary to construct
the enzyme–substrate complex. As a result of the reaction, the end product is separated
from the enzyme structure and the enzyme returns to its initial state. A certain site of an
enzyme, where the substrate is bound and catalytic activity occurs, is called the active site
(Yeşilada 2010).

7.1.1 Enzyme Classification

In the systematic nomenclature suggested by the International Union of Biochemistry and
Molecular Biology (IUBMB), the enzymes are divided into six large groups by the Enzyme
Commission (EC), and each group has subclasses according to the type of catalyzed reac-
tion. Enzymes are systematically identified with an EC number. This number has four
descriptions that are used to categorize the enzymes according to the property of converting
substrate to product.

Green Synthesis of Nanomaterials for Bioenergy Applications, First Edition. Edited by Neha Srivastava,
Manish Srivastava, P. K. Mishra and Vijai Kumar Gupta.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
166 7 Nanomaterials for Enzyme Immobilization

EC 1 Oxidoreductases: Catalyze the reactions in which a substrate gives one or more

­electrons to an electron acceptor and is oxidized during the process (e.g. dehydrogenases,
­oxidases, ­oxygenases, peroxidases).
EC 2 Transferases: Catalyze the reactions in which a chemical group is transferred from a
donor substrate to an acceptor substrate (e.g. fructosyltransferases, transketolases, acyltrans-
ferases, transaminases).
EC 3 Hydrolases: Catalyze the reactions in which a bond in a structure of the substrate is hydro-
lyzed to create two fragments (e.g. proteases, amylases, acylases, lipases, phosphatases, cutinases).
EC 4 Lyases: Catalyze the non-hydrolytic reactions in which a chemical group is removed
from a substrate leaving a double bond (e.g. hydratases, dehydratases, pectate lyases, fumar-
ase, decarboxylases, argino succinase).
EC 5 Isomerases: Catalyze the isomerization reactions (one-substrate/one-product reac-
tions) (e.g. isomerases, epimerases, racemases).
EC 6 Ligases: Catalyze the reactions to bring together two or more molecules due to the
hydrolysis of ATP or a similar molecule (e.g. synthetases, ligases) (Cornish-Bowden 2014).

7.1.2 Enzyme Sources and Their Application Fields

Enzymes can be obtained from animals (chymosin, pepsin, trypsin, etc.), plants (ficin,
papain, bromelain, etc.), and microorganisms (protease, α-amylase, pectinase, cellulase,
etc.). Terrestrial and aquatic microorganisms are the most dynamic producers of enzymes
and have been used in various industries for more than 50 years. In principle, any enzyme
similar to enzymes found in animals and plants can be obtained from microorganisms.
From the biotechnological and industrial perspective, the general tendency is the use of
microbial enzymes (Vaidya et al. 2015).
Microorganisms are ideal organisms with many advantages (such as easy availability, fast
reproduction and capacity to produce many enzymes under desired conditions using cheap
nutrients, ease of genetic modification to improve the enzyme production capacity, short
fermentation times, etc.) for the production of industrial enzymes (Illanes et al. 2012;
Kalpana and Pandian 2014; Singh et al. 2016). Also, microorganisms might be cultured
and manipulated in large quantities to meet the rising demand with recombinant DNA
technology and protein engineering techniques (Liu et al. 2013).
The utilization of enzymes in diverse industrial processes is increasing rapidly due to
high catalytic activity/stability, high efficiency and quantity, short processing time, nontox-
icity, eco-friendly characteristics, cost-effectiveness, and low energy demand (Li et al. 2012;
Choi et al. 2015; Singh et al. 2016). Also, increasing desire for consumer goods, the require-
ment for reducing costs, depletion of natural resources, and ensuring environmental safety
are the driving forces in the use of microbial enzymes in industrial processes (Choi et al.
2015; Singh et al. 2016).
The technical application of isolated enzymes in the industry is possible provided that
the enzymes are stabilized in the presence of salts and against extreme pH, temperature,
alkalis, and surfactants. The main applications of enzymes are carried out at high tempera-
ture (e.g. starch gelatinization at 100 °C, washing at 60–70 °C, textile de-sizing at 80–90 °C),
alkaline conditions, high salt concentrations, and in the presence of surfactants (Iyer and
Ananthanarayan 2008).
 7.1 ­Enzyme 167

7.1.3 Economic Importance of the Industrial Enzymes

Today it is known that there are about 4000 enzymes and some 200 microbial varieties are
used commercially among them. However, approximately 20 kinds of enzymes are pro-
duced in the industrial size (Li et al. 2012; Kumar et al. 2014).
Investments made in various industries to perform industrial processes easily and in a
short time increased utilization rate of enzymes. From the early nineteenth century
onwards, enzymes have been regarded as biological catalysts that accelerate the reaction by
decreasing the activation energy.
The isolated enzymes were primarily used in 1914, and their protein structures were dem-
onstrated in 1926. In the 1960s, the large-scale production of microorganisms started. The
industrial enzyme studies are continuously growing through production technologies, engi-
neered enzyme features, and new application fields of need (Iyer and Ananthanarayan 2008).
The catalytic activity of the enzyme provides the opportunity for its usage in different
fields. Thanks to this feature, the enzymes have a crucial role in various industrial and
research applications. Their effects are crucial in genetic engineering and other molecular
biological research. With the improvements in the enzyme technology, important work is
being done to develop new enzymes with enhanced functionalities.
Enzyme technology, defined as a considerable factor of sustainable industrial develop-
ment by the Organisation for Economic Cooperation and Development (OECD), is a mul-
tidisciplinary field of study. The applications in this field extend from basic industrial
processes to pharmaceutical exploration and improvement. Several examples of enzyme
applications in different industrial fields are given in Table 7.1.
The rapid progress of genetic engineering, high-efficiency scanning, and existing and
developing technologies contribute to the economics of current industrial processes and
offer opportunities for new application areas (Iyer and Ananthanarayan 2008).
Comprehensive developments and investigations in bioprocess technology and the use of
recombinant DNA methods have led to increased fermentation efficiency, stability, speci-
ficity, and selectivity of enzymes (Kumar et al. 2014). Many industries are utilizing the
enzymes to generate ecofriendly products on account of limiting the use of chemicals.
Increasing demand for enzymes in different industries along with the widespread use of
genetic engineering are the main factors that promote the growth of the global enzyme
market (van Beilen and Li 2002).
Enzymes have an extensive range of applications in industrial fields, such as food and
beverage, brewing, dairy, food processing, animal feed, pharmaceutical, pulp/paper, textile,
and leather. Among the main sectors using industrial enzymes are the drugs (41%), food
and feed (17%), detergents (17%), leather/paper (17%), and textile (8%). The distribution of
the worldwide enzyme market in diverse application areas is indicated in Figure 7.1.

7.1.4 Advancements in Enzyme Engineering

Numerous companies around the world have acknowledged that bio-based techniques are
an important constituent in the acceleration of global growth. Nonetheless, biological pro-
cessing is often considered only when the chemicals required for the synthesis of the target
molecule are scanty. The main reason for this is that the enzyme does not have the expected
168 7 Nanomaterials for Enzyme Immobilization

Table 7.1 Enzymes and their benefits in different industrial applications.

Application
field(s) Enzyme(s) Technical benefit(s) Reference(s)

Pulp and Amylase Deinking, drainage Li et al. (2012),

paper improvement Kumar et al. (2014),
industry Lipase Pitch control Singh et al. (2016)

Cellulase Deinking, drainage

improvement
Laccase Bleaching for improve
brightness
ß-xylanase Bleach boosting
Mannanase Degrading the residual Li et al. (2012),
glucomannan to increase Kumar et al. (2014)
brightness
Textile Amylase Desizing Li et al. (2012),
industry Cellulase Cotton softening, denim Kumar et al. (2014)
finishing
Pectinase Bio-scouring of cellulosic
fabrics under alkaline
conditions
Laccase Non-chlorine Bleaching, fabric Li et al. (2012), Singh
dyeing et al. (2016)
Glucoseoxidase Creating bleaching agent to Li et al. (2012)
whiteness
Laundry Protease Protein spot elimination Li et al. (2012),
detergents Lipase Fat spot elimination Kumar et al. (2014),
Singh et al. (2016)
Amylase Starch spot elimination
Cellulase Color clarification
Cutinase Triglyceride removal Singh et al. (2016)
Mannanase Mannan spot removal
Dairy Lipase Maturing of the blue mold Li et al. (2012),
industry cheese Kumar et al. (2014),
ß-galactosidase, Convert lactose to galactose Singh et al. (2016)
Lactase and glucose in milk to prevent
lactose intolerance
Lysozyme, Cheese fabricating Li et al. (2012),
Chymosin, Kumar et al. (2014)
Catalase
Acid proteinase Milk coagulation Singh et al. (2016)
Baking α-amylase Flour setting, bread softness Li et al. (2012),
industry ß-xylanase Dough conditioning Kumar et al. (2014),
Singh et al. (2016)
 7.1 ­Enzyme 169

Table 7.1 (Continued)

Application
field(s) Enzyme(s) Technical benefit(s) Reference(s)

Lipase Making better stability of the

gas cells in paste
Oxidoreductase Giving increased gluten Li et al. (2012),
strength Kumar et al. (2014)
Protease Reducing the protein in flour
Pentosanase Breaking down Pentosans/ Kumar et al. (2014)
Hemicellulose matter during
food Processing
Glucose oxidase Dough strengthening Singh et al. (2016)
Transglutaminase Laminated dough strength
Juice Amylase Breaking down starch into Li et al. (2012),
industry glucose, Kumar et al. (2014),
Clarifying cloudy juice Singh et al. (2016)
Pectinase Depectinization
Cellulase Fruit liquefaction
Hemicellulase Fruit liquefaction Li et al. (2012),
Glucoamylase Reducing starch to glucose Kumar et al. (2014)
Clarifying blurred juice
Laccase Promoting the sensibility of
browning along the storage
Naringinase, Debittering
Limoninase
Starch and α-amylase Separating α-1,4-glycosidic Li et al. (2012),
sugar bonds of the starch Kumar et al. (2014)
industry
Pullulanase breaks the α-1,4 and α-1,6
bonds in polysaccharides that
consist of glucoses
Neopullulanase, Acting on bothα-1,6- and
Amylopullulanase α-1,4-linkages
Glucoamylase Break α-1,6-linkages and
α-1,4-linkages from non-
reducing points to
releaseβ-d-glucose
Isoamylase Hydrolyzing α-1,6-linkages in
amylopectin and glycogen
Glucose Isomerization of glucose to
isomerase fructose

(Continued)
170 7 Nanomaterials for Enzyme Immobilization

Table 7.1 (Continued)

Application
field(s) Enzyme(s) Technical benefit(s) Reference(s)

Glycosyltransferase Increasing the number of

branch points to obtain
modified starch with improved
functional properties (e.g. a
higher solubility, lower
viscosity, and limited
retrogradation)
ß-amylase Separating α-1,4-linkages of Li et al. (2012)
glycogen, amylopectin, and
amylose
Dextranase Hydrolyze the dextran which is Kumar et al. (2014)
an unwanted compound in
sugar manufacturing
(promotes the viscosity of the
flow and reduces the industrial
recovery)
Xylanase Significant for hydrolysis of
Xylan (especially, in cereal
grains and lignocellulosic
biomass); obtaining short
chain xylo-oligosaccharides of
variable lengths
Animal feeds Xylanase Functions as degrading fiber in Li et al. (2012),
industry viscous solutions Kumar et al. (2014)
Phytase Degrades phytic acid to release
phosphorus, calcium, and
magnesium ions
Protease Hydrolyzes proteins
(subtilisin)
α- amylase Hydrolyzes starch
ß-glucanase Digestive aid Li et al. (2012),
Kumar et al. (2014),
Singh et al. (2016)
Cellulase Breaks down Cellulose/ Kumar et al. (2014)
Hemicellulose
Organic Lipase, Acylation, deacylation, Li et al. (2012), Singh
synthesis Nitrile hydratese, enantioseparation et al. (2016)
industry Esterase,
Nitrilase,
Amidase
Alcohol Degredation of C─O and C─C Li et al. (2012)
dehydrogenase, bonds
Lactate
dehydrogenase
 7.1 ­Enzyme 171

Table 7.1 (Continued)

Application
field(s) Enzyme(s) Technical benefit(s) Reference(s)

Monooxygenase, Oxidation of alchohols and

Dehydrogenase oxigenation of C─H and C─C bonds
Fructose C─C coupling
1,6-bisphospate
aldolase,
Diels-Alderase
α-fucosidase, Glycosidic bonds
Sialidase
Cosmetics Oxidase, Changing hair color Li et al. (2012)
industry Peroxidase,
Polyphenol
oxidase
Protein disulfide Hair waving
isomerase,
Glutathione
sulfhydrl, oxidase
Transglutaminase,
Papain
Promelain, Giving soft peeling effects in
Subtilisin skin care
Amyloglucosidase, In oral/dental care (in
Glucose oxidase toothpastes and gargles)
Superoxide Free radical scavenging, skin Singh et al. (2016)
dismutase care
Laccase Hair dyeing
Lipase Skin care
Endoglycosidase Teeth and gum tissue care
Leather Alkaline and Dehairing Kumar et al. (2014),
industry acid protease Singh et al. (2016)
Alkaline and Degreasing
acid lipase
Waste Amidase Degradation of nitriles Singh et al. (2016)
management containing wastes
Amylase Bioremediation of vegetable
wastes
Amyloglucosidase Breaking down of starch for
bioremediation
Lipase Disruption of raw oil
hydrocarbons
Nitrile hydratase Disruption of nitriles including
wastes

(Continued)
172 7 Nanomaterials for Enzyme Immobilization

Table 7.1 (Continued)

Application
field(s) Enzyme(s) Technical benefit(s) Reference(s)

Cutinase Disruption of plastics,

polycaprolactone
Laccase Disruption of waste including
phenolic compounds and
polyurethane
Protease Bioremediation of containing
keratinous wastes
Manganese Distruption of phenolic
peroxidase, compounds
Lignin
peroxidase
Oxygenase Degradation of halogenated
contaminants
Pharmaceutical L-Asparaginase, Anti-tumor Singh et al. (2016)
industry L-glutaminase,
L-tyrosinase,
Galactosidase
Superoxide Anti-inflammatory
dismutase,
Serrapeptase
ß-Lactamase Antibiotic resistance
Ribonuclease, Antiviral
Serrapeptase
Uricase Gout
a-amylase, Digestive disorders
Lipase
Rhodanase Cyanide poisoning
Brewing α- amylases Hydrolyze starch to decrease Li et al. (2012)
industry viscosity
Enhance glucose and maltose
content
Pullulanases Hydrolyze the α-1,6 branches
of starch.
Providing of maximal
fermentability
ß-glucanases Hydrolyzation of glucans into
soluble oligomers to obtain a low
viscosity and better filterability
α-acetolactate- Conversion of α-acetolactate to
decarboxylases acetoin
(ALDC) Reduction of fermentation
time by preventing diacetyl
formation Provide the desired
taste of beer
 7.1 ­Enzyme 173

Table 7.1 (Continued)

Application
field(s) Enzyme(s) Technical benefit(s) Reference(s)

Amyloglucosidases Increase the glucose content Li et al. (2012),

Proteases Increasing soluable protein Kumar et al. (2014)
and free amino‑nitrogen
Developing yeast growth
Malt development
Pentosanases, Hydrolyze pentosans of barley,
Xylanases malt, and wheat
Developing of extraction and
beer filtration

Textile
8%
Leather and paper
Drugs
17%
41%

Food and Feed

17%
Detergents
17%

Figure 7.1 Industrial demand for enzymes.

characteristics in order to efficiently catalyze the reaction. The common purpose of mod-
ernization in the enzyme manufacturing industry is the improvement of enzyme proper-
ties and production processes (Li et al. 2012).

7.1.5 Global Enzyme Demand

Today, enzymes are routinely used in many fields such as fine-chemicals, tanning, textiles,
detergent, laundry, cosmetics, pharmaceuticals, food, and feed industries and classified
based on particular applications fields. The most important source of enzymes used in
these industrial fields is microorganisms (Adrio and Demain 2014; Kumar et al. 2014).
These kinds of industrial applications are responsible for over 80% of the worldwide
enzyme market (van Oort 2010; Kumar et al. 2014). More than 500 industrial products are
being produced using enzymes.
174 7 Nanomaterials for Enzyme Immobilization

While worldwide sales of enzymes were around US$1.5 billion in 1998 (van Beilen and Li
2002), this value reached US$6.57 billion in 2017. In addition, this figure is expected to be
US$10.63 million by 2025. Global enzyme demand is met by approximately 12 main manu-
facturers and 400 small suppliers. About 75% of all available enzymes are manufactured by
well-known firms such as Novozymes (Denmark based) and DuPont (USA based) and
Roche (Switzerland based).

7.2 ­Conventional Enzyme Immobilization Methods

Enzymes are natural and highly active biological molecules that increase the speed of
­catalytic reactions (Cui and Jia 2017). They have significant potency for use in many bio-
technological and industrial fields (Vaz et al. 2016) owing to advantages such as activity at
diverse pHs and temperatures, enantioselectivity (Cui and Jia 2017), high efficiency, easy
production, high substrate specificity, and selectivity, green chemistry, environmental sus-
tainability in applications, biodegradability, and biocompatibility (Vaz et al. 2016; Cui et al.
2018; Nadar and Rathod 2018). Due to these kinds of features, enzymes are becoming pop-
ular alternatives to classic chemical catalysts under moderate and environmentally friendly
circumstances (Feng and Ji 2011; Cui and Jia 2017).
Besides all these benefits, industrial applications of soluble enzymes are often forced due
to low operational/chemical stability, storage stability, difficult recovery and reusability,
substrate and product inhibitions, and high production cost (Cui and Jia 2017; Cui et al.
2018; Nadar and Rathod 2018). These kinds of drawbacks are hardly economical situations
and can be overcome by the enzymatic technologies, e.g. immobilization techniques.
Enzymes are naturally bound to the cell membrane in their natural environment and are
stable and active in this state. The immobilization process is similar to this structure.
Therefore, the enzymes immobilized to the solid support are more stable than the free
enzymes and resistant to environmental changes. More importantly, the heterogeneous
system formed by the immobilization of enzymes allows reuse of enzymes, easy recovery
of the enzyme, continuous production, easy termination of the reaction, and various reac-
tor designs. Furthermore, immobilization prevents products from inhibiting the enzyme
and provides better functional properties (Krajewska 2004).
Enzymatic technologies are important tools to achieve high-value-added products (Brady
and Jordaan 2009; Goncalves et al. 2019). They require more stable enzymes and economi-
cal processes for the industry. Numerous industrial operations occur under harsh condi-
tions such as extreme pH and temperature and form inhibitory end products for enzyme
activities (Vaz et al. 2016). Free enzymes cannot stay stable at high temperatures and wide
pH range (acidic to alkaline) in toxic reagents and organic/inorganic solvents and are also
relatively costly and cannot be reused (Mohamad et al. 2015; Voberkova et al. 2018).
Besides, enzymes as biological molecules lose activity during the reactions, so that replace-
ment with fresh enzymes is necessary after a while (Fraas and Franzreb 2017). The use of
free or solubilized enzymes leads to failure in enzyme reuse and/or product recovery, high
price, and low stability. These drawbacks don’t let down the commercialization of enzymes
and enzymatic products but the enzyme immobilization is an effective way of minimizing
and solving these kinds of problems (Cao et al. 2016; Goncalves et al. 2019).
 7.2 ­Conventional Enzyme Immobilization Method 175

Enzyme immobilization is a considerable way for the entrapment of soluble/free enzymes

to a solid insoluble phase (support, matrix) different from a substrate or product to compose
insoluble and reusable enzymes with high stability and activity (Bernal et al. 2018). This
method accomplishes most of the restraints of industrial processes, such as loss of catalytic
activity, low pH and thermal stability, inhibition of the enzyme by substrates or byproducts,
reduction of the enzyme during product separation, and trouble in the purification.
Immobilization increases the pH and thermal stability of the enzyme at diverse pHs and
extreme temperatures by forming a more rigid structure for binding of the enzyme. Hereby,
the actions of high heat and/or harsh pH values in the unfolding of secondary/tertiary struc-
tures or in the breaking of interactions of enzymes are diminished (Vaz et al. 2016).
The first industrial immobilized enzyme called aminoacylase from Aspergillus oryzae
was developed in the 1960s (Tosa et al. 1966; Bilal et al. 2018). The immobilized aminoac-
ylase provided the resolution of synthetic racemic D-L amino acids. From that time, differ-
ent techniques have been reported to improve the benefits of immobilized enzymes like
efficient catalytic potency, enhanced performance of industrial processes, induced stability,
and reduction in cost (Asgher et al. 2014; Bilal et al. 2017; Bilal et al. 2018).
After the enzymes are immobilized, their features such as reusability, stability, activity,
and selectivity are improved (Cui et al. 2018). Thus, immobilized enzymes have become
reusable and stable catalysts against temperature and pH changes, inhibitors, organic sol-
vents, and denaturants under storage and reaction conditions (Bornscheuer 2003; Sheldon
2007; Voberkova et al. 2018).
In comparison with free enzymes, immobilized enzymes have different advantages, such
as improved thermal and pH stability, the possibility of recovery and reuse of the enzymes,
enhanced enantioselectivity, easy applications of continuous enzymatic processes, easier
reactor operation, low product cost, product separation, and environmentally friendly
catalysis (Ansari and Husain 2012; Vaz et al. 2016; Bilal et al. 2019). Immobilization eases
the enzyme separation and prevents or minimizes the protein contamination in a reaction
medium (Nadar and Rathod 2018). Lack of biocatalyst in product flow makes an industrial
application more economical (Cui and Jia 2017; Cui et al. 2018). Herewith, the process cost
reduces and the industrial applicability of the enzyme increases (Nadar and Rathod 2018).
The main aim of enzyme immobilization is the reusability of enzymes in many reaction
cycles and biotechnological applications for a long time (Voberkova et al. 2018). Because
the reusability provides higher catalyst efficiency.
Recently, a number of new methods have emerged for enzyme immobilization that has
higher efficiency and a wide range of applications in multidisciplinary fields. Immobilized
enzymes also find significant applications in bioethanol and biodiesel synthesis, food
industry, drug metabolism, antibiotic production, biosensor production, and bioremedia-
tion (Zucca and Sanjust 2014; Sirisha et al. 2016). The widespread use of immobilized
enzymes is due to the fact that they are ecofriendly, cost-effective, and much simpler to use
(Sirisha et al. 2016).
An enzyme immobilization process should generally satisfy the following conditions: the
enzyme support material needs to be reusable, biocompatible, and have a high specific
surface area (Ansari and Husain 2012) for a suitable enzyme loading. Furthermore, it
should be simple and mild. Potential use in biotechnological and industrial applications,
catalytic efficiency, and stability are the expected properties of the immobilized biocatalyst
176 7 Nanomaterials for Enzyme Immobilization

(Cao et al. 2016). Enzymes for immobilization should be resistant to denaturation,

­aggregation, degradation, the various pHs, and heat. However, in obtaining an immobi-
lized enzyme with optimum biocatalytic activity, the method of immobilization should be
biocompatible and provide the enzyme to reach the substrates and cofactors throughout
the reaction (Bilal et al. 2018).
The immobilization method physically confines the enzymes in a definite region or
attaches them to insoluble support to keep their catalytic activities against challenging
environmental conditions such as extreme temperature, pH, and nonaqueous solvents
(Bernal et al. 2018; Bilal et al. 2018).
Some desired features of support carriers are reusability/recyclability, insolubility, ther-
mal and chemical stability, high binding capacity, functional groups availability, ease of
fabrication and tunability, and cost-effectiveness. Choosing a proper support is directly
related to the type of enzyme and bioprocess of the catalytic system. Also, the characteris-
tics of support materials are of vital importance for efficient immobilization (Bilal et al.
2018). Organic (particularly polysaccharides, polyvinyl, and polyacrylic) and inorganic
(particularly silica or other metal oxide-based) carriers have been defined as effective sup-
port materials for the immobilization of enzymes. Inorganic supports provide excellent
mechanical, thermal, and microbial resistance (Sheldon 2007; Magner 2013; Zucca and
Sanjust 2014). Insoluble support is convenient for the practical applications of the enzyme
immobilization due to ease of use and separation of enzymes from a reaction mixture, high
stability, recovery and reusability, prevention of interactions with interfaces and separation
of subunits, rigidification via multipoint covalent attachment, improved activity, specific-
ity, resistance to inhibitions, selectivity, and purity (Bilal et al. 2018; Sheldon and Woodley
2018). Diverse advantages and disadvantages of enzyme immobilization technology are
shown in Figure 7.2.
Choosing a proper immobilization method is of great importance in determining the
enzyme activity in a reaction. There are two main groups of immobilization methods:
chemical and physical (Figure 7.3). Weaker and noncovalent interactions such as hydro-
phobic interactions, hydrogen bonds, ionic binding of an enzyme to a matrix, and van der
Waals forces or mechanical hold of the enzyme within the support are the principal proper-
ties of physical methods. In chemical methods, covalent bonds linking enzyme and matrix
are formed by means of ether, thioether, amide, or carbamate bonds (Chiou and Wu 2004;
Voberkova et al. 2018).

7.2.1 Physical Methods

7.2.1.1 Adsorption
Adsorption is an easy and useful technique for immobilization. Some methods, such as
physical adsorption, ion adsorption, and affinity adsorption are included in the adsorption
methods. In this immobilization technique, usually used support carriers are cation and
anion exchange resins, silica gel, controlled pore glass, alumina, and ceramics. Enzymes
are adsorbed on the solid support materials by van der Waals forces, hydrogen bondings,
hydrophobic or electrostatic interactions (Bastida et al. 1998; Quilles Junior et al. 2016), or
immobilized in the pores of mesoporous materials (Fan et al. 2003; Pang et al. 2016). For
instance, Bastida and colleagues (1998) reported that the lipase was immobilized on the of
 7.2 ­Conventional Enzyme Immobilization Method 177

-Recovery/Reusability
-Matrice/carrier limitations
-Low or no contamination
ADVANTAGES

-Surface functionality of support

-High enzyme-substrate ratio
material
-Process control
-Partially high cost
-Continuous operation
-Limitations in implementation

DISADVANTAGES
-The rapid ending of enzyme assay
-Inactivation by the heat generated in
-High catalytic activity in process
the systems
-pH and thermal stability
-High functional efficiacy
-High product stability
-Eco-friendly
-Shelf life, storage stability

Figure 7.2 Advantages and disadvantages of enzyme immobilization technology. (Bilal et al. 2019).

Enzyme

Covalent bond

Support Support

Adsorption Covalent Bonding

Matrix Semi-permeable
membrane Enzyme
Immobilization
Cross links

Support Support

Entrapment/Encapsulation Cross Linking

Figure 7.3 Conventional enzyme immobilization methods. (Nguyen and Kim 2017).

Phenyl-Sepharose CL-4B and Octyl-Sepharose CL-4B resins by hydrophobic adsorption.

Another enzyme laccase was adsorbed on the surfaces and into the pores of micro-
mesoporous Zr-metal organic frameworks (Zr-MOFs) (Pang et al. 2016). Because no extra
coupling agents and modifications are required for immobilization, this method is a low-
cost application. Also, the support carriers can be regenerated. Weak and reversible links
178 7 Nanomaterials for Enzyme Immobilization

between enzyme and support matrix cause low stability of an immobilized enzyme.
Because these linkages are sensitive to pH, heat and ionic strength (Liu et al. 2018)

7.2.1.2 Entrapment
In the entrapment method, enzymes are encapsulated in an organic/inorganic polymer
with a low cost. The aggregation tendency of enzymes reduces the catalytic activity of
them. The entrapment is a convenient and practical method for preventing the precipita-
tion of enzymes. Chitosan, collagen, calcium alginate, cellulose triacetate, polyacrylamide,
agar, gelatin, silicone rubber, polyvinyl alcohol, and polyurethane can be used as a support
material for this technique. According to some research, enzymes were entrapped within
chitosan and calcium alginate beads or polyvinyl alcohol-alginate beads for dyes degrada-
tion. In this method, while the enzymes are arrested in the networks, the direct passage of
the substrates and products are provided (Liu et al. 2018).

7.2.2 Chemical Methods

7.2.2.1 Covalent Attachment
In this method, supports and enzymes’ side chain amino acids such as cysteine, lysine, or
glutamic and aspartic acid residues form the covalent bonds through chemical reactions
(Cirillo et al. 2014; Liu et al. 2018). Amino, imidazole, carboxylic, indolyl, and phenolic
hydroxyl groups are convenient for the formation of covalent bonds (D’Souza 1999; Liu
et al. 2018). Covalent bonds prevent the infiltration of enzymes from the support material.
In this way, the enzyme remains more stable (Liu et al. 2018). Covalent attachment allows
the enzyme to get a more rigid structure (Goncalves et al. 2019).
The shortness of the covalent bond constituted between the enzyme and the support mate-
rial and the excess number of bonds increase the durability of the enzyme. In this type of
covalent binding model, the enzyme becomes conformationally more stable, and the stability
of the enzyme against thermal, pH and organic solvents increases (Mateo et al. 2000, 2007).

7.2.2.2 Crosslinking
The crosslinking occurs between bifunctional reagents (crosslinking agent) and the
enzyme, and the reaction depends on intermolecular reactions (Goncalves et al. 2019). In
this method, enzymes are immobilized tightly to develop stability and reusability with
covalent bonds. However, the enzymes might lose their activities in the crosslinking
method. Isocyanate, N, N′-ethylene bis maleimide, and glutaraldehyde are the generally
used bifunctional reagents in this immobilization method (Liu et al. 2018). The toxicity of
such reagents is a restricting element in implementing this method to many enzymes.
Benefits and drawbacks of the most common methods of enzyme immobilization are
listed in Table 7.2.

7.3 ­New Generation Immobilization Methods

Free enzymes in solution are more sensitive to environmental changes compared to immobi-
lized enzymes. Above all, the heterogeneity of immobilized enzymes allows for multiple reuses
of enzymes, easy recovery of enzymes and products, continuous enzymatic processes, rapid
 7.3 ­New Generation Immobilization Method 179

Table 7.2 Benefits and drawbacks of the most common methods of enzyme immobilization.
(Zucca and Sanjust 2014; Bilal et al. 2017).

Immobilization
Method Benefits Drawbacks

Adsorption Easy, simple and cheap to implement Lower efficiency

No need to reagents Desorption
Short time Changes in optimum pH and pH
No pore diffusion limitations stability of the enzyme due to
Protect the enzymes from nproteolysis, deterioration of kinetics
aggregation, and mhydrophobic Non-specific adsorption
Higher yields/greater stability
High affinity between the enzyme and
support
Entrapment Needs to mild conditions Leakage of enzyme
Faster method Pore diffusion restriction
Easy to carry out at small scales Sourced from microorganisms
Inexpensive contamination hazard
Less conformational changes Lower industrial application
High surface area and adsorption
capacity
Uniform and tunable pores
No chemical changing of the natural
enzyme
Covalent Wider applicability Competitive inhibition problems
attachment Long life and reusability Chemical chancing of enzymes
Easier method Functional conformation loss of
Minimization of catalyst leakage enzymes
Stabilization of enzymes
Different supports/carriers can be
utilized
Robust binding of enzymes to supports
High residual activity owing to
increased surface area and porosity
Crosslinking No support is needed The necessity to poly-functional
Fast, simple and cheap method reagents
Minimization of catalyst leakage Risk of structural modification
Preferability in industrial applications or denaturation
Stabilization of the enzyme

reactions, and a wider diversity of bioreactor designs. These properties have made the
­immobilized enzymes great candidates for sustainable and green applications. Two well-known
main methods, immobilization and chemical modification, have been used to improve the sta-
bility and the activity of the enzyme. Although chemically modified enzymes have shown high
stability, they have not exhibited a significant increase in catalytic activity. Moreover, the time-
consuming and labor-intensive synthetic procedures have limited application of the chemical
modification method (Gron et al. 1990; Davis et al. 1999; Matsumoto et al. 2002).
180 7 Nanomaterials for Enzyme Immobilization

In this case, many enzyme immobilization techniques have high potential both industri-
ally and scientifically due to their economic and technological importance (Ge et al. 2012;
Zhu et al. 2013; Ocsoy et al. 2015; Somturk et al. 2015). For example, Zare and colleagues
focused on an approach for the synthesis of immobilized enzymes in the form of nanoflow-
ers (NFs) with enhanced catalytic and activity stability (Ge et al. 2012; Zhu et al. 2013)
(Figures 7.4 and 7.5). They also reported the synthesis of a membrane containing laccase
NFs for the detection of phenol (Zhu et al. 2013).
In following works, Zeng and collaborators used the same synthetic method to produce
CaHPO4-α-amylase-based NFs (Wang et al. 2013) and Yang and colleagues utilized enzyme-
inorganic NFs as sensors for detection of hydrogen peroxide and phenol (Lin et al. 2014).
Recently, Somturk et al. (2015) worked on the preparation, stability, and catalytic activity
of a hybrid nanoflower (HNF) composed of copper ions (Cu2+) and horseradish peroxidase
(HRP) enzyme (Figure 7.6). They evaluated the structure of HNFs as a function of the con-
centrations of chloride (Cl−) and copper (Cu2+) ions and HRP enzyme, the temperature of
the reaction, and the pH of the buffer solution (Somturk et al. 2015).
They also studied the synthesis of urease HNFs and their improved catalytic properties
and found the effects of reaction parameters on stability, activity, and morphology of HNF
(Somturk et al. 2016). Ocsoy et al. (2015) argue that the drastic rise in HNF activity is
related to the high in-nanoscale local HRP concentration dimension, appropriate HRP

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

Figure 7.4 Shows the scanning electron microscopy (SEM) images of HNFs. a–l, Column 1,
a-lactalbumin; column 2, laccase; column 3, carbonic anhydrase; column 4, lipase; at protein
concentrations of 0.5 mg ml–1 (a–d), 0.1 mg ml–1 (e–h) and 0.02 mg ml–1 (i–l). The morphological
changes with concentration conditions are clearly evident from this figure. (Ge et al. 2012).
 7.3 ­New Generation Immobilization Method 181

Filter Filter with nanoflowers Sample OH

H3C N N O

H3C NH2

Wash

N O
H3C N

Nanoflowers on filter H3C N O

Figure 7.5 The membrane with integrated laccase NFs is manufactured, used, washed, and reused.
Phenols and ortho-, meta-, and para-substituted phenols carrying groups of carboxy, halogen,
methoxy, or sulfonic acid react to colored compounds with 4-aminoantipyrine, which is then easily
and quickly detected. (Zhu et al. 2013).

I II III
2+
Figure 7.6 Possible HRP-Cu HNF formation mechanism consists of three parts: (I) nucleation,
(II) growth and (III) completion of the entire HNF formation. The dots indicate the copper
phosphate complex (Somturk et al. 2015).

conformation, fewer limitations on mass transfer, and the role of Fe2+ ion as an HRP acti-
vator (Figure 7.7). In another work, the synthesis of bovine serum albumin-Cu (II) hybrid
nanoflowers (BSA-NFs) and their use as adsorbents for lead and cadmium ions is reported
through the building blocks of BSA-Cu2+ ions in phosphate buffered saline (PBS) (Yilmaz
et al. 2016). Another report is available as a green approach to the synthesis of lactoperoxi-
dase (LPO) enzyme and metal ions HNFs and to investigate mechanisms underlying the
formation and improved stability and activity under various experimental parameters by
Altinkaynak and colleagues (2016a). A novel organic/inorganic nanobio/antimicrobial
agent called “nanoflowers” (NFs) from Camellia sinensis (L.) Kuntze extracts and their
major components was produced for the first time, and Baldemir and colleagues (2017)
explained the increase in antimicrobial activity of the NFs.
182 7 Nanomaterials for Enzyme Immobilization

(a) (b)
20000
18000
OH
OCH3 16000

Activity (U/mg)
OCH3
14000
Nanoflower hNFs + 4˚C
2 + H2O2 O O
12000
Free HRP
10000
OCH3
guaiacol 3, 3-dimethoxy-4, 4-diphenoquinone 8000
6000
4000
2000
0
0 10 20 30 40 50
Time (minute)

(c) (d)
100 100

80 80
Relative Activity (%)

Relative Activity (%)

hNFs + 4˚C
60 Free HRP + 4˚C 60 hNFs + 4˚C
hNFs RT hNFs RT
40 Free HRP RT 40 Free HRP

20 20

0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Time(Day) Time(Day)

Figure 7.7 NFs stability and activity and free HRP enzyme toward guaiacol. (a) Formation of the
“3,3-dimethoxy-4,4-diphenoquinone” orange quinone type structure from HRP nanoflower
catalyzed guaiacol reaction with H2O2. (b) Increased activity of synthesized NFs at +4 ° C and free
HRP measurements. (c) Stability of synthesized NFs at +4 °C (asterix line) and room temperature
(RT) (dotted line) and free HRP enzyme at +4 °C (thin solid line) and RT (short dashed line). (d)
Stability of synthesized NFs at +4 °C (dotted line), RT (asterix line) and free HRP (long dashed line)
measured at RT (Somturk et al. 2015).

In recent years, Ildiz et al. (2017) have claimed that the production of new organic–inor-
ganic nano biocatalytic and antimicrobial agents called “nanosnowballs” (NSBs) with a
rational design has elucidated the increase in antimicrobial and catalytic activity. In
another recent work, the researcher focused on a hierarchical assembly of flower-like
Turkish black radish peroxidase-Cu2+ nanobiocatalyst and its effective use in decoloration
of the dye. And they found that the peroxidase-Cu2+ HNFs showed increased activity and
stability toward different pHs and provided great decoloration efficiency for Victoria blue
(VB) dye with more than 90% within one hour (Altinkaynak et al. 2017). As a result, new
techniques for enzyme immobilization are collected under two main titles, which are
hybrid NFs incorporated in enzymes and hybrid NFs incorporated in non-enzymes.

7.3.1 Enzyme Incorporated Hybrid Nanoflowers

The new generation immobilization methods, NFs, were discovered by Zare and colleagues
(Ge et al. 2012; Zhu et al. 2013). With this an encouraging breakthrough in the synthesis of
flower-shaped hybrid nanostructures called NFs immobilized enzymes and showed greatly
enhanced catalytic activities and stabilities. The NFs consist of two major organic and
 7.3 ­New Generation Immobilization Method 183

i­ norganic components. While the enzymes/proteins have been used as an organic part,
metal ions, especially copper ion (Cu2+), act as the inorganic part for the formation of NFs. In
a typical NF synthesis procedure, Cu2+ reacts with solvent-accessible amine groups of ­protein
in the PBS to form NFs. The coordination chemistry between Cu2+ and the amine group of
amino acid residues for the formation of complexes is well documented (Smith et al. 1985;
Rulisek and Vondrasek 1998). In the potential NF formation mechanism, in the first step,
Cu2+ reacts with phosphate ions (PO43−) to form Cu2+-PO43− complexes as primary nanocrys-
tals (Cu3(PO4)2). In the second step, the Cu3(PO4)2 nanocrystals interact with amine groups
in the backbone of proteins through Cu to create Cu3(PO4)2-protein molecule conjugates as
called “petal.” The last step, the combination of petals owing to adhesive properties of protein
to complete the synthesis of NFs.
The immobilized enzymes in the NF form exhibit much-enhanced activities compared to
conventionally immobilized or free enzymes due to a number of potential reasons:
1) The favorable enzyme conformation in the NFs.
2) The high surface to volume ratio leading to low mass-transfer limitations.
3) The porous structure of the NFs.
4) Synergistic effects in the NFs.
5) The presence of metalloenzymes in the NFs.
For instance, Zare and colleagues demonstrated that laccase, copper-containing enzymes,
in the NF form, provided rapid oxidation and much-increased activity for norepinephrine
(~450%) and dopamine (~480%), respectively. Carbonic anhydrase, zinc-containing
enzyme, showed ~260% higher activity in the hydration of CO2 than the free enzyme.
However, lipase, metal-free enzymes, even in the NFs, showed the almost same hydrolytic
activity compared to free lipase. The examples proved that the activity and stability of met-
alloenzyme-incorporated with NFsare drastically increased.
Since the discovery of the NFs in 2012, various enzymes and metal ions have been uti-
lized in different experimental conditions for the production of the NFs and examination
of the catalytic activities and stabilities (Lin et al. 2014; Sun et al. 2014; Batule et al. 2015;
Ocsoy et al. 2015; Somturk et al. 2015; Yin et al. 2015; Somturk et al. 2016; Yilmaz et al.
2016; Altinkaynak et al. 2016b, 2017; Celik et al. 2018). For instance, Ocsoy et al. (2015)
produced HRP, Cu2+, and Fe2+ ions at two different reaction temperatures +4 °C and
RT(20 °C) and showed comparative catalytic activities. In addition to that, instead of com-
mercial enzymes, isolated and purified LPO from bovine milk was successfully reacted
with Cu2+ to synthesize the NFs and it was investigated how some experiment conditions,
including pH and storage temperatures, affect the stability and catalytic activity of the NFs
in the presence of hydrogen peroxide (H2O2).
Additionally, Hou and colleagues introduced the synthesis of CaHPO4-α-amylase NFs
with various morphologies such as nanoplates, NFs, and parallel hexahedrons. And then,
the increase in activity of the CaHPO4-α-amylase NFs was explained based on the allosteric
effect and morphologies (Wang et al. 2013).

7.3.2 Non-Enzyme Incorporated HNFs and Their Enzyme Mimic Activity

The NFs have also been produced using amino acid, standard plant molecule, and whole
plant extracts as organic parts. We claim that the same formation mechanism as proposed
184 7 Nanomaterials for Enzyme Immobilization

in enzyme incorporated NFs, Cu2+, reacts with amine or carboxyl groups of those organic
molecules to initiate the formation of NFs. The researchers reported that those NFs acted
as Fenton reagents and exhibit peroxidase-mimic activity in the presence of H2O2 toward
corresponding substrates. In the Fenton reaction, Cu2+ in the NFs give interaction with
H2O2 to create the Cu2+ ions, which react with H2O2 to produce highly reactive hydroxyl
radical. Then, peroxidase-like activity is observed oxidation of the substrates by hydroxyl
radicals.
For instance, 20 natural amino acids acted as organic parts and Cu2+ ion preferentially
bind to carboxyl and amine groups of amino acids for the formation of NFs (Wu et al.
2016). In the presence of H2O2, amino acid NFs showed peroxidase-like activity toward
Rhodamine B and ABTS as the substrates.
Additionally, standard plant molecule and whole plant extracts have been used for the
first time as organic part to synthesize NFs. In this NFs concept, Viburnum opulus (VO)
extract reacts with Cu2+ in the PBS and results in the production of flower or snowball-like
hybrid nanostructures as shown in Figure 7.8 (Ildiz et al. 2017). A similar strategy was uti-
lized using the green tea (GT) extracts (from ethanol and water and standard molecules)
catechin and caffeine to synthesize NFs (Baldemir et al. 2017). The report demonstrated
that while GT ethanol extract and ethanol soluble molecule catechin give NFs with similar
morphology, the NF is also quite similar in shape and size with the use of GT water extract
and water-soluble molecule caffeine. It is worth mentioning that both reports showed that
plant extracts or standard molecules-incorporated NFs exhibited peroxidase mimic activi-
ties based on Fenton reaction.

PBS
solution CuSO4

Natural Synthetic nanosnowball

Snowball viburnum viburnum

100
80
Enzymatic 60
40
Activity 20
0
0 100

Antimicrobial
Activity

Figure 7.8 Graphic abstract of plant extract preperation VO-inorganic hybrid NSBs. (Ildiz
et al. 2017).
 References 185

7.4 ­Conclusion

Enzymes have been in used as biological catalysts in several different scientific and indus-
trial fields. Although enzymes in free form exhibit high and specific catalytic activities,
short lifetime, rapid enzyme degradation in some experimental conditions and lack of
reusability of enzymes strictly limit their use. To overcome the limitations of free enzymes,
conventional methods of immobilization were developed by immobilizing enzymes on/in
several external supports. However, conventionally immobilized enzymes showed high sta-
bility but low catalytic activity compared to free enzymes.
Discovery of new generation immobilization methods lead to flower-shaped hybrid
nanostructures–NFs–consisting of enzymes and metal ions with greatly enhanced catalytic
activity and stability. In addition to that, various organic molecules except enzymes have
been used to form NFs and show peroxidase mimic activities in the presence of H2O2
through a Fenton-like reaction. We consider that NFs with their unique catalytic activities
can widen their use in a variety of scientific and industrial fields.

­Acknowledgment

We thank Nihan Arabacı and Tuna Karaytuğ for writing sections 7.1, 7.1.1, 7.1.2, 7.1.3, 7.1.4,
7.1.5, 7.2, 7.2.1, 7.2.1.1, 7.2.1.2, 7.2.2, 7.2.2.1, and 7.2.2.2. We also appreciate the help of Ayşe
Demirtas, Ismail Ocsoy, and Ahmet Katı in writing sections 7.3, 7.3.1, 7.3.2, and 7.4. We
would like to thank the Erciyes University Scientific Research Office for supporting this
research with the FCD-2018-8242 coded project.

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 191

Nanomaterial Biosynthesis and Enzyme Immobilization

Methods and Applications
Indu1, Ankush Yadav1, Mrinal Kanti Mandal2, and Kashyap Kumar Dubey1
1
Bioprocess Engineering Laboratory, Department of Biotechnology, Central University of Haryana, Mahendergarh, Haryana, India
2
Department of Chemical Engineering, NIT Durgapur, Durgapur, West Bengal, India

8.1 ­Introduction

Nanotechnology comprises nanomaterials that clarify the construction and exploitation of

bulk materials in range of nanometer size (1 nm = 10−9 m), (John et al. 2010; Rao et al.
2006). It has great importance as an interdisciplinary science due to its potential for wide
application in different industries, including cosmetics, clothing, food industry, medical
industry, household appliances, and renewable energies. Along with these applications,
other untouched areas of advantage for nanomaterials are also available. Nanotechnology
becomes popular and fascinating, with a wide range of applications. These include the
interaction of nanomedicine with biological molecules and application in green technol-
ogy for environmentally friendly and renewable energy solutions for capturing, reserving,
and transferring energy (Ghoranneviss et al. 2015).
The theme of nanotechnology is miniaturization and the nanometer-scale structures can
lead to a science comprising new devices and technologies. Nanotechnology is a promising
technology that exploits nanometer-sized materials. Nanofilteration is used for separation
of divalent ions (Ankush et al. 2019). Nanomaterials hold great importance because of
their unique electrical, optical, magnetic properties and their huge influence in biofuels,
electronics, medicines, and other fields. In the growing era of nanotechnology, biocompat-
ible nanomaterials are being used to replace natural materials in living systems. This chap-
ter explains the manufacture and assembly of nanostructures and their applications in
engineering and biology (Prashant et al. 2008).
Immobilization defines the cell or enzyme static to a matrix or support. At industrial
level, the demand for increasing shelf life and production of enzymes is growing continu-
ously. In the near future, engineered enzymes will be accessible for all industrial and phar-
maceutical processes (Dubey et al. 2019). Therefore, immobilization plays an important
role in the availability of enzymes for a continuing a chemical reaction. Immobilization
provides the base for the enzyme–substrate reaction, which leads to increased reaction

Green Synthesis of Nanomaterials for Bioenergy Applications, First Edition. Edited by Neha Srivastava,
Manish Srivastava, P. K. Mishra and Vijai Kumar Gupta.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
192 8 Nanomaterial Biosynthesis and Enzyme Immobilization

efficiency (Sheldon 2007). This is a very old technique and amino acylase of Aspergillus
oryzae was the first immobilized enzyme for production of L-amino acids. A suitable matrix
for immobilization may be natural or synthetic and it should be cheap, easily available, and
non-reactive in nature. In addition to facilitating easy handling of an enzyme, immobiliza-
tion minimizes the product contamination by facile separation of products. Consequently,
immobilization of enzymes increases the functional efficiency of products, stability, eco-
nomic growth, purity, recovery of product, etc. (Datta et al. 2013).

8.2 ­Types of Nanomaterials

As already mentioned, nanotechnology contributes a wide range of applications in the cos-

metic, food, clothing, and household appliances industries (Ghoranneviss et al. 2015; Kim
et al. 2018; Rao et al. 2006). Different types of nanomaterials are shown in Table 8.1.

8.3 ­Size and Forms of Nanomaterials

According to Siegel, their size ranges from one dimension to three dimensions: one dimen-
sion includes surface films, two dimensions include strands or fibers, and three dimensions
include particles. Figure 8.1 shows the following examples of nanomaterials: nanotubes,
nanowires, nonporous solids, surface and thin films, dendrimers and quantum dots, etc.
Nanomaterials exist in different forms such as single, aggregate, fused, spherical, tubular,
and irregular forms. In regard to their different dimensions: zero-dimensional nanomateri-
als contain spheres, filaments, and atomic clusters; one-dimensional nanomaterials con-
tain multilayers; two-dimensional nanomaterials contain an over or buried layer; and
three-dimensional nanomaterials contain nano-phase material (Alagarasi 2011).

Table 8.1 Types of nanomaterials on the basis of size and material.

Sr. No. Types of Nanomaterial Size (diameter) Materials Used

1 Nanoparticle 1–100 nm Ceramic oxides

2 Nanowires nitrides 1–100 nm Metals, semiconductors, oxides,
sulfides
3 Nanotubes 1–100 nm Carbons, layered metal
chalcogenides
4 Nanocrystal and 1–10 nm Metals, semiconductors,
clusters magnetic materials
5 Nanoporous solid 0.5–10 nm Zeolites, phosphates
6 Surface and thin film 1–1000 nm A variety of materials
7 Two dimensional 2 nm–2 μm Metals, semiconductors,
magnetic
8 Three-dimensional Several nanometer metals, semiconductors,
material size in magnetic
Three dimensional
 8.5 ­Methods for Nanomaterial Biosynthesi 193

(a) (b)

(c) (d)

Figure 8.1 The Classification of Nanomaterials, (a) clusters and spheres; (b) rods, one-dimensional
nanofibers and wires; (c) plates, two-dimensional films and networks; (d) three-dimensional
nanomaterials. Source: Modified from Alagarasi 2011.

8.4 ­Properties of Nanomaterials
Some nanoparticles are found in nature and some are man-made, designed for particular
interest and used in commercialized industry. Nanoparticles exhibit particular properties
different from other materials because of their nanoscale size. When a crystalline solid is
broken into small pieces, the different range of materials is obtained. However, the broken
pieces of the same solid do not show similar properties corresponding to a large solid (Rao
et al. 2006). Nanomaterials show a number of properties, i.e. optical, magnetic, mechani-
cal, thermal, and electrical, which are size dependent (Prashant et al. 2008). These proper-
ties are listed in Table 8.2.

8.5 ­Methods for Nanomaterial Biosynthesis

8.5.1 Mechanical Grinding

This is the simplest method of nanomaterial production. It involves the grinding of large
crystalline solids into nano-sized particles. It is easy, simple, inexpensive, and applicable for
the synthesis of all classes of materials. The demerits of using grinding mechanism are con-
194 8 Nanomaterial Biosynthesis and Enzyme Immobilization

Table 8.2 Types of properties of nanomaterials.

Sr.
No. Property Concept Examples References

1 Optical These properties depend on Laser, imaging, sensor, Alagarasi (2011)

property the size, surface solar cell, aoptical-
characteristics, shape, detector, display, photo
doping, and interaction with catalysis, photo
surrounding electrochemistry and
biomedicine, etc.
2 Electrical Nanotubes with different Nanotubes, nanowires, Alagarasi
property lengths and fiber bundles Nano-rods (2011), John
with increasing protrusion et al. (2010),
and carbon nanotubes, will Shim et
touch the surface of mercury
droplets and contribute
electrical energy
3 Mechanical It relates to porosity, grain Polymer-ceramic Alagarasi
property size, ceramic materials, nanocomposites (2011),
plasticity, filled polymers, Christian
nanocomposites filled with (2009);
platelets, and carbon Patterson (2004)
nanotube-based composites
4 Magnetic This property also depends Hard disk coating of Christian
properties on size and structural silver, cobalt, platinum, (2009),
changes and tantalum Alagarasi (2011)

tamination from the atmosphere and the powder product becoming hard. Mechanical grind-
ing is achieved by using planetary balls, energy shakers, and tumbler mills, as shown in
Figure 8.2. Grinding speed depends upon the number of balls, size, speed, atmosphere, and
time of milling, and the ratio of the ball powder mass. This method is suitable for amorphous,
crystalline, and compound powder and unsuitable for non-oxide materials (Alagarasi 2011).

8.5.2 Thermolysis, Photolysis, and Sonolysis

After mechanical grinding these are the simplest methods. Nanoparticles are driven either by
photolysis (light), thermolysis (heating), or sonolysis (sound). These are the factors that also
control the growth of nanoparticles. Organometallic compounds are in close proximity to each
other so after decomposition of these compounds the controlled crystalline and morphology is
formed. Decomposition in the presence of capping agents, polymers, and structural hosts pro-
vides additional control over the nucleation process. For example, capping preventing coalesce
and agglomeration. If carbonyl is decomposed in the presence of polymers, the spherical nan-
oparticles are formed. Similarly, by the addition of surfactant into nanoparticles, the themor-
phology of particles is changed. To execute controlled growth and rapid nucleation, the
carbonyl is injected into the hot solution (Christian 2009; John et al. 2010).
The nucleation process is performed for designing alloys and core-cell structures.
Transmetalation is defined as a process in which alloys can grow on nuclei and it is used for
alloy formation. For core material synthesis, first nuclei are allowed to react with surface;
 8.5 ­Methods for Nanomaterial Biosynthesi 195

Figure 8.2 Mechanical milling. Source:

Modified from Alagarasi (2011).

Refractory
or
Steel Ball

Preferably Inert
Atmosphere

then the temperature is lowered; and later, a second precursor is added into it. When the
ultrasonic waves are used for disintegration of nanoparticles, the powder forms of ­materials
are produced, which are usually agglomerated, amorphous, and porous. To reach the crys-
talline phase, the powder form of substances is annealed. After sonication, if a stabilizer is
added to powder, the colloidal metal particles with large surface area and amorphous state
are formed and later used for catalytic reactions (Gedanken and Mastai (2004).

8.5.3 Top–Down Approach

Within this approach, large bulk materials are broken down into nanoscale-size material by
performing the physical method in aqueous solution. For powder formation, the methods
are milling, devitrification, and plastic deformation. Methods for nanocrystalline films are
laser ablation, sputtering, and thermal vaporization (Christian 2009; Mendes et al. 2004).

8.5.4 Bottom–Up Approach

In this process the small molecules are used for preparation of particles. The principle
behind this bottom up method was super-saturation. It is defined as the point where a
­solution contains more dissolved material than the solution saturation point of dissolving
solute. For example, productions of calcium sulfate precipitate by adding sulfate into
­calcium ion solution (Christian 2009; Zaizer and Lamber 1948).

8.5.5 Sol-Gel Process

Sol-gel process or liquid mix process was developed by Maggio O. Pechini in 1967. It
involves formation of colloidal and gelation suspension of solution to form networks in a
continuous liquid phase (Alagarasi 2011; Massart 1981). It also involves hydrolysis and
condensation of alkaloid-based precursors such as Si(OEt)4, e.g. hydrolysis and condensa-
tion of metal alkaloids M(OR)z:

MOR H 2 0 MOH ROH hydrolysis

MOH ROM M O M ROH condensation

196 8 Nanomaterial Biosynthesis and Enzyme Immobilization

Sol-gel is a widely accepted process for nanomaterial biosynthesis. It involves a series of

steps to prepare oxide materials as shown in Figure 8.3 (Mohanpuria et al. 2008):

Step 1: Different stable solutions of the alkoxide or solvated metal precursors are formed.
Step 2: A polycondensation reaction will increase viscosity of solution.
Step 3: The gel ages, which takes about seven days and involves consumption of
smaller particles by larger particles leading to syneresis.
Step 4: The drying of the gel by four sub-steps, i.e. the constant rate period, critical
point, falling rate period, and the second falling rate period.
Step 5: Dehydration leads to stabilization of gel.
Step 6: The decomposition of gel with high temperature of 8000 °C.
(Alagarasi 2011).

8.5.6 High-Temperature Nanomaterial Biosynthesis

This process is very simple, that is why it has been scaled up from the laboratory to industry
(Shaw et al. 2003). It involves the heating of the desired material in a heating crucible at
2000 °C. It mainly condenses particles with high vapor pressure. Evaporated material from
the cluster may be inert or reactive. Clusters rapidly grow in the super-saturation region
and are removed by a carrier gas. Metal with a low vapor process forms organometallics,
which later condense in the furnace. Three parameters—rate of evaporation, rate of con-
densation, and rate of gas flow—control the cluster size and its distribution in the furnace

Precursor

Dissolve

Dipping Calcine Thin Film Coating

nning
Spi

Solution
Dehydration
Calcine Powder
Reaction

Surfactant Calcine
Dense Ceramic
Gel Organic
Xerogel
Rapid Drying

Aerogel

Figure 8.3 Different steps involved in the sol-gel process. Source: Modified from Alagarasi (2011).
 8.7 ­Nanomaterials-Immobilized Enzymes toward Biofuel and Bioenergy Productio 197

(Alagarasi 2011; Feng and Xu 2001; John et al. 2010). Finally, nanoparticles are collected on
a cold plate, shown in Figure 8.4.

8.5.7 Flame-Assisted Ultrasonic Spray Pyrolysis

This comprises the burning of unwanted components to get wanted material, e.g. Zr(CH3
CH2 CH2O)4 is convert to ZrO2. For silica preparation, SiCl4 is heated in an oxyhydrogen
flame to give silica in a dispersed form 7–40 nm in size. At industrial level, for production
of fumed silica, carbon black, and titanium oxide in powder form, the combustion flame
method is used. The burning of a mixture of oxygen and hydrogen, oxygen and acetylene,
provides the energy for initiation of pyrolysis of precursor material (Network et al. 2012).
The main aim of using the low-pressure combustion synthesis is to extend the range of
pressure and avoid agglomeration. Low-pressure flame is widely used in aerosol particle
formation (Alagarasi 2011, John et al. 2010). A layout of flame-assisted ultrasonic spray
pyrolysis is shown in Figure 8.5.

8.6 ­Applications of Nanoparticles

A number of areas include various applications of nanoparticles shown in Figure 8.6.

8.7 ­Nanomaterials-Immobilized Enzymes toward Biofuel

and Bioenergy Production

Nanotechnology is advancing due to various optical, chemical, magnetic, mechanical, and

electronic properties (Biswas et al. 2012; Wu et al. 2008). It is a rapidly growing field with
nanoscale range components and dimensions comparable to the biological molecule
(nucleic acid, enzymes) (Stark 2011). Immobilization by different processes such as

Figure 8.4 Representation of different steps In Inert Atmosphere

inside heated crucible. Source: Modified from
Alagarasi (2011).
Nanoparticles Collected on a
Cold plate

Clusters

Evaporated Matter

Materail to be Evaporated

Heated Crucible
198 8 Nanomaterial Biosynthesis and Enzyme Immobilization

Droplet Containing
Precursor

Clusters

Fog
Flame
Carrier Gas
Precursor Nebulizer
Nanomaterial
ate
bstr
Su

Piezoelectric
Transducer

Figure 8.5 Diagram representing flame-assisted ultrasonic spray pyrolysis. Source: Modified from
Alagarasi (2011).

Energy
Optical Biofuel
Engineering Production

Defense and
Nano-Fabrics
Securtiy

Medical and Nano-

Drugs Technology

Nano-Device

Figure 8.6 Applications of nanotechnology.

g­ lutaraldehyde activation, silanization, polyethylene glycol (PEG), polyvinyl alcohol (PVA)

­spacing, and ­natural/synthetic polymer bring enzymes to the nanomaterials. Nanomaterial
types such as nanotubes, nanosheets, nanofibers, nanoporous, and nanocomposite have
large surface area, which improves the preparation and revolution of enzymes (biocatalyst)
(Verma et al. 2014). The main problem in today’s world is the depletion of global
­nonrenewable fossil fuel at unpredicted rate and this directs our focus toward a tenable
 8.7 ­Nanomaterials-Immobilized Enzymes toward Biofuel and Bioenergy Productio 199

source of alternative fuels. Biofuel is defined as nontoxic, biodegradable fuel produced

from vegetable oils, different feedstock, and biomass. By utilizing these domestic, readily
available renewable energy sources we were able to satisfying future energy requirements
(Pugh et al. 2011; Puri et al. 2012). The high temperature tolerance and environmentally
friendly nature of enzymes means that the use of enzyme-based processes for biofuel pro-
duction have increased (Du et al. 2005; Kumari et al. 2009).
The main enzymes used for energy production from industrial biomass are lipases and
cellulases (Verma et al. 2014). The chief limitation of using enzyme-based biofuel produc-
tion is the high cost of enzymes. Use of novel nanomatrices may lead to efficient immobili-
zation of enzymes and multiple reuses of magnetic nanomaterials or nanocomposites
significantly lowers the cost of enzymatic processes (Cox et al. 2004; Schmid et al. 2002).
Nanomaterials represent several key factors such as more surface area, reduced fouling
effect, mass transfer resistance, and specific nonchemical separation from the mixture
(Mahmood and Hussain (2010). All of these factors enhance the efficiency of enzymes (Puri
et al. 2013). The many benefits related to nanotechnology-based products include the fol-
lowing: increased electrical storage capacity, high efficiency of lighting and heating, less
pollution from renewable energy, etc. (Hussein 2015; Brennan and Owende, 2010).
Bioenergy is the cheapest alternative source of energy. Different types of bioenergy
sources include biofuels, biogas, biodiesel, bio-oil, vegetable oil, etc. Synergy between
Nanotechnology and biotechnology gives a new sub-field called nanobiocatalysis, which
explores nanostructured material (Kim et al. 2008; Schmid et al. 2001; Thomas et al. 2002).
Due to demand for fossil fuel production on a large scale, the concern about lipase-medi-
ated biodiesel production is increasing. Biodiesel is produced by chemical or lipase medi-
ated transesterification reaction and the chemical catalysis is highly accepted at industrial
level due to high reaction rates (Iso et al. 2001). Although, other factors, i.e. environmental
issues and downstream process cost, become an issue with biofuel production. To over-
come these issues an ecofriendly production method comes into light and enzyme-medi-
ated biodiesel production becomes advantageous over chemical catalysis (Gupta et al.
2012; Puri et al. 2011). So, lipase-mediated enzymatic method is employed for biodiesel
production with high activity and stability for cost-effective commercialization (Verma
et al. 2013).
Biofuels are the alternative source of renewable energy. Excellent biofuels produced by
yeasts, i.e. ethanol and methanol, are highly flammable. Bioethanol is the most commonly
used fuel in vehicle engines due to its auto-ignitability properties. It burns in a very clean
and environmentally friendly manner (Owende et al. 2010; Pittman et al. 2011).
Biogas is used as a gas for cooking and heating and for transportation at commercialized
level (Fontes 2010). Vegetable oils have a high heat point for vaporization, energy density,
and balanced air–fuel ratio. Biogas contains a significantly decreasing concentration of car-
bon monoxide, sulfur oxides, smoke, and noise (Chen et al. 2013).
Bio-oil is a dark brown, viscous, corrosive fuel with a smoky odor that is used for diesel
engines, furnaces, gas turbines, and stationary engines. It contains a complex composition
of lignocellulosic biomass such as aliphatic alcohol/aldehyde, pyranoides, fatty acids, fura-
noids, and large molecular-weight hydrocarbons (Naik et al. 2010). The use of nanosized
cerium oxide particles as the additive reduces the emission of hydrocarbons and nitric
oxide (NOX) components (Sajith et al. 2010).
200 8 Nanomaterial Biosynthesis and Enzyme Immobilization

8.8 ­Immobilization

The enzyme—also called the biocatalyst—is a major discovery in science. It has great
advantages in different sectors because of its easy production, substrate specificity, and
green chemistry (Patel et al. 2001; Schoemaker et al. 2003; Straathof et al. 2002; Liese and).
However, the production of biocatalysts at commercialized level becomes difficult and this
leads to decline in economic growth at industrial level. The demands for enzyme produc-
tivity at industrial level are increasing day by day; therefore, we have to develop some
novel technique for increasing the shelf life of enzymes. One of the major limiting applica-
tions of enzymes is instability; and enzyme immobilization increases the availability of
enzymes to the substrate and makes it stable. Immobilization refers to the static of enzymes
at the typical support surface. This attachment of a cell or enzyme with the surface aids in
the exchange of substrate from the matrix (surface) and helps the immobilized enzyme
function efficiently, enhancing the productivity. There are different types of matrixes,
which may be natural or synthetic (Adamczak and Krishna 2004; Bornscheuer 2003;
Krajewska 2004; Tischer and Kasche, 1999).

8.9 ­Matrix for Immobilization

A matrix is used to hold an enzyme properly for chemical reactions. An ideal matrix has the
following characteristics: inertness, re-generatability, physical strength, stability, enzyme
specificity/activity, and reduced nonspecific adsorption. It should be least reactive in nature
and available at low cost. Different types of matrixes include synthetic polymer, natural
polymer (biopolymer), inorganic support, and smart polymer (Datta et al. 2013; Sheldon
2007; Huisman and). Different matrixes or supports that may be natural, synthetic, or
­inorganic are shown below in Table 8.3.

8.9.1 Natural Polymers

Natural polymers include a variety of biopolymers such as starch, cellulase, pectin, alginate,
gelatin, collagen, chitin, and carrageenan. Water soluble polymers are agarose, cellulose,

Table 8.3 Matrix or support for immobilization.

Matrix

Natural Polymer Synthetic Polymer Inorganic Polymer

Alginate, DEAE, Zeolites,

Chitosan and chitin, Cellulose, Celite,
Collagen, Polyvinyl chloride, Ceramics,
Carrageenam, Polyethylene glycol, Silica,
Gelatin, Cyclodextrin glucosyl transferase, Glass,
Cellulose, Polyaniline Activated carbon,
Starch, Charcoal,
Pectin Diatomaceous
 8.10 ­Methods of Enzyme Immobilizatio 201

and starch; and protein polymers include gelatin, collagen, and albumintic (Krajewska
2004; Lozinsky and Plieva 1998; Lozinsky et al. 2003; Gao et al. 2004).

8.9.2 Synthetic Polymers

These are the insoluble matrixes with porous surface, used mainly for ion exchange. They
trap and immobilize an enzyme. Examples are DEAE cellulose, PVC, and UV-activated
PEG. (Alkan et al. 2009; Dutta et al. 2009; Huang and Cheng 2008; Kumari and Kayastha
2011; Kahraman et al. 2007;Khan et al. 2006; Xing et al. 2000).

8.9.3 Inorganic Support

Inorganic support may be used for immobilization of enzymes. It includes zeolites, ceramics,
silica, glass, charcoal, activated carbon, and diatomaceous earth (Lalena and Cleary, 2010;
Alkan et al. 2009; Huang and Cheng 2008; Kahraman et al. 2007; Khan et al. 2006;
Sheldon 2007; Xing et al. 2000).

8.10 ­Methods of Enzyme Immobilization

8.10.1 Adsorption
Adsorption is an old and simple method of enzyme immobilization, shown in Figure 8.7.
In 1916, Nelson and Griffin used charcoal as a support material for enzyme adsorption. The
interaction between enzyme and support may be ionic, physical (hydrogen bonds and Van
der Waals forces), and covalent (Dey et al. 2002; Hernandez et al. 2007; Karagulyan et al.
2007). Covalent interaction is stronger than ionic, while ionic interactions are stronger
than Van der Waals forces. Support material may protect the enzyme from proteolysis and
aggregation. Researchers used coconut fiber as a support material because of its good
water-holding capacity and high cation-exchange property. Salinized molecular sieve,
octyl-agarose, and octadecyl-sepabeads are examples of physical interaction. Polypropylene
is attached to the enzyme with hydrophobic interactions (Usuki et al. 1993). Recently,
3-hydroxybutyrate-co-hydroxyvalerate is being used instead of polyhydroxybutyrate1,

Figure 8.7 Adsorption of enzymes.

Enzym En
e zy
me

Enzyme
Support
Enzyme

Enzyme
Enzyme
202 8 Nanomaterial Biosynthesis and Enzyme Immobilization

4-Butenediol di-glycidyl, because of its up to 12-hour operable cycles (Cabrera-Padilla et al.

2012; Cunha et al. 2009). Types of adsorption method include static process, dynamic batch
process, reactor loading, electron position, etc. (Brígida et al. 2010; Huang et al. 2011;
Mitchell and Pérez-Ramírez 2011).

8.10.2 Covalent Binding

This is a widely used method for enzyme immobilization that involves the formation of a
covalent bond between chemical groups of enzymes and chemical groups of support, as
shown in Figure 8.8 (Brown et al. 1996). Chemical groups are imidazole, indolyl, hydroxyl,
carboxyl, thiol, guanidyl, phenolic, etc. (D’Souza 1998; Singh 2009). Improvement in covalent
binding was achieved by “peptide-modified surfaces” to make binding more specific and sta-
ble (Fu et al. 2011). The commonly used supports for covalent binding are polyacrylamide,
agarose, cellulose, DEAE cellulose, amino benzyl cellulose, collagen, gelatin, silica, chitosan,
porous glass, etc. (Dutta et al. 2012). In the field of biocatalyst immobilization, the nanodia-
metric supports reach a turning point because of greater residual activity (further increase in
surface area and porosity) (Zaks, 2001). Methods of covalent bonding include peptide bond-
ing, diazoation and use of bi or multifunctional reagents. Crosslinking and bifunctional
nature of glutaraldehyde make it water soluble and form stable covalent bonds in water
(Huang et al. 2008; Li et al. 2007; Kim et al. 2006; Ren et al. 2006; Sakai et al. 2010; Wu et al.
2005). A major application of covalent binding in pharmaceutical industry is that different
orientations of an immobilized enzyme lead to enhanced longevity, operational stability, and
reusability.

8.10.3 Copolymerization (Crosslinking)

In this method, the immobilized enzyme is attached to enzymes by covalent binding via
bifunctional or polyfunctional reagents. Unlike other methods, there is no support or

Enzyme
Enzyme

Covalent bond between

Support and Enzyme
Enzyme
Support
Enzyme

Enzyme e
Enzym

Figure 8.8 Covalent bonding of enzyme.

 8.10 ­Methods of Enzyme Immobilizatio 203

matrix involved in this method (Tischer and. In previous years, crosslinked enzymes
(CLEs) were ­produced by the reaction between surface NH2 groups with chemical crosslink-
ers, as shown in Figure 8.9 (Brown et al. 1966). However, this method of producing CLE
had several ­limitations such as poor reproducibility, mechanical instability, and low activ-
ity retentions. To overcome these limitation CLEs switched to carrier-bound enzymes and
further crosslinked enzyme crystals (CLECs) are used for stabilization of enzymes from
heat ­denaturation (Cao et al. 2000; Cao, 2005). The limitations of using CLECs are that we
need enzymes in crystal form, which requires enzymes in highly pure form. So, copolym-
erization is used for direct isolation of an enzyme in purified form from the crude fermen-
tation broth (e.g. penicillin G amidase). The greater demerit of this method is denaturation
of crosslinking and loss of catalytic properties of enzymes.

8.10.4 Entrapment and Encapsulation

Entrapment is the caging of an enzyme in a polymer network by covalent or noncovalent
bonds (Singh 2009), while encapsulation is defined as enclosing the enzyme in a mem-
brane capsule (Figure 8.10. The membrane is made up of nylon, nitrocellulose, and
­alginate-gelatin–‑calcium hybrid. The use of nanofibers revolutionized the world of
enzyme immobilization (Dai and Xia 2006; Kim et al. 2006; Wang et al. 2009; Wen et al.
2011; Tischer and Kasche,1999). Commonly used matrixes for entrapment are agar,
­gelatin, alginate, polyacrylamide gels, cellulose triacetate, and carrageenan. The main dis-
advantage of entrapment is the leakage of an enzyme (Shen et al. 2011). For restriction of
leaching, delicacy, and escalation of entrapped efficiency, chitosan is used as a matrix
(Chen et al. 2011). Chitosan is nontoxic, biocompatible, and amenable to chemical modi-
fications. Entrapment or encapsulation by porous silica imputes properly pore distribu-
tion, high surface area, and continuous pore distribution with high adsorption capacity
(Hsieh et al. 2000). The advantages of entrapment are that it is cheap, fast, and easy to
practice and there is less chance of change in catalytic site enzymes (Jegannathan et al.
2010; Tümtürk et al. 2007).

Enzyme Enzyme Enzyme

Cross Links

Enzyme Enzyme

Enzyme Enzyme Enzyme

Figure 8.9 Crosslinking (copolymerization) of enzyme.

204 8 Nanomaterial Biosynthesis and Enzyme Immobilization

Enzyme Enzyme Enzyme

Enzyme
Enzyme
Matrix
Semi permeable Enzyme
Membrane Enzyme Enzyme Enzyme

Enzyme Enzyme

Enzyme Enzyme Enzyme

Encapsulation

Entrapment

Figure 8.10 Encapsulation and entrapment of enzymes.

8.11 ­Merits of Immobilization

Immobilization has a number of merits, including the following:

●● In the food industry, immobilization of pectinases and cellulases on a suitable carrier
leads to production of jams, jellies, and syrups.
●● It helps in production of antibiotics, amino acids, and beverages.
●● Immobilized enzymes are used to detect and treat various diseases.
●● In different techniques of immunology, immobilization of enzymes increases efficiency
of processes.
●● Production of biodiesel from vegetable oils.
●● It helps in treatment of sewage and industrial wastes.
●● IT helps in removal of dirt in the detergent industry.

8.12 ­Immobilization of Enzymes Beneficial for Biofuel

Production

In Europe and the USA, biodiesel is produced chemically from vegetable oils. The various
advantages of the enzymatic process over the chemical process are stereo specificity,
­substrate specificity, and functional group specificity (Chen et al. 2001; Laumen et al. 2002).
Two enzymes—cellulase and lipase—are primarily used for biofuel production at indus-
trial level. Enzyme hydrolysis is based on three factors, i.e. efficiency, thermal stability, and
reusability of enzymes and all are assisted by immobilization of enzymes (Puri et al. 2013).
However, due to the high cost of enzyme catalysts the enzymatic method was not adopted
at industrial level. For utilization of an enzyme catalyst repeatedly with low cost, the immo-
bilization process is very helpful. Immobilization helps in exposing the active site of the
enzyme and makes it more effective; and after reaction the immobilized enzyme is easily
separated from the matrix by the process of “decantation” (Chen et al. 2001). Immobilization
using nanostructured material improves biocatalytic efficiency of high-cost enzymes for
 References 205

industrial applications. Later, co-immobilization is achieved by nanomaterials for biofuel

production (Facin et al. 2019; Puri et al. 2013).

8.13 ­Conclusion

As we know, in today’s world the demand for biofuels is increasing and nanomaterials
contribute to biofuels by utilization of waste as substrate. Nanoparticle properties are
dependent on size, shape, structural changes, and composition. This chapter defines differ-
ent suitable methods and applications of nanomaterial biosynthesis and their wider appli-
cations in different industries. The demands for enzyme productivity at industrial level are
increasing day by day. Therefore, we have to develop some novel technique for increasing
the shelf life of enzymes. One of the major limiting applications of enzymes is instability,
and immobilization increases the availability of enzymes to the substrate so that enzymes
function efficiently, which later enhances the productivity.

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 211

Carbon Nanotubes for Hydrogen Purification and Storage

Pietro Bartocci1, Giovanni Russo1, Haiping Yang2,3, Song Hu2,3,
Øyvind Skreiberg4, Liang Wang4, Fausto Gallucci5, Gianni Bidini1,
and Francesco Fantozzi1
1
Department of Engineering, University of Perugia, Perugia, Italy
2
State Key Laboratory of Coal Combustion, Huazhong University of Science and Technology, Wuhan, Hubei, China
3
China-EU Institute for Clean and Renewable Energy, Huazhong University of Science and Technology, Wuhan, Hubei, China
4
SINTEF Energy Research, Trondheim, Norway
5
Inorganic Membranes and Membrane Reactors, Department of Chemical Engineering and Chemistry, Eindhoven University
of Technology, Eindhoven, the Netherlands

9.1 ­Production and Structure of Carbon Nanotubes

9.1.1 Introduction to Carbon Nanomaterials and Their Synthesis

In 1985, a new material—namely, fullerene— was discovered through unexpected results
on the mass spectra of samples of evaporated carbon (Kroto et al. 1985; Aqel et al. 2012).
Fullerenes can be defined as a type of carbon allotropes (see Figure 9.1). An allotrope is a
different form in which the carbon element can be found at the same physical state; this is
due to its ability to form many types of bonds between carbon atoms. The structure of
graphite is made by several sheets of linked hexagonal carbon rings. One single sheet is
named “graphene.” The structure of fullerenes is formed by pentagonal or heptagonal
rings. The particular structure of fullerenes prevents them from being planar. Carbon
nanotubes (CNTs) can be considered as cylindrical fullerenes.
The CNT is characterized by a nanometer‐sized diameter and a micrometer‐sized length.
The atoms are arranged in hexagons similarly to graphite. Different types of CNTs exist,
depending on the number of layers of graphene that are rolled: single‐walled carbon nano-
tubes (SWCNTs), double‐walled carbon nanotubes (DWCNTs), and multiwalled carbon
nanotubes (MWCNTs) (Jacobsen et al. 2017), see Figure 9.2.
SWCNTs are formed by the rolling of a graphene layer. The length to diameter ratio of
CNTs is usually about 1000 and more, which implies that they can be considered very simi-
lar to one‐dimensional structures. SWCNTs are a very important type of CNTs and they are
characterized by important electric properties which are not possessed by e.g. MWCNTs.
This means that SWCNTs can be very good conductors, while MWCNTs cannot. On the

Green Synthesis of Nanomaterials for Bioenergy Applications, First Edition. Edited by Neha Srivastava,
Manish Srivastava, P. K. Mishra and Vijai Kumar Gupta.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
212 9 Carbon Nanotubes for Hydrogen Purification and Storage

(a) (b) (c)

(d) (e) (f)

(g) (h)

Figure 9.1 The structure of eight carbon allotropes (a) diamond, (b) graphite, (c) lonsdaleite,
(d) C60 or bukyball, (e) C540 fullerene, (f) C70 fullerene, (g) amorphous carbon, (h) single-walled
carbon nanotube. Source: Aqel et al. (2012).

(a) (b)

Figure 9.2 Illustration of multiwalled (a) and single-walled (b) CNT. Source: Reprinted with
permission from Zhao and Fraser Stoddart (2009).
 9.1 ­Production and Structure of Carbon Nanotube 213

other hand, the production of single‐walled CNTs is still very expensive (see Collins and
Avouris 2000). SWCNTs usually are produced with a cap at both sides, represented by a half
fullerene (C60) sphere. An oxidation process can be used to remove the caps at the end and
so opening the CNT.
MWCNTs are given by multiple layers of graphene that are wrapped together; and they
usually have different diameters, depending on the number of layers. The length, diameter,
and properties of these structures are different from those of SWCNTs (Iijima and Ichihashi
1993). The distance between the different layers that compose the MWCNTs is generally
about 3.3 Å. This distance is similar to that of the layers of graphene that make up graphite
(Aqel et al. 2012). A particular type of MWCNTs are the DWCNTs, which have technical
properties and morphology that are more similar to SWCNTs than to MWCNTs.
As reported in Prasek et al. (2011), CNTs can be produced using the following five meth-
ods: (i) arc discharge; (ii) laser ablation; (iii) sono‐chemical; (iv) hydro‐thermal; and (v)
chemical vapor deposition (CVD). Each method has some advantages and disadvantages,
resulting in different final properties. CVD in particular can be performed on a hot fila-
ment, or can be water assisted or oxygen assisted; it can be based on microwave plasma,
radio frequency, thermal processes, or it can be plasma enhanced (PE).
The most important high temperature processes used to prepare CNTs are arc discharge
and laser ablation. They were the first to be developed to produce CNTs, but they are also
very difficult to scale up. Currently, the most used method is CVD. This can be performed
in different ways, as mentioned earlier, but has the advantage to operate at a temperature
lower than 800 °C. CVD is preferred nowadays because better quality CNTs can be obtained,
sensible improvements on CNTs orientation, alignment, and purity have been reached, and
the length, diameter, and density of the CNTs have improved (He et al. 2010). Other tech-
niques are in development, experimental tests have been performed on liquid pyrolysis and
other techniques. The obtained CNTs often contain many impurities, depending also on
the adopted technique. Most of the methods, together with CNTs, also produce powders,
which contain several impurities, such as carbon particles (which can be made up by
graphite, amorphous carbon, or fullerenes) and metals (e.g. iron, cobalt, molybdenum, or
nickel) that have been used as catalysts during the synthesis. It is very important to perform
some efficient purification techniques, after the synthesis of the CNTs (Kruusenberg et al.
2011). Usually purification is performed through acid treatment of the obtained CNTs
(Mubarak et al. 2011).

9.1.2 Structure of the CNTs

As discussed earlier, the structure of a CNT is usually obtained by rolling one or more gra-
phene layer to produce SWCNTs, DWCNTs, and MWCNTs. The rolling action can be per-
formed in different ways and following different directions along the graphene sheet. To
distinguish the different directions and the type of rolling, a vector, called the chiral vector,
is considered (see Figure 9.3).
The chiral vector is practically identified by two integers: “n” and “m.” First, two atoms
of carbon in the plane of the graphene sheet are chosen: one represents the origin of the
vector and the other represents the other extreme. In this way a vector C can be defined, as
in Eq. (9.1):
214 9 Carbon Nanotubes for Hydrogen Purification and Storage

Zigzag
(8.0)

Tube axis
(7.0) (8.1)
acc
(6.0) (7.1)
(5.0) (6.1) (7.2)
(4.0) (5.1) (6.2)
(3.0) (4.1) (5.2) (6.3)
(2.0) (3.1) (4.2) (5.3)
(1.0) (2.1) (3.2) (4.3) (5.4)
n ar (0.0) (1.1) (2.2) (3.3) (4.4)
Armchair

C
Θ
a1
ma2

a2 Semiconductor
Metal

Figure 9.3 The two-dimensional graphene sheet diagram. Source: Aqel et al. (2012).

C na1 ma2 (9.1)

where the aforementioned n and m are integers; while a1 and a2 represent the number
of unit cell vectors. The axis of the obtained CNT is perpendicular to the direction of the
chiral vector. The angle between C (chiral vector) and ma2 (i.e. the zig zag tube axis) is
named the chiral angle (θ). Based on this, three types of nanotube structures can be
described (Aqel et al. 2012):
1) zig zag tubes: (m = 0) and (θ = 30°),
2) armchair tubes: (m = n) and (θ = 0°),
3) chiral tube: (n m) and (0° < θ < 30°).
The values of n and m, which determine the chirality of the CNT deeply influence opti-
cal, mechanical, and electronic properties of CNTs. CNTs can be of two types, based on the
value of the absolute difference: |n‐m|. If |n‐m| = 3i the CNT is considered as metallic (e.g.
(10,10) tube), this means that the CNT is a highly conducting material. If |n‐m| = 3i ± 1 the
CNT is considered semiconducting (e.g. (10,0) tube).

9.1.3 CNT Production: Arc Discharge and Laser Ablation

Arc discharge is a high‐temperature method (temperatures higher than 1700 °C are used).
Usually, with this method CNTs with few structural defects are produced, so the number of
structural defects is lower than that obtained with other techniques. The technique is based on
 9.1 ­Production and Structure of Carbon Nanotube 215

the use of an electric arc, that is placed between two carbon electrodes, which are very closely
spaced. The distance between the two is about 1 mm. The electrodes are placed into a chamber,
which is filled with inert atmosphere composed by helium or argon (Journet and Bernier
1998). The pressure inside the chamber is about 50–700 mbar (Yan et al. 2015). A high‐tem-
perature (>3000 °C) plasma is generated by a direct current of intensity comprised between 50
and 120 A and generates a potential of about 30 V. The high temperature gradient contributes
to the evaporation of the carbon contained in the graphite anode. The vaporized carbon rap-
idly condenses on the cathode, which is at a lower temperature. In this way, CNTs are formed
together with other products. A scheme of the arc discharge system is shown in Figure 9.4.
The basic principle of the laser ablation method is similar to that of the arc discharge.
High temperature is created by a laser, instead of an electric arc, and carbon is vaporized in
this way. Laser ablation usually obtains a standardized chirality (10,10) of the CNTs.
Arc discharge and laser ablation usually produce CNTs with superior structure (Journet
and Bernier 1998), compared to CVD.
The most important characteristics of arc discharge, laser ablation, and CVD are the fol-
lowing (Yan et al. 2015):
●● Arc discharge is performed at temperatures higher than 3000 °C; pressure is comprised
between 50 and 7600 Torr; and it is difficult to scale up at industrial level.
●● Laser ablation is performed at temperatures higher than 3000 °C; pressure is comprised
between 200 and 750 Torr; SWCNTs (10,10) can be formed; and it is also difficult to scale up.
●● CVD is based on the decomposition of a precursor using a transition metal (TM) as a
catalyst; temperature is comprised between 500 and 1200 °C and pressure is comprised
between 760 and 7600 Torr; it is easier to scale up; templates can be used; on the other
hand the obtained CNTs can have poor quality.

Figure 9.4 A scheme of the arc discharge system. Source: Ando

et al. (2004).

Graphite
rod – C-soot

Deposit Graphite
rod +

+
DC arc Gas
discharge
power
source Vacuum
– pump
216 9 Carbon Nanotubes for Hydrogen Purification and Storage

9.1.4 Chemical Vapor Deposition

Catalytic chemical vapor deposition (CCVD) is now the state‐of‐the‐art method for CNT
production. It can be either thermal (Ando et al. 2004) or Plasma-Enhanced. Other tech-
niques always based on CVD are water‐assisted CVD (Smajda et al. 2009; Steiner et al.
2009; Tempel et al. 2010), oxygen‐assisted CVD (Patole et al. 2008), hot filament (HFCVD)
(Byon et al. 2007; Chen and Zhang 2011), microwave plasma (MPECVD) (Varshney et al.
2010; Kim et al. 2011), and radiofrequency chemical vapor deposition (RF‐CVD) (Brown
et al. 2011). The CNT growth model is still a subject of research. Two models have been
developed until now: the base‐growth model and the tip‐growth model (Yan et al. 2015)
(see Figure 9.5). The typology of the growth mechanism (either tip growth or base
growth) can be explained by referring to bulk diffusion, in the first case, and surface dif-
fusion, in the second case. It depends also on the interaction between metal and
support.
In the CVD process, the catalyst is used first for the decomposition of the carbon
source, then for its nucleation, so that CNTs can be formed. Usually, Transition Metals
such as iron, cobalt, or nickel are used as catalysts (Lee et al. 2010 n.d.). Dealing with the
carbon source, usually this is represented by pure hydrocarbons (e.g. methane (Palizdar
et al. 2011), ethane (Tomie et al. 2010), ethylene (Narkiewicz et al. 2010), acetylene (He
et al. 2011), xylene (Atthipalli et al. 2011; Shirazi et al. 2011), and eventually their mix-
ture (Li et al. 2010), isobutane (Santangelo et al. 2010) or ethanol (Hou et al. 2011; Yong
et al. 2011).
A particular application of the CVD method regards the production of vertically aligned
CNTs. This represents a method that can grant very high yields. Nanoporous anodized
aluminum oxide (AAO) is frequently used as a template for CNTs growth. Other common
processes to produce CNTs are liquid pyrolysis, solid state pyrolysis, flame pyrolysis, and
bottom–up assembly approach. The latter is based on the synthesis of templates, based on
aromatic macrocyclic molecules and the promotion of polymerization reactions, to increase
the length of the nanotube.

(a) (b)
CxHy
CxHy CxHy

CxHy Fe CxHy

Base-grown CNT Tip-grown CNT

CxHy CxHy
CxHy Fe CxHy

Strong substrate-catalyst interaction Weak substrate-catalyst interaction

Figure 9.5 (a) Base-growth mechanism and (b) tip-growth mechanism. Source: Ando et al. (2004).
 9.2 H2 Separation Using Carbon Nanotubes 217

9.2 ­H2 Separation Using Carbon Nanotubes

9.2.1 Introduction to H2 Separation

Currently, hydrogen can be purified through three main processes (Ockwig and Nenoff
2007):
1) Pressure swing adsorption (PSA) (Stocker et al. 1998; Sircar and Golden 2000);
2) cryogenic methods; and
3) separation through membranes (Bredesen et al. 2004; Adhikari and Fernando 2006).
The first two methods are currently at the commercial stage, but they still have some
disadvantages: they are expensive, have high energy demand, and do not guarantee suffi-
cient hydrogen purity. Membrane separation can be a competitive technology, based on its
advantages: low energy requirements, possibility to be operated continuously, and low
costs of investment.
Membranes can be made of metals (pure metals or alloys), porous carbon, various inor-
ganic oxides, pure organic polymers, and numerous types of composites (i.e. metal–organic
frameworks, cermets, etc.) (Lin 2001). All these materials perform different functions in
the separation process. The US Department of Energy (DOE) has identified six perfor-
mance targets for H2 separation. The membranes should first grant a high H2 flux rate.
They should be made by low‐cost materials and be highly durable. They should consume
reduced amount of energy and have low production costs.
Membrane targets for hydrogen separation, as reported by the US Department of Energy,
Office of Hydrogen, Fuel Cells & Infrastructure Technologies (2004), regard six key areas,
which change over time (2003–2015): costs, operating temperature, operating difference of
pressure, H2 recovery, H2 purity, and durability (see Table 9.1).
Hydrogen separation is mainly based on five mechanisms (see Figure 9.6) (Koros and
Fleming 1993; Kluiters 2004): (i) Knudson diffusion (Malek and Coppens 2003); (ii) surface
diffusion; (iii) capillary condensation; (iv) molecular sieving; and (v) solution diffusion
(Jaguste and Bhatia 1995; Noble and Stern 1995).
These mechanisms can also act in parallel. Two important parameters regulate membrane
activity in the gas separation process. One is permeance (or flux) and the other is selectivity.

Table 9.1 Membrane targets for hydrogen separation.

Property 2003 2007 2010 2015

Cost (US$/ft2) 178 150 100 <100

Operating temperature (°C) 300–600 400–700 300–600 250–500
Operating Delta P (MPa) 0.69 1.38 2.75 2.75–6.90
H2 recovery (% gas processed) 60 70 80 90
H2 purity (% dry gas) >99.99 >99.99 >99.95 99.99
Durability (years) <1 1 3 >5

Source: US Department of Energy Office of Hydrogen, Fuel Cells & Infrastructure Technologies (2004).
218 9 Carbon Nanotubes for Hydrogen Purification and Storage

Figure 9.6 Five hydrogen separation mechanisms., Source: Adapted from Ockwig and Nenoff
(2007).

The “flux” is defined as the amount of gas that passes through the membrane. It is
expressed in kilograms (or moles) per unit of surface area (generally cm2) per second.
The permeability coefficient is defined by the transport flux of gas through the mem-
brane, per unit of driving force per unit of membrane thickness. The selectivity is the
parameter that expresses the capacity of the membrane to separate (Mulder and Mulder
1996).
Based on the mechanism used for separation the carbon‐based membranes can be
divided into the following three classes:
1) molecular sieves,
2) selective surface flow (SSF™) membranes, and
3) CNT membranes.
While molecular sieves use the molecular sieving mechanism, SSF membranes use the
surface diffusion mechanism and CNT membranes use an improved Knudson diffusion
mechanism.
The molecular sieves’ advantages are that they have porosity dimensions at molecular
level (which is optimal) and high pore volume.
SSFs are used for example for hydrogen purification. In this process, SSFs work in the
opposite way, compared to other inorganic membranes. In fact, since adsorption occurs
on the side that is subjected to high‐pressure conditions, the adsorbed component partial
pressure can be low. In this case, the driving force of the adsorption ­process (and so of the
mass transfer process) is not the gradient of partial pressure but is the gradient of con-
centration of the species. For example, if SSFs are used after the methane reforming
process, hydrogen remains in the high‐pressure side, while other species pass through
the membrane. This means the purified hydrogen is already compressed and ready for
further applications. Besides this, the process has the advantage that selectivity and
capacity increase with the decrease of the temperature. In this way operational costs can
be reduced.
 9.2 H2 Separation Using Carbon Nanotubes 219

9.2.2 Carbon Membranes Production

The separation ability of carbon membranes depends on both the material chemistry and the
design of the module. If compared with polymeric membranes, carbon membranes can cost
from one to three times more, while they do not always have better performances in separa-
tion. Usually carbon‐based membrane production is based on a precursor, namely polyimide,
which has high manufacturing costs. To reduce costs, some attempts to use polyacrylonitrile
have been made, but this has led to the obtainment of low‐performance membranes (Saufi
and Ismail 2004). The precursors can be considered as organic polymers, which are converted
to pure carbon by carbonization. The carbon‐based membranes can be produced in two
forms: supported and unsupported. These two forms can also be realized with different con-
figurations. Unsupported membranes can be realized in three possible configurations: flat
(film), hollow fiber, and capillary (Ockwig and Nenoff 2007). Supported membranes can be
realized in two possible configurations: flat and tube (Ockwig and Nenoff 2007). The produc-
tion of supported membranes is performed to reduce the problem of brittleness of unsup-
ported carbon membranes. Supported membranes are usually produced by coating of the
membrane with supports made by a polymeric layer, which is usually quite thin.
Usually, six steps are necessary to produce carbon‐based membranes:
Step 1: the precursor is selected.
Step 2: a polymeric membrane is prepared.
Step 3: the precursor is pretreated.
Step 4: the pyrolysis process is performed.
Step 5: post‐treatment is performed.
Step 6: the module is constructed.
Possible substrates to be pyrolyzed as precursors are: thermosetting resin, graphite, coal,
pitch, and biomass (Liang et al. 1999). Also, synthetic precursors can be used, such as poly-
mide and derivatives, PAN (polyacrylonitrile), phenolic resins, PFA (polyfurfuryl alcohol),
PVDC‐AC (polyvinylidenechloride‐acrylate terpolymer), phenolformaldehyde, cellulose,
etc. The pretreatment of the precursor can be mechanical and physical. Oxidation can also
be used as a pretreatment. Pyrolysis is a thermal treatment in inert atmosphere or under
vacuum conditions, which produces three main products: a gas (pyrogas), a liquid (biooil),
and a solid (char or charcoal). The volatiles contained inside the precursor during pyrolysis
or carbonization are liberated by the substrate and pass through it, creating pores. During
the pyrolysis process, the temperature and the pressure should be adjusted to obtain an
optimal size of the pores. Also, the shape and connectivity of the pores have to be opti-
mized, if the final solid product has to be used to build carbon‐based membranes. Pyrolysis
final temperature is between 500 and 1000 °C (Geiszler and Koros 1996; Suda and Haraya
1997). The post‐treatment processes include (Yong et al. 2011) oxidation, CVD, post‐­
pyrolysis, and coating. Dealing with the module construction, it has to be taken into con-
sideration that to increase the rate of permeation it is preferable to use an asymmetric
structure or hollow fiber c­ onfigurations (Petersen et al. 1997).
As defined by Ismail and David (2001), carbon‐based membranes can be produced in
different configurations (see Figure 9.7). The unsupported membranes can be produced in
three configurations: flat, hollow fiber, and capillary (Ismail and David 2001).
220 9 Carbon Nanotubes for Hydrogen Purification and Storage

Figure 9.7 Scheme of the composite membrane structure. Source: Adapted from Ockwig and
Nenoff (2007).

The supported membranes can be produced in two configurations: flat and tube. Flat
membranes, both supported and unsupported, can be used as molecular‐sieving carbon
membranes (MSCMs) or as SSF™ membranes (Ismail and David 2001). SSFs are nanopo-
rous membranes that use the surface diffusion mechanism to separate mixtures of different
gases. They are composed of two layers: a thin (2–5 μm) carbon layer containing nanopores
(with an effective diameter of about 5–6 Å) and a layer containing mesopores, which is
used as inert support (made of graphite or alumina with effective pore diameter of about
0.3–1.0 μm) (Ismail and David 2001). Carbon membranes can be supported on tubes of
porous support; in this case they continue to act as molecular sieves and can increase the
selectivity of the support for gaseous species like hydrocarbons, CO2, and CH4. A capillary
module membrane is similar to a tubular membrane but, while a tubular membrane has
bigger dimensions, outer diameter of 5–25 mm, a capillary membrane has an inner diam-
eter of 0.5–5 mm and an outer diameter of 0.7–8 mm. The hollow fiber membrane generally
has even lower dimensions: outer diameter is 0.08–0.8 mm and inner diameter is 0.05–
0.5 mm (Balster 2013).

9.2.3 Hydrogen Separation Using CNT-Based Membranes

The use of CNTs in gas separation was initially studied using molecular dynamic simula-
tions (Bernardo et al. 2009). These studies considered a SWCNT with diameter of about
1 nm and predicted that the transport of gases inside it was several orders of magnitude
faster compared to other nanoporous materials, such as zeolites (Skoulidas et al. 2002;
Sokhan et al. 2002; Chen and Sholl 2006; Chen et al. 2006). The advantage of CNT‐based
membranes compared with other membranes made, for example, from ceramic materials
and characterized by atomic scale pores are the following (Sholl and Johnson 2006):
CNT‐based membranes have a smooth inner surface; the graphene plane makes them
very rigid; they have a nonpolar nature, because of the sp2‐­hybridized carbon network
(Verweij et al. 2007). Holt et al. (2006) and Hinds et al. (2004) have produced carbon
membranes based on SWCNTs and DWCNTs with diameters of 1.3–2 nm and also
MWCNTs with diameters of 6–10 nm. Diffusion tests of light gases and hydrocarbons
have shown transport rates one to two orders of magnitude larger that Knudson ­diffusion
 9.3 ­Carbon Nanotubes for Hydrogen Storag 221

prediction (Jones and Thomas 2007). Despite the very fast mass transport, the selectivity
of CNTs has to be improved. Further research is needed to decrease C fouling, while
improving flux and selectivity. Also, technical problems have to be solved, e.g. SWCNTs
to be used in membranes must be all vertically aligned. A scheme of a possible mem-
brane structure is proposed in Figure 9.7. As it can be seen, in this case the polymer is all
around the CNTs.

9.3 ­Carbon Nanotubes for Hydrogen Storage

9.3.1 Introduction to Hydrogen Storage

As already mentioned earlier in this chapter, hydrogen storage currently plays a key role in
the development of the hydrogen economy, especially in the transport sector (Schlapbach
2002; Report of the Basic Energy Science Workshop on Hydrogen Production 2003; Jones
and Thomas 2007; Read et al. 2007). The US DOE Hydrogen Plan has established a set of
targets for hydrogen reversible adsorption and storage in solid state. There are two main
targets or benchmarks: the target weight ratio of H2 with respect to the total weight (which
is set to be 6.5 wt% hydrogen) and the target volumetric density of hydrogen, with respect
to the volume of the storing solid (which is set to be about 62 kg H2 m−3). The above men-
tioned targets also have to consider that a fuel cell vehicle can require more than 3.1 kg of
H2 for a 500 km range (Dillon et al. 1997). Figure 9.8 shows that currently it is very difficult
to reach these targets (Dillon et al. 1997).
Recently, great attention has been given to the possibility of storing H2 in CNTs and other
low‐dimensional carbon materials. These materials are considered very promising because

80
C/polymer
Fibreglass/Al
Volumetric energy density (kg H2 m–3)

70
Iron cycle
Liquid H2
60 AC
SWNTs
20 Å
50 Goals
Metal 16.3 Å
hydrides
40
13.6 Å
60 MPa
30 60 MPa
40 MPa
40 MPa
20
12.2 Å
20 MPa
10 24.8 MPa 24.8 MPa
20 MPa

0
0 2 4 6 8 10 12
Gravimetric energy density (wt%)

Figure 9.8 Energy density for different H2 storage technologies to be adopted in the transport
sector. Source: Adapted with permission from Cheng et al. (2001).
222 9 Carbon Nanotubes for Hydrogen Purification and Storage

of their great porosity and pore surface area. There are about nine properties that should be
optimized to find the best solid state H2 storing material:
1) The material needs to be light in weight
2) It needs to have a reduced cost and great availability.
3) It needs to have high volumetric and gravimetric density of H2 (as reported earlier).
4) Adsorption and desorption kinetics have to be fast.
5) The material should be easily activated (it should operate below 100 °C for H2 delivery
(Niemann et al. 2008)).
6) It should have a low dissociation/decomposition temperature.
7) Appropriate activation energy is required for the adsorption/desorption process
(30–55 kJ mol−1 H2 (Niemann et al. 2008)).
8) The material should have a long term cyclability.
9) A high degree of reversibility (~1000 cycles (Niemann et al. 2008)) is required.
There are basically six methods to store hydrogen in a reversible way with a possibly high
volumetric and gravimetric density (Züttel 2014):
●● storage in gaseous form,
●● storage in liquid form,
●● physisorption,
●● metalhydride,
●● complex hydrides, and
●● chemical hydrides.
Usually the volumetric H2 density is calculated with Eq. (9.2):

V mH / V (9.2)

where ρV is the volumetric H2 density, expressed in [kg∙m−3] and calculated by dividing

the mass of hydrogen stored by the volume of the storing solid material.
The gravimetric hydrogen density can be calculated with Eq. (9.3):

m mH / m (9.3)

where ρm is the gravimetric H2 density, expressed in [mass%] and calculated by dividing

the mass of hydrogen by the mass of the storing material.
Carbon‐based materials are considered promising for hydrogen storage, because they
have many positive characteristics: they have an important volume of pores, inside
which H2 can be stored (so they have an interesting storage capacity); they have a high
specific surface area; they are a micro‐porous material; and they have low density.
Despite the numerous studies on hydrogen storage in carbonaceous materials, the real
mechanism of this process is still not clear. It may be due to Van der Waals forces of
attraction, which are at the basis of the physisorption process. Or it may be due to the
attraction between carbon molecular orbitals and the electronic wave function of the H2
electron. The overlapping of carbon highly occupied orbitals with the occupied elec-
tronic wave function of the hydrogen molecule is at the basis of the chemisorption pro-
cess. If the only mechanism responsible for binding H2 to carbon is physisorption, then
less than one hydrogen atom per two carbon atoms can be linked (this gives a mass ratio
 9.3 ­Carbon Nanotubes for Hydrogen Storag 223

between H2 and C of about 4.2). This ratio changes if chemisorption is considered and
we obtain a ratio of two hydrogen atoms per one carbon atom (Nijkamp et al. 2001;
Sudan et al. 2003; Viswanathan et al. 2003) (here the maximum mass ratio that has been
calculated through DFT is about 7.7%). The binding energy of physiosorbed H2 is nor-
mally about 0.1 eV, while the binding energy of chemisorbed H2 is the same as C─H
covalent bonds, i.e. equal to more than 2–3 eV.

9.3.2 H2 Storage in SWCNTs

The work of Dillon et al. (1997) was the first experimental campaign performing H2 adsorp-
tion in SWCNTs. To perform these tests, they have developed a particular analysis, which is
now known as temperature programmed desorption (TPD). A mass spectrometer was used
to measure the H2 released by the carbon sample, while it was heated. The procedure
entails the following steps:
Step1. The sample adsorbs H2 at room temperature and pressure of 300 Torr (which can be
approximated to 0.4 bars) then is cooled to 133 K.
Step 2. The sample then is cooled to 90 K and pressure is brought to <5 × 10–8 Torr.
Step 3. TPD is performed using a heating rate of 1 K s−1. The maximum temperature reached
during the experiment is about 500 K.
Based on the results of these tests, Dillon et al. (1997) have assumed that SWCNTs could
reach a hydrogen gravimetric storage density of around 5–10%. The optimal nanotube
diameter was identified to be about 2 nm. The main mechanism of adsorption discussed by
Dillon et al. (1997) is physisorption, the desorption peak was measured at 150 K. A second
less important peak was measured at 300 K.
Ye et al. (1999) performed H2 adsorption using laser‐generated SWNTs. The CNTs were
purified and sonicated before performing the adsorption tests. They obtained a hydrogen
adsorption rate of 8 wt% at about 40 atm and 80 K.
In the beginning, the hydrogen storage results using SWCNTs were quite optimistic
and this rose many expectations. Nevertheless, further experiments have shown that
at room temperature CNTs’ capacity to store hydrogen is not so promising (Tibbetts
et al. 2001; Hirscher et al. 2002; Ritschel et al. 2002; Kajiura et al. 2003). Nowadays,
it can be estimated that at room temperature SWCNTs, MWCNTs, and other carbona-
ceous material have a storage capacity that is lower than 1.7 wt% at the pressure of
120 bars (Liu et al. 2010). These conclusions have been drawn by considering the
trend of the data reported in different research over time; as reported in Figure 9.9.
The research clearly shows that CNTs alone cannot reach the targets identified by the
US DOE.
If CNTs are not effective alone, this means that they can be used as additives to increase
the kinetics and capacity of other hydrogen storage materials (e.g. MgH2/SWNT composite
materials (Wu et al. 2006a), NaAlH4 with CNTs as catalysts (Chen et al. 2007)). Also, doping
of SWCNTs with alkali or TMs can be an effective strategy. Doping with TMs may promote
non‐dissociative binding of hydrogen in a way that is more efficient, compared to the Van
der Waals forces. Such particular Dihydrogen complexes take the name of Kubas complexes
(Kim et al. 2006). In Kim et al. (2006), it was shown that C54Be can store 1.7 wt% of H2 in a
224 9 Carbon Nanotubes for Hydrogen Purification and Storage

16
Hydrogen storage Capacity (wt.%)

12

0
1998 2000 2002 2004 2006 2008 2010
Year

Figure 9.9 Hydrogen storage capacities of CNTs reported in the literature. Source: Liu et al. (2010).

reversible way under ambient temperature conditions. Another technique to take into
account is the storage of hydrogen as an atom. In this approach a metal (such as palladium)
is used to catalyze the dissociation of the hydrogen molecule (Lachawiec et al. 2005).

9.3.3 Hydrogen Storage in Multiwall Nanotubes

MWCNTs are extensively studied materials because the adsorption process can be per-
formed in different sites: external surface, interstitial space, and inside the channel
(Suttisawat et al. 2009; Park and Lee 2010; Reyhani et al. 2010). The first studies have
already demonstrated that MWCNTs cannot store hydrogen (Gangu et al. 2019) without
being modified through decoration or doping with metal and metal oxides (Lueking and
Yang 2002; Reyhani et al. 2009; Ravindra and Bhat 2012). The improved H2 adsorption,
which can be noted after doping, can be explained with several mechanisms such as the
creation of new adsorption sites, surface defects, spillover mechanism (dissociation, migra-
tion, and diffusion of H2), and stronger substrate–hydrogen interactions (Chang et al. 2009;
Reyhani et al. 2011; Chen et al. 2012). For an effective deposition of the metal on the CNT
surface, different methods can be used such as ball milling, reduction, impregnation, and
sputtering (Kukovecz et al. 2005; Nikitin et al. 2008). Rather and Hwang (2016) compared
H2 adsorption performances of Ti‐MWCNT composite materials using two different
mechanical processes to prepare them: sputtering and ball milling. Adsorption tests carried
our using a Sievert’s volumetric apparatus have demonstrated that by using Titanium,
adsorption of hydrogen was 0.43–2.0 wt%. These results were obtained at 298 K and 16 atm
and were respectively 5 and 25 larger than those obtained with pristine MWCNTs. In
Rather (2017), Co and Cu were used to decorate multiwalled CNTs to improve H2 storage
abilities; the hydrogen uptake was respectively about 0.8 wt% and 0.9 wt% at a temperature
of 25 °C and at the pressure of 23 atm. The retention of hydrogen increased ten times, com-
pared to the value obtained without metal oxides.
In Han and Park (2017), Ni nanoparticles were used as a dopant for MWCNTs. The basic
mechanism that promotes hydrogen storage in this case was found to be the “dissociative
chemisorption”, operated by the nickel nanoparticle.
 9.3 ­Carbon Nanotubes for Hydrogen Storag 225

The final mass percentage of stored hydrogen was about 0.87 wt% at 298 K and 100 bar.
Another interesting work is Silambarasan et al. (2017), in which MWCNTs have been
irradiated with γ‐rays at different doses: 25, 50, 100, 150, and 200 kGy. This irradiation was
proved to have physical effects on the CNTs and created several defects, improving H2 stor-
age ability. A hydrogen uptake of 1.2 wt% was obtained with MWCNTs irradiated with a
dose of 150 kGy.
Muthu et al. (2016) prepared a A‐MWCNT/h‐BN nanocomposite following these steps:
Step 1: After heating the MWCNTs at 300 °C for three hours, they were mixed with H2SO4
and HNO3 in a ratio of three to one.
Step 2: Ultrasonication was performed for six hours.
Step 3: Washing and drying was performed.
Step 4: The acid treated MWCNTs and boron nitride (h‐BN) were grounded and then mixed
in different weight ratios (0, 1, 3, and 5 wt%).
Step 5: The mixture was dispersed in a dimethylformamide (DMF) bath and kept in a ultra-
sonication bath for three hours.
Step 6: DMF was evaporated in successive steps, to obtain dried A‐MWCNT/h‐BN (see
Figure 9.10).
After hydrogenation of the nanocomposite, a H2 uptake of 1.4, 1.6, and 2.3 wt% was
measured respectively for the samples prepared with 1, 3, and 5 wt% of h‐BN. The desorp-
tion analysis shows a loss of weight equal to 2.3 wt% in the temperature range 120–410 °C.
Muthu et al. (2016) also present an interesting table (Table 9.2), showing a comparison of
hydrogen uptake from different composite materials based on MWCNTs.
As shown in Table 9.2, the best result obtained until now is that presented by Yuan et al.
(2014). They produced a composite Mg95‐Pd/MWCNT, preparing it in two steps:
Step 1: A chemical reduction method was applied to produce Pd supported MWCNTs.
Step 2: The Mg95‐Pd material was added through hydriding combustion synthesis (HCS)
and mechanical milling (MM).
The good results obtained can be explained with the synergies between magnesium and
the carbon scaffold represented by the multiwalled CNTs.
COOH

COOH
COOH

COOH
COOH

COOH

COOH COOH

COOH COOH
3:1 h-BN

COOH COOH COOH COOH

H2SO4:HNO3 Ultrasonication
COOH COOH
COOH

COOH
COOH

COOH
COOH

COOH

C
h-BN

Figure 9.10 A-MWCNT/h-BN nanocomposite preparation. Source: Muthu et al. (2016).

226 9 Carbon Nanotubes for Hydrogen Purification and Storage

Table 9.2 Hydrogen storage in different MWCNTs composites.

Composite H2 (wt%) Conditions Source

Ni‐MWCNT 0.4 Room temperature, ambient pressure Reyhani et al. (2011)

Fe‐MWCNT 0.75 Room temperature, ambient pressure Reyhani et al. (2011)
Ca‐MWCNT 1.05 Room temperature, ambient pressure Reyhani et al. (2011)
Mg‐MWCNT 1.5 300 °C Reyhani et al. (2012)
Co‐MWCNT 1.51 Room temperature, ambient pressure Reyhani et al. (2011)
MWCNT/h‐BN 2.3 100 °C Muthu et al. (2016)
PdNi/MWCNT 2.3 1.5 MPa and Room temperature Jianwei et al. (2006)
MWCNT‐TiO2 2.5 77 K and 25 bar Mishra et al. (2008)
MWCNT‐Ni 2.8 330–520 K Kim et al. (2005)
MWCNT‐DyNi2 3.5 143 K and 75.4 bar Rakhi et al. (2008)
Pt/MWCNT 3.1 298 K and 90 bar Reddy and
Ramaprabhu (2007)
Pd/Ni‐MWCNT 6.6 573 K Gao et al. (2010)
Mg‐Pd/MWCNT 6.67 473 K Yuan et al. (2014)

Source: Muthu et al. (2016).

Another possible synergy between magnesium and CNTs is that presented by Tan et al.
(2015). In their study, they prepared a Mg95Ni5‐TiO2/MWCNT composite material, which
performed an uptake of hydrogen of 5.60wt% at 373 K.

9.3.4 Some More Insights on the Spillover Mechanism

As already stated, at room temperature (300 K) and pressure no greater that 100 bars, car-
bon nanostructures can hardly store more than 2 wt% of hydrogen (Ritschel et al. 2002;
Panella et al. 2005; Jorda‐Beneyto et al. 2007; Klechikov et al. 2015). This is probably due to
the low binding energies associated with physisorption. To improve adsorption effective-
ness TMs can be mixed with CNTs either through mechanical mixing or via chemical wet
impregnation. In this case, it has been shown that the uptake of hydrogen can increase
100–300% with respect to the uptake of undoped CNTs (Zielinski et al. 2005; Wang and
Yang 2008; Huang et al. 2011; Wang et al. 2011). The key mechanism, which is at the basis
of this improvement, is called “spillover.” Spillover is defined as “the transport of an active
species adsorbed or formed on a first surface onto another surface that does not, under
these conditions, adsorb or form the active species” (Conner Jr and Falconer 1995). If we
are dealing with hydrogen storage:
●● H2 represents the active species,
●● the first surface is the surface of the TM, and
●● the second surface is the surface of the CNT.
Hydrogen spillover, as it is shown in Figure 9.11, comprises three main phases:
Molecular H2 dissociation into atomic H2. The atomic H2 is then linked to the TM
nanoparticle.
 9.3 ­Carbon Nanotubes for Hydrogen Storag 227

2 2 3
3
3

Figure 9.11 Hydrogen spillover mechanism: (1) dissociation; (2) migration; (3) diffusion. Source:
Pyle et al. (2016).

Atomic H2 migration from the TM surface to the CNT surface.

Diffusion of the atomic H2 along the surface of the carbon, until a stable C—H bond is
formed.
It is important to notice that for effective H2 uptake the C—H binding energies have to be
higher than 1/2EH2 = ~2.3 eV (Lin et al. 2008). This means that in order to adsorb signifi-
cant quantities of hydrogen on the CNT surface the leading mechanism has to be chem-
isorption instead of physisorption. Dealing with the role of TMs in the spillover mechanism
it has to be considered that some studies on catalysis have demonstrated that Pt and Pd are
able to dissociate H2 to atomic hydrogen already at room temperature (Sermon and Bond
1974; Conner et al. 1986; Teichner 1990). This is why in hydrogen storage experiments via
spillover Pd and Pt are usually used as TMs (Jain et al. 2007; Contescu et al. 2011; Vinayan
et al. 2011; Wang and Yang 2012; Geng et al. 2014; Giasafaki et al. 2014; Dibandjo et al.
2013).
In conclusion, what can be said about the spillover mechanism is that the TMs
­certainly increase hydrogen storage capabilities, compared to pure CNTs. However,
while the theoretical limit of hydrogen storage per carbon structure (like graphene) is
about 7.7%, this could be decreased by impregnating CNTs with TMs. This is because the
TM nanoparticles act in the spillover mechanism by dissociating molecular hydrogen
into atomic hydrogen but do not take an active part in binding or storing hydrogen. So,
while recent experiments introducing lattice dopants and oxygen functional groups have
reached results of 3 wt% uptake at 15 °C and 40 bar (Parambhath et al. 2011), the possibil-
ity of reaching 5.5 wt% is still to be demonstrated. Further work is required to optimize the
percentage weight of TM, TM nanoparticle size, the level of oxygen functionalization, and
the use of lattice dopants.

9.3.5 Are Nanomaterials Really Necessary for H2 Storage?

Given the high costs of production of SWCNTs a and MWCNTs, it is reasonable to ask our-
selves if there are some other carbonaceous materials that can provide the same perfor-
mance at lower cost. An example of these materials can be activated carbons. To this end,
Rossetti et al. (2015) have prepared a super‐activated carbon, which they have doped using
Cu, Ni, Pt, Pd, and Rh metals. The loading of the metals was done through two techniques:
wet impregnation (IMP) and CDV.
228 9 Carbon Nanotubes for Hydrogen Purification and Storage

Hydrogen sorption tests were performed at two main temperatures (77 K and 273 K) and
two main pressure ranges: 5–20 bar at the temperature of 77 K and 5–100 bar at the tem-
perature of 273 K. The results obtained at the temperature of 273 K, which is close to room
temperature, are reported in Table 9.3. The quantity of TM expressed in mass percentage
varies between 0.5 wt% and 2 wt%.
The tests reported by Rossetti et al. (2015) confirm that at ambient temperature activated
carbon cannot achieve the targets identified by the US DOE.
So, maybe the use of nanomaterials in hydrogen storage is of paramount importance to
obtain the identified targets. The use of carbon nanomaterials together with TMs and mag-
nesium can be interesting. See e.g. Yao et al. (2007), in which it is stated that carbon nano-
materials can be used to overcome the following two problems of magnesium when used
as a hydrogen storage material: (i) the elevated operational temperature and (ii) the slow
hydrogenation kinetics.
CNTs in this context can be useful to increase hydrogen diffusion in MgH2‐C systems
(Orimo and Fuji 2001; Kiyobayashi et al. 2004; Wu et al. 2006b). In that way, they can pro-
mote an important working temperature reduction and increase hydrogenation kinetics.
For this reason, Yao et al. (2007) prepared a MgH2‐VTi‐CNT composite were the mass frac-
tion of V and Ti is 5 wt% and the mass fraction of CNTs is another 5 wt%. Hydrogen uptake
was measured through a Sieverts apparatus. Three different adsorption temperatures were
tested: 300, 200, and 150 °C under the pressure of 20 bar. The hydrogenation process was
assumed to be realized in three steps:
Step 1: Dissociation of the molecule of H2 on the Mg surface.
Step 2: Diffusion of atomic H2 along the boundaries of the Mg grain, facilitated by CNTs (in
this phase the CNTs help to produce more hydrides).

Table 9.3 Hydrogen storage (wt%) in activated carbon doped with different transition metals.

Sample 5.9 bar 20.6 bar 40.2 bar 98.8 bar

AC 0.11 ± 0.02 0.26 ± 0.05 0.51 ± 0.09 0.82 ± 0.04

Cu0.5IMP 0.29 ± 0.06 0.56 ± 0.10 1.29 ± 0.06 1.63 ± 0.09
Ni0.5IMP 0.13 ± 0.04 0.51 ± 0.07 1.33 ± 0.05 2.63 ± 0.06
Cu2.0IMP 0.11 ± 0.02 0.50 ± 0.05 0.79 ± 0.06 1.18 ± 0.17
Ni2.0IMP 0.09 ± 0.07 0.43 ± 0.05 0.69 ± 0.04 0.99 ± 0.03
Pt0.5IMP 0.13 ± 0.02 0.43 ± 0.08 0.62 ± 0.10 0.8 ± 0.2
Pd0.5IMP 0.21 ± 0.00 0.461 ± 0.011 0.69 ± 0.02 1.5 ± 0.5
Rh0.5IMP 0.0501 ± 0.0012 0.192 ± 0.012 0.450 ± 0.010 0.67 ± 0.05
Cu0.5CVD 0.41 ± 0.08 0.72 ± 0.13 1.36 ± 0.14 2.040 ± 0.010
Ni0.5CVD 0.15 ± 0.04 0.58 ± 0.04 1.25 ± 0.14 2.69 ± 0.05
Pt0.5CVD 0.060 ± 0.011 0.29 ± 0.02 0.472 ± 0.002 0.68 ± 0.02
Pd0.5CVD 0.11 ± 0.09 0.171 ± 0.011 0.38 ± 0.02 0.67 ± 0.05
Rh0.5CVD 0.062 ± 0.015 0.23 ± 0.05 0.52 ± 0.05 0.74 ± 0.05

Source: Rossetti et al. (2015).

 9.3 ­Carbon Nanotubes for Hydrogen Storag 229

Step 3: V hydrides formation and subsequent dissociation of the H2 molecule into H2 atoms,
which are adsorbed by magnesium atoms forming MgH2 around the V site.
Figure 9.12 shows the behavior of different composite materials based on magnesium. It
is shown that the best performance at 150 °C is that of MgH2‐VTi‐CNTs. The performance
is very close to the required parameters of the US DOE.
So, we conclude that given their costs, CNTs may not be the main material to realize
hydrogen storage but that they could be important catalysts to increase the performance of
other cheaper materials like magnesium or other hydrides.

9.3.6 CNTs’ Influence on Hydrogen Storage Performance of Hydrides

Given what has been said in the previous paragraphs, it appears interesting to consider the
catalytic action of CNTs on H2 storage process when hydrides are used. Given that CNTs
contain delocalized electrons, due to the sp2 hybridization and the fact that one C atom is
linked with three other C atoms, CNTs can have an important catalytic effect on various
hydrogen storage materials such as Mg, boro‐hydrides, ammonia borane, alanates, and
metal‐N‐H systems. CNTs can also act as carbon scaffolds for the above mentioned materi-
als (Wu and Cheng 2010). Hydrides can be classified into three main types:
1) Conventional metal hydrides (e.g. LaNi5H6 or MgH2).
2) Complex metal hydrides (e.g. NaAlH4 or LiBH4).
3) Chemical hydrides (e.g. NH3BH3).
The positive effect of CNTs on the first two classes of hydrides can be synthesized into:
increase of hydrogen storage capacity and increase of hydrogenation and dehydrogenation
kinetics. The chemical hydrides benefit most from CNTs when they are used as carbon
scaffolds.

7
US DOE
6
IEA
5 MgH2-VTi-CNTS
Capacity, wt%

4
MgH2-VTi
3
MgH2-FeTi
2
MgH2-V
1 MgH2-FeTi-CNTs
MgH2-CNTs
0 MgH2

0.0 0.2 0.4 0.6 0.8 1.0

Hydrogenation rate, wt% per minute

Figure 9.12 Maximum capacity and average hydrogenation kinetics of different magnesium-
based nanocomposites at 150 °C. Source: Yao et al. (2007).
230 9 Carbon Nanotubes for Hydrogen Purification and Storage

4.5

4.0
H-removal energy (eV)

3.5 graphene
(4,4)
3.0 (5,5)
4.0 (8,0) C60
2.5
∆E (eV)

3.0

2.0 2.0
(5,0)
1.0
0 1 2 3 4
1.5 q-EA

1.0
0 1 2 3 4
electron affinity (eV)

Figure 9.13 Correlation between carbon electron affinity and hydrogen removal energy. Source:
Berseth et al. (2009).

A particularly interesting aspect is how the carbon materials influence the kinetics of H2
release from hydrides. In Berseth et al. (2009), NaAlH4 crystals were prepared to under-
stand, through modeling and experimental tests, how NaAlH4 clusters interact with carbon
materials. It was found, as shown in Figure 9.14, that 3.8 eV are required to remove a H
atom from the NaAlH4 cluster, while, when the NaAlH4 cluster is supported respectively
on (5,0) SWCNT and C60, only 2.0 eV and 2.85 eV are needed. The electron affinity increases
with the curvature of the carbon catalyst. So, basically, the carbon catalyst can weaken the
Al─H bond and decrease dehydrogenation temperature.
Unfortunately, what is valid for complex metal hydrides is not valid for metal hydrides. In
fact, the interaction between C and Mg is not strong enough to dissociate the H2 molecule,
which is linked with Mg (see Figure 9.14). The use of CNTs as catalysts improves the hydrogen
storage capacity of Mg, acting on the diffusion mechanism, but it does not have a big effect on
hydrogen molecule dissociation. To improve this aspect, CNTs have to be used together with
TMs and metal oxides. These can effectively decrease the hydrogen dissociation energy.
In this way, a similar mechanism to that shown in Yao et al. (2007) can be reproduced.

9.4 ­Conclusion

In this chapter, different types of CNTs are described: SWCNTs, DWCNTs, and MWCNTs.
Attention is focused on their structure and properties. Also, the most frequently used pro-
duction techniques are described: CVD, arc discharge, and laser ablation. The most impor-
tant properties and functioning principle of molecular sieves, SSF membranes, and CNTs
membranes are described. While the separating action of molecular sieves is mainly based
on the molecular sieving mechanism, SSFs basically use the surface diffusion mechanism
and CNT‐based membranes use an improved Knudson diffusion mechanism. It has to be
taken into account that molecular dynamic simulations have demonstrated that transport
of gases inside SWCNTs with diameter of about 1 nm happens with orders of magnitude
 ­Reference 231

1.2
1.0 Pure Mg
0.8
0.6
Relative Energy (eV)

0.4
0.2
0.0
–0.2
C-incorporated Mg
–0.4
–0.6 Ti-incorporated Mg
–0.8
–1.0
–1.2
0 5 10 15 20 25 30
Image Number Index

Figure 9.14 H2 dissociation at the Mg surface: energy barrier and energy pathway for pure
magnesium and C-incorporated magnesium. Source: Yao et al. (2006).

faster than any other material with nanoscale pores (e.g. zeolites). Despite this advantage,
an improvement in the selectivity of the CNT‐based membranes is needed. Dealing with
hydrogen storage in CNTs, it has been demonstrated that at room temperature SWCNTs,
MWCNTs, and other carbonaceous materials have a storage capacity lower than 1.7 wt% at
pressure of 12 MPa. This clearly indicates that CNTs alone cannot reach the benchmarks
set by the US DOE. Possible solutions could be:
Doping the CNTs with TMs, to obtain a higher hydrogen spill‐over effect.
Using CNTs to catalyze the hydrogen storage activity of metal hydrides and complex
hydrides.
Effective strategies to increase the performance of CNTs are doping with TMs, to obtain
the hydrogen spillover effect, and also the use of CNTs to catalyze the hydrogen storage
activity of metal hydrides and complex hydrides.

­Acknowledgments
The authors acknowledge the financial support from the Research Council of Norway and
a number of industrial partners through the project BioCarb+ (“Enabling the biocarbon
value chain for energy”).

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 239

Index

Note: Page references followed by ‘f’ refer to Figures; those followed by ‘t’ refer to Tables

a Aspergillus flavus 131t

Acacia nilotica 151f Aspergillus fumigatus 110, 131t
biosynthesis of silver nanoparticles Aspergillus oryzae 175, 192
from 150–2 Aspergillus sp. 36
collection of plant material 150–1 atomic force microscopy (AFM)
preparation of leaf extract of 151 42, 56, 57f
preparation of plant materials 151 attrition 132
Achillea biebersteinii 108t auger electron spectra (AES) 91
actinomycetes 130
Agathosma betulina 51f b
algae 110, 130–1 Bacillus cereus 131t
algae‐mediated synthesis 131 Bacillus licheniformis 131t
algal biomass 147 Bacillus methylotrophicus 36
allotrope 211 Bacillus mycoides 36
Amaranthus polygonoides 37 Bacillus subtilis 36
amorphous carbon 212f bacteria 130
A‐MWCNT/h‐BN nanocomposite bacterial‐induced synthesis 109
preparation 225f baking industry 168–9t
anaerobic digestion 6–8 ball milling process for nanomaterial
Andrographis paniculata 109t fabrication 29
animal feeds industry 170t Beer’s law 46
annual global biomass production 1 BET analysis 92, 94
annual photosynthesis yield 1 biocatalyst 200
anodized aluminum oxide (AAO) 216 biochemical conversion technologies
applications of nanoparticles 197, 198f (BCCTs) 2, 3t, 4, 6
applications of nanotechnology 83, biodiesel 2, 9–12, 13–14t, 147–8
84f,198f bioenergy 148, 199
arc discharge 84, 213, 215–16 bioethanol 2, 6, 148, 199

Green Synthesis of Nanomaterials for Bioenergy Applications, First Edition. Edited by Neha Srivastava,
Manish Srivastava, P. K. Mishra and Vijai Kumar Gupta.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
240 Index

biofuel energy 147–50 capillary membranes 219

biofuels 1–15, 199 Capparis deciduas 108t
production 1, 2–4, 3f, 10–11t, 197–9 carbon allotropes 211, 212f
biogas 7t, 199 carbon‐based membrane structure 220f
biological reduction 36–7, 37f carbon black 85
biomass as power source supply 148 carbon nanomaterials 84‐5, 211–13
biomass conversion, role of catalysts in carbon nanotubes (CNTs) 25, 84–5, 127
4–5, 4t H2 separation using 217–21
biomass feedstocks 1 in hydrogen storage 221–30, 224f
biomineralization 109 membranes 218, 219–20
biomolecule‐directed synthesis of production 215–16
nanomaterials 105f structure 213–14
biomolecule–nanoparticle hybrid catalytic chemical vapor deposition
systems 112 (CCVD) 216
bio‐oil 199 cellulosic ethanol 5
biopolymers 129 cerium dioxide (CeO2) nanoparticles 128
biosensors 29 cetyltrimethylammonium bromide
biosynthesis 27, 90, 94 (CTAB) 101
nanomaterials 193–7 characterization of nanomaterials 42–64,
of silver nanoparticles from Acacia 91–94, 92t, 93f
nilotica 150–7 composition 93
bottom–up approach 32–42, 28f, 88–9, 99, concentrations 94
100f, 133, 134f, 147, 195, 216 crystallography 94
bovine serum albumin‐Cu (II) hybrid silver nanoparticles (AgNPs) 152
nanoflowers (BSA‐NFs) 181 size 92
Bragg angle 48 surface area 92–3
Bragg’s law 47 surface charge 94
Brevibacterium casei 131t surface morphology 93–4
brewing industry 172–3t chemical conversion technologies
Brownian motion 63 (CCTs) 2, 4
Brunauer–Emmett–Teller Physisorption chemical precipitation 28
(BET) method 63 chemical properties of nanomaterials 88
Brust–Schiffrin two‐phase process 35 chemical reduction 34–6, 35f, 132
buckyballs 127, 212f chemical vapor deposition (CVD) 28,
32–3, 33f, 84, 89, 134, 135, 213, 215,
c 216, 219
cadmium sulfide nanoparticles Chlorococcum humicola 36
(CdSNPs) 128 Citrus limon 108t
CaHPO4‐α‐amylase NFs 180, 183 classification of nanomaterials 23, 84–7,
Calophyllum tomentosum 109t 85f, 126–9, 193f
Camellia sinensis 108t, 181 based on materials 84–5
Candida sp. 36 basis of dimension 86–7, 86f
Candida utilis 131t composite‐based nanomaterials 86
 Index 241

condensation particle counter (CPC) 94 electrospinning 27, 30–1, 31f

Cooper pairs 59 energy dispersive X‐ray (EDX)
co‐precipitation 40–2 spectroscopy 51, 52f, 152, 154–6
Coriandrum sativum 37 Enterobacter sp. 131t
Corynebacterium glutamicum 131t Enterococcus faecalis 36
cosmetics industry 171t environmental scanning electron
covalent bonding of enzyme 202, 202f microscopy (ESM) 54
crosslinked enzyme crystals (CLECs) enzyme immobilization 191–205
203 conventional 177f
crosslinked enzymes (CLEs) 203 enzymes in biofuel production
Croton caudatus 108t 204–5
merits 204
d methods 201–204
dairy industry 168t adsorption 201–2
definition of nanomaterial 145 benefits and drawbacks 179t
dendrimers 127 copolymerization
Desulfovibrio desulfuricans 131t (crosslinking) 202–3
diamond 212f covalent binding 202
differential mobility analyzer (DMA) 93 entrapment and encapsulation 203
differential scanning calorimetry (DSC) technology 177f
61, 62 enzyme immobilization, nanomaterials
dimensionality‐confined nanomaterials 197–9
23–6, 24f adsorption of 201f
one‐dimensional nanomaterials 25–6, conventional methods 174–8
86, 192 chemical methods 178
three‐dimensional nanomaterials 26, physical methods 176–8
87, 192 covalent bonding of 202f
two‐dimensional nanomaterials 26, crosslinking (copolymerization)
87, 192 of 203f
zero‐dimensional nanomaterials 24–5, economic importance of industrial
86, 192 enzymes 167
dissociative chemisorption 224 encapsulation and entrapment 204f
Dodonaea viscosa 108t enzyme classification 165–6
dopamine‐PEG‐COOH 101 enzyme engineering 167–73
double‐walled carbon nanotubes enzyme sources and their application
(DWCNTs) 211–13, 220 fields 166
dynamic light scattering (DLS) 63–4 global enzyme demand 173–4, 173f
matrix for immobilization 200–1, 200t
e inorganic support 201
electrical properties 87, 194t natural polymers 200–201
electrochemical storage systems 138 synthetic polymers 201
electrochemical synthesis 136 new generation methods 178–84,
electron probe microanalysis (EPMA) 91 180–2f
242 Index

enzyme incorporated hybrid nanoflowers graphite 212f

182–4 green chemistry principles 135t
enzymes, industrial applications green nanomaterials
of 168–72t green synthesis of 132–8, 146–7
Eryngium campestre 37, 43, 46, 47f, 55 natural sources for 129–32
Escherichia coli 36, 131t green nanotechnology principles 135t
etch mask patterning 29
Eucalyptus globulus 108t h
extended X‐ray absorption fine structure Heriteira fomes 109t
(EXAFS) 91 heterogeneous catalysts 4t
Hevea brasiliensis 108t
f Hibiscus rosa sinensis 37
fabric sciences 148 high‐resolution transmission electron
fatty acid ethyl esters (FAEE) 9 microscopy (HRTEM) 56
fatty acid methyl esters (FAME) 9, 113 high‐temperature nanomaterial
Fermi level energy 101 biosynthesis 196–7, 197f
field‐cooled (FC) magnetization hollow fiber membranes 219
measurement 59 hollow spheres 86
field emission scanning electron homogeneous catalysts 4t
microscopy (FESEM) 54, 55f horseradish peroxidase (HRP)
flame‐assisted ultrasonic spray pyrolysis enzyme 180
197, 198f hot filament (HFCVD) 216
flame pyrolysis 216 hybrid nanoflower (HNF) 180, 180f,
flat membranes 219, 220 182–3
Fourier decomposition 63 hybrid nanomaterials 26–27
Fourier transform infrared (FT‐IR) hybrid organic–inorganic nanocomposites
spectrometer 42–3, 44f 26–27
Fourier transform infrared spectroscopy hydrazine 132
152, 154, 155f hydrazine hydrate 35
Froriepia subpinnata 37, 46, 47f, 55 hydrogen storage, carbon nanotubes in
fuel cells 138 221–30
Fullerenes 84, 127, 211, 212f in activated carbon doped with different
fungi 110, 130 transition metals 228t
Fusarium oxysporum 110, 131t hydrogen separation mechanisms
Fusarium sp. 36 217t, 218f
influence on hydrogen storage
g performance of hydrides 229–30
gallium nitride (GaN) nanowires 26 in multiwall nanotubes 224–6
gasification 8 in MWCNTs composites 226t
Gibbs free energy 101 need for nanomaterials in 227–9
gold (Au) nanoparticles (AuNPs) 107, spillover mechanism 226–7, 227f
112, 128 in SWCNTs 223–4
graphene 26, 84 hydrolases 166
 Index 243

hydrophilic nanomaterials, synthesis of liquid crystals 127

102–13 liquid mix process 195
hydrophobic nanomaterials, phase liquid pyrolysis 216
transfer into aqueous Litchi chinensis 109t
solutions 102, 102f localized surface plasmon resonance
hydrothermal synthesis 134, 213 (LSPR) 106
3‐hydroxybutyrate‐co‐hydroxyvalerate lonsdaleite 212f
201–2 low energy electron diffraction
(LEED) 91
i lyases 166
immobilization 191–92
inorganic‐based nanomaterials 85 m
inorganic nanoparticles 27 magnetic force microscopy (MFM) 42,
interferometer 43 59–61
isomerases 166 magnetic MgO/MgFe2O4, 9
magnetic nanoparticles (MNPs) 112–13
j magnetic properties 87, 1944t
Jatropha curcas 37 magnetite nanoparticles (Fe3O4 NPs)
juice industry 169t 6, 40
magnetosome 109
k magnetotactic bacteria 107, 109
Knudson diffusion 218, 221 mass spectrometry ion
Kubas complexes 223 chromatography 93
mechanical grinding 193–4
l mechanical milling 28–9, 90, 195f
Lactobacillus acidophilus 36 mechanical properties 87, 194t
LaMer theory 101, 101f membrane separation 217
laser ablation 29–30, 84, 91, 136, 195, 213, Memecylon edule 37
215–16 metal nanoparticles 128
laser‐by‐laser method 27 formation on graphene oxide 103, 104
laser diffraction technique 92 metal oxide nanoparticles (MO‐NPs)
laundry detergents 168t 107–11
leather industry 171t metallic hybrid nanoparticles 111–12
Leucasaspera 108t metallic nanoparticle synthesis 130, 132,
ligases 166 133f, 134f
light emitting diodes (LEDs) 138 methoxy‐polyethylene glycol 132
lignocellulosic biomass 149 microorganisms 110, 129
lignocellulosic biomass feedstocks microwave‐assisted techniques in
(LCB) 5 nanoparticle synthesis 136–7
lignocellulosic feedstocks 2 microwave plasma (MPECVD) 216
lipase immobilized functionalized Mimosa pudica 37
magnetic nanocatalyst 12 molecular electronics 29
lipid deoxyribonucleic acid (DNA) 101 molecular sieves 218
244 Index

molecular‐sieving carbon membranes Nephelium lappaceum L., 108t

(MSCMs) 220 Neurospora crassa 131t
monochrometer 42, 43 nuclear magnetic resonance spectroscopy
Morinda pubescens‐blended AgNPs 154 (NMR) 51–3, 53f, 93
multiwalled carbon nanotubes (MWCNTs)
211–13, 212f, 220, 223 o
Ocimum santcum 154
n octadecyl‐sepabeads 201
nano CaO 12 octyl‐agarose 201
nano composites 105f octyl‐sepharose CL‐4B resin 177
nano lenses 86 one‐dimensional nanomaterials 25–6,
nanocatalyst synthesis 15f 86, 192
nanocatalysts, application of 5–12 optical properties 87, 194t
biomass pretreatment 5–6 organic nanoemulsions 26
biochemical conversion methods 6–8 organic nanomaterials 26–7, 85
thermochemical conversion organic synthesis industry 170–1t
methods 8–9 oxidoreductases 166
nanoclusters 24–5 oxygen‐assisted CVD 216
nanocoating 87
nanocomposites 87 p
nanocrystalline films 195 Papaver somniferum 37
nanofibers 86, 87 paper industry 168t
nanofilms 87 paramagnetic‐based nanocatalysts 6
nanofilteration 191 PEG‐phospholipids 101
nanoflowers (NFs) 180, 181 Penicillium sp. 36
nanoimprint lithography 29 Persea americana 108t
nanoimprinting 29 Phaenerochaete chrysosporium 131t
nanolayers 87 pharmaceutical industry 172t
nanolithography 90 phenyl‐sepharose CL‐4B resin 177
nanoparticles 24, 39 Phormidium tenue 36
size 83 photocatalysis 136, 137
synthesis of 138, 139f, 147 photolysis 194–5
nanorods 39, 86 photon correlation spectroscopy 92
nanoscale 24 photonics 29
nanosheets 26, 39 photoreduction 36
nanosnowballs (NSBs) 182 physical properties 87
nanospheres 39 physical vapor deposition 33, 34f
nano‐SQUID 59 phyto‐synthesis of nanomaterials 36
nanotechnology application range 139f Pichia pastoris 36
nanotechnology chart ruler 128f Piper nigrum 109t
nanotubes 25, 39, 86 Pisonia grandis 108t
nanowaste generation 15f plant extract‐directed metal oxides
nanowires 25–6 107–11
 Index 245

plant‐mediated synthesis of metal Raman scattering spectroscopy 43–6

nanoparticles 133, 133f ZnO nanoparticles 45, 45f
plasmonic nanoparticles (PNPs) 103–7, Rhizophora mucronate 108t
108t Rhodococcus sp. 131t
Plectonema boryanum 131t Rhodopseudomonas capsulata 131t
poly(analine) 101 Rhodotorula sp. 36
poly(ethylene glycol (PEG) diacrylates
(PEGDA) 29 s
poly(ethylene glycol (PEG) dimethacrylates Saccharomyces boulardii 36
(PEGDMA) 29 Saccharomyces cerevisiae 36, 110
poly(ethylene imine) 102 salinized molecular sieve 201
poly(glycolic acid) 102 Salvia officinalis 109t
poly(lactic acid) 102 Sansevieria raxburghiana 108t
poly(methylidene malonate) 101–102 Sargassum wightii 36, 131t
poly(pyrrole) 102 scanning electron microscopy (SEM) 42,
polyacrylonitrile 219 53–4, 91, 92, 94, 152, 157, 158f
polyethylene glycol (PEG) block scanning mobility particle size
copolymer 101 (SMPS) 92
Polygala tenuifolia 108t Scherrer equation 48
polyhydroxybutyrate 1, 4‐butenediol secondary ion mass spectroscopy
di‐glycidyl 202f (SIMS) 91
polyimide 219 selective surface flow (SSF™)
polyvinyl pyrrolidone 132 membranes 218, 220
potassium bitartrate 132 self‐assembly method 27
powder formation 195 Shewanella algae 132t
pressure swing adsorption (PSA) 217 Shewanella oneidensis 132t
primary nanocrystals (Cu3(PO4)2) 183 Shewanella sp. 132t
properties of nanomaterials 87–8, 193, 194t silica‐coated magnetic nanoparticles
Prunus domestica 109t (SiM NPs) 6
Pseudomonas aeruginosa 131t, 158 silver nanoparticles (AgNPs) 106–7, 128,
Pseudomonas cepacia 113 145–7
Pteristriparita 108t absorption spectra of 154f
Pt‐nanoparticles (PtNPs) 128 activity on microorganisms 146
pulp industry 168t antimicrobial mechanism 111f
Pyrobaculum Islandicum 131t biosynthesis from Acacia
pyrolysis 8–9, 90, 132, 134, 219 nilotica 150–7
characterization of 152
q EDX graph 157f
quantum dots 25, 86, 112 FT‐IR spectra of 155f
green synthesis of 151–2
r single‐walled carbon nanotubes (SWCNTs)
radiofrequency chemical vapor deposition 211–13, 212f, 220, 221
(RF‐CVD) 216 size of nanomaterials 192, 192t
246 Index

sodium ascorbate 35 Terminalia catappa 109t

sodium borohydride 35, 132 Terminalia mellueri 109t
sodium citrate 35 tetraethoxysilane (TEOS) 102
sodium dodecyl benzyl sulfate 132 textile industry 168t
sol‐gel 28, 40, 41f, 88–9, 134, 136, thermal decomposition 34, 35f, 91
195–6, 197f thermal vaporization 195
solid state pyrolysis 216 thermochemical conversion technologies
solvothermal method 39–40, 136 (TCCTs) 2, 3t, 4, 6, 8
Sonneratiaa petala 109t thermogravimetric analysis (TGA) 61–2
sono‐chemical methods 136, 137–8, 213 thermolysis 194–5
sonolysis 194–5 three‐dimensional nanomaterials 26,
spillover mechanism 226–7, 227f 87, 192
spinneret 31 Ti‐MWCNT composite materials 224
spinning 89 Tinospora cordifolia 153
spinning disc reactor (SDR) 89 titanium dioxide nanoparticles 110
Spirulina platensis 36 titanium dioxide nanotube 6, 127
spray drying 37–8, 38f top–down method 28–31, 28f, 90–91, 99,
spray hydrolysis 135–6 100f, 133, 134, 134f, 147, 195
spray pyrolysis 39 Trachyspermum ammi 37
sputtering 91, 195 transferases 166
starch industry 169–70t transmetalation 194
step and flash imprint lithography (S‐FIL) transmission electron microscopy (TEM)
29, 30f 42, 54–5, 55f, 91, 92, 94, 152, 157,
Stokes–Einstein equation 63 159f
Streptacidiphilus durhamensis 64, 65f Trianthema portulacastrum 109t
sugar industry 169–70t Trichoderma sp. 36
superconducting quantum interference Trichoderma viride 132t
device (SQUID) 42, 57, 58–9 Trifolium resupinatum 108t
superconductor–insulator–superconductor Tropaeolum majus 108t
(SIS) Josephson tunnel two‐dimensional graphene sheet
junction 59 diagram 214f
superparamagnetic nanoparticles 126–7 two‐dimensional nanomaterials
surface‐modified nanoparticles 26, 87, 192
138, 139f
surface plasmon reverberation u
(SPR) 153–4 ultrasonic irradiation 136, 137
Syzygium aromaticum 37 ultraviolet‐visible spectroscopy 46, 47f,
152, 153
t Ureibacillus thermosphaericus 132t
temperature programmed desorption
(TPD) 222 v
Terminalia bellerica 109t vegetable oil 129
Terminalia bentazoe 109t Verticillium luteoalbum 132t
 Index 247

Verticillium sp. 132t y

vibrating sample magnetometer Yarrowia lipolytica 36, 132t
(VSM) 42, 57, 58f yeast‐based biosynthesis processes 130, 132t
Viburnum opulus (VO) 108t, 184
viruses 130 z
vitamin B 129 Zea mays 108t
vitamin C 129 zero‐dimensional nanomaterials 24–5,
vitamin E 129 86, 192
zero‐field‐cooled magnetization (ZFC)
w 59, 60f
waste management 171–2t zeta‐potential (ξ‐potential) 64, 65f
water‐assisted CVD 216 zeta potentiometer 94
Wheeler equation 63 zinc oxide (ZnO) nanoparticles 45, 45f,
128, 137–8
x zinc oxide (ZnO) nanowire 26
X’Pert HighScore plus software 48 Zingiber officinale 108t
X‐ray diffraction 46–8, 48f, 94 zirconium dioxide (ZrO2)
X‐ray fluorescence 48–9, 49f nanoparticles 129
X‐ray photoelectron spectroscopy (XPS) zirconium‐metal organic frameworks
49–51, 50f, 51f, 93 (Zr‐MOFs) 177
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