Process Safety and Environmental Protection 146 (2021) 190–200

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Process Safety and Environmental Protection

journal homepage: www.elsevier.com/locate/psep

Bio-ethanol production: A route to sustainability of fuels using

bio-based heterogeneous catalyst derived from waste
Minakshi Gohain a,∗ , Maskura Hasin a , Khalifa S.H. Eldiehy b,c , Pritam Bardhan b ,
Khairujjaman Laskar d , Hridoyjit Phukon e,f , Manabendra Mandal b , Dipul Kalita e,f ,
Dhanapati Deka a
a
Department of Energy, Tezpur University, 784028, Napaam, India
b
Department of Molecular Biology and Biotechnology, Tezpur University, 784028, Napaam, India
c
Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, 71524, Assiut Branch, Egypt
d
Golaghat Engineering College, Golaghat, 785702, Assam, India
e
Cellulose Pulp and Paper Group (Material Sciences and Technology Division), North East Institute of Science and Technology, Jorhat, 785006, Assam, India
f
Academy of Scientific and Innovative Research, CSIR-NEIST Campus, India

a r t i c l e i n f o a b s t r a c t

Article history: Microalgae have been accepted as a potential feedstock for biofuel production due to their high oil content
Received 6 July 2020 and rapid biomass production. In this study, deoiled Scenedesmus obliquus (SO) was used for evaluating
Received in revised form 6 August 2020 whether deoiled algal biomass residue is potential as an alternative energy resource for bio-ethanol
Accepted 31 August 2020
production with different heterogeneous catalysts. The SO biomass was examined for its physiochemical
Available online 5 September 2020
properties and also evaluated using FTIR, XRD, and TGA techniques. The successful hydrolysis of SO was
performed employing different eco-friendly bio-based heterogeneous catalysts and hydrolysate thus
Keywords:
obtained was then subjected to fermentation using Saccharomyces cerevisiaeand was analyzed through
Biomass
Scenedesmus obliquus
HPLC and GC which resulted in the production of bio-ethanol with the highest yield of 68.32 % at 8.24 g/L
Heterogeneous catalysts concentration.
Saccharomyces cerevisiae © 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Bio-ethanol

1. Introduction and Vassileva, 2016). The high carbohydrate content, fast growth
rate along with no competition with the conventional plant crops
Countries all around the globe are primarily concerned with and simpler processing steps in comparison with the lignocellulosic
energy security and climate change. Furthermore, the over-reliance biomass make microalgal biomass the most sought after biomass
on conventional sources of fuel as coal and crude oil adds to the for alternative energy source (Phwan et al., 2018; Tan et al., 2019;
issues of air pollution and depletion of non-renewable resources. Martín-Juárez et al., 2017). Microalgae being composed of car-
For reducing our dependency on fossil fuels, an alternate energy bohydrates (5–23 %), lipids (7–23 %) and proteins (6–52 %) can
source i.e. bio-ethanol has been adopted as the most suitable be treated as model feedstock for commercially significant prod-
liquid transportation fuel (Bardhan et al., 2019a). Moreover, bio- ucts that can be used in agriculture, cosmetic, and pharmaceutics
ethanol can either be blended with existing gasoline fuel or used (Chandra et al., 2019, 2014). Various microalgal species has been
as pure-ethanol to limit the exhaustion of pollutants. However, utilized for biodiesel and bio-oil production (Hu et al., 2020; Wang
the predominant usage of terrestrial crops such as maize and sug- et al., 2019a; Silitonga et al., 2017). Scenedesmus sp. has been exten-
arcane for producing bio-ethanol have garnered public criticism sively studied for fatty acid methyl esters (FAME) synthesis for its
owing to food security and declining arable land area concern ability to accumulate larger quantities of lipids. Apart from lipids,
(Ge et al., 2011). In this context, non-food feedstock, particularly Scenedesmus sp. also contains carbohydrates as cellulose and starch
microalgal biomass is among the most potential alternative renew- (Sivaramakrishnan and Incharoensakdi, 2018).
able feedstock for third-generation biofuel production (Vassilev The algal biodiesel industry produces surplus amount of deoiled
microalgal biomass as a by-product that is economically feasible
to be utilized as a substrate for bioethanol production. Deoiled
∗ Corresponding author. microalgal biomass contains carbohydrates, proteins and miner-
E-mail addresses: [email protected], min [email protected] als as the molecular composition that is suitable as a feedstock for
(M. Gohain).

https://doi.org/10.1016/j.psep.2020.08.046
0957-5820/© 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
 M. Gohain et al. / Process Safety and Environmental Protection 146 (2021) 190–200 191

producing bio-ethanol (Maurya et al., 2016). Baker’s yeast or Sac- Gohain and Deka, 2018; Gohain et al., 2020a, b; Gohain et al., 2020c)
charomyces cerevisiae is used commonly for bio-ethanol production without any pre-treatment step for bio-ethanol production. The
due to high yield and ethanol tolerance. However, fermentation of CBPA, CWH, CCPS, CTGL has been prepared without any chemi-
only simple sugars into bio-ethanol can be done by Saccharomyces cal functionalization, hence making them eco-friendly and green
cerevisiae. Thus, a pre-treatment step (either acid/alkali or enzy- in nature. These bio-based catalysts have been efficiently utilized
matic hydrolysis) is included to convert the cellulosic material of for biodiesel production. Hence, herein we focus on production of
the microalgae into simple sugars. bio-ethanol from Scenedesmus obliquus deoiled biomass (SO). For
Studies are reported with various individual pre-treatment the suitability of bio-ethanol production, the biomass properties
methods like physical (microwave, sonication, high pressure, of SO including calorific value, physico-chemical characteristics
hydrothermal), chemical (acid, base) and enzymatic (amylase, have been evaluated. We also report bio-ethanol production by
pectinase, cellulases, mixture of enzymes, etc.), that primarily assist an indigenously isolated yeast strain (Saccharomyces cerevisiae iso-
breakdown of the cell structure and allows extraction and struc- late BY01) from traditional rice beer starter culture of Assam. Thus,
tural modification of carbohydrate moieties (Phwan et al., 2018; the present protocol is focused on utilization of waste biomass for
Velazquez-Lucio et al., 2018; Kumar et al., 2020). Among them, production of bio-ethanol paving its route to a sustainable future.
physical method is easy to operate but results in lower sugar
yield. Alternatively, chemical method is suitable for effective degra- 2. Materials & methods
dation of polysaccharides with the use of liquid acid catalysts
(Morschbacker, 2009) which was practiced in earlier days, but later Fig. 1 shows the process to convert deoiled Scenedesmus obliquus
the technique was discarded due to the cost and environmental residue into bio-ethanol production without any pre-treatment
challenges associated with homogeneous acid catalysis. However, step. The first stage involves the culture of Scenedesmus obliquus
recent advancement in biomass pre-treatment and hydrolysis by followed by lipid extraction. Saccharification was carried out using
solid or heterogeneous catalyst overcomes these shortcomings and various green catalysts. The second stage involves conversion of
opens up with suitable opportunities to produce various types fermentable sugars into bio-ethanol by Saccharomyces cerevisiae
of hydrocarbons (Galadima and Muraza, 2015). Solid acid cata- isolate BY01, isolated from local rice beer starter.
lysts posses special characteristics such as higher Brønsted acid
sites, better reactant substrate affinity, high surface area, good 2.1. Algal strains and culture maintenance
thermal stability, high catalyst porosity, and highly water stable
that makes the process efficient in biomass hydrolysis (Tan et al., The algal strain (Scenedesmus obliquus) was obtained from the
2019; Guv et al., 2012). However, the acidic conditions may lead Department of Biotechnology, Gauhati University, India. The stock
to decomposition of sugars into other unwanted compounds like cultures were maintained and propagated in 500 ml of Erlen-
furfural and 5-hydroxymethylfurfural that might inhibit the fer- meyer flasks with autoclaved BG11 medium (Rippka, 1988) which
mentation process, and also require costly downstream treatment contained (g/L) NaNO3 , 1.5; MgSO4 .7H2 O, 0.075; Ferric ammo-
of the waste (Ho et al., 2013; Mussatto et al., 2010). Hyper thermal nium citrate, 0.006; Citric acid, 0.006; Na2 CO3 , 0.02; K2 HPO4 , 0.04;
acid hydrolysis is conducted to minimize the degradation of sugars CaCl2 .2H2 O, 0.036; Na2 EDTA, 0.001; and one mL of a microele-
into inhibitory products, which is again energy expensive process ment solution consisting of (g/L) H3 BO3 , 2.86; ZnSO4 .7H2 O, 0.22;
(Sukwong et al., 2018). The acids or bases utilized for ethanol pro- Na2 MoO4 .2H2 O, 0.39; Co(NO3 )2 .6H2 O, 0.05; CuSO4 .5H2 O, 0.08;
duction are chemically synthesized and are not environmentally MnCl2 .4H2 O, 1.81 and buffered at pH 7.0 ± 0.1. The culture was
friendly. In contrast, when enzymatic hydrolysis of carbohydrate incubated under continuous illumination 98 ␮mol photon m−2 s-1
degradation is compared with acid hydrolysis, it is a much slower and constant feeding 2% CO2 at 30 ± 1 ◦ C. For the experiments
process and also more expensive. Another drawback is the require- on the influence of various nitrogen concentrations on the growth
ment of energy consuming pre-treatment steps to enhance the parameters, three nitrogen concentrations [control (1.5 g L-1 ), − 50
hydrolysis efficiency, but, enzymes are environment friendly (Ho % (0.75 g L-1 ), and -100 % (0 g L-1 )] was studied.
et al., 2013; Rastogi and Shrivastava, 2017).
Alkaline hydrolysis has several advantages like no furans forma- 2.2. Photobioreactor setup
tion, low sugar degradation and possibility of recovering caustic
salts (Balat, 2011). Certain biomass is rich in alkali, alkali metals The 20 L of transparent bottle made from polyethene tereph-
like Na, K, and Ca which can be converted into their corresponding thalate used as indoor photobioreactors. The photobioreactor
oxides (Sharma et al., 2012; Chouhan and Sarma, 2013; Betiku et al., was inoculated by axenic microalgae strain, which previously
2016; Gohain et al., 2017; Gohain and Deka, 2018; Gohain et al., propagated with an inoculum size 0.12 g L−1 . The operation of
2020a, b). Catalyst developed from biomass and bio-waste owns photobioreactor was done at 30 ± 1 ◦ C, pH 7.0 ± 0.1, continu-
potential advantage, as biomass is renewable, abundant and low- ous illumination provided via six cool-white fluorescent tube light
cost. Some deoiled biomass has also been activated chemically for (14 W; BAJAJ CO, India) fixed on both the sides of a photobioreac-
production of base functionalized catalysts (Gohain et al., 2020c; tor, and continuous injection for prefiltered air (Moxcare, 0.22 ␮m
Hameed et al., 2009; Baroutian et al., 2010; Konwar et al., 2012). pore size) was provided by air pump to the agitation of container’s
They are most promising heterogeneous catalysts as they have contents with an airflow rate 3.2 SLPM (Gas Mass Flow Meters,
sufficiently strong basic sites and they are non-corrosive as well Alicat Scientific, Inc, Tucson, USA) without adding any outside car-
as non-toxic. These catalysts have shown good catalytic efficacy bon dioxide source to the photobioreactor. At the beginning of the
towards transesterification reaction. However, mixed oxide cata- stationary phase (after day 15), the microalgae liquid sample was
lyst derived from waste, has not been studied for saccharification collected to evaluate the dry cell biomass, lipid and carbohydrate
reaction. content.
In this context, present study is an attempt to investigate
the hydrolysis capacity of waste derived catalyst namely calcined 2.3. Determination of algae biomass concentrations
Musa balbisiana colla peel (CBPA), calcined water hyacinth (CWH),
calcined Carica papaya stem (CCPS), Calcined Tectona grandis The growth of microalgae was determined daily by the optical
leaves (CTGL) and potassium impregnated Rhodotorula mucilagi- density measurement at 560 nm (i.e., OD560 ) (Wetherell, 1961; El-
nosa deoiled biomass activated carbon (K-RAC) (Gohain et al., 2017; Sheekh et al., 2013), using UV–vis Spectrophotometer (Shimadzu
192 M. Gohain et al. / Process Safety and Environmental Protection 146 (2021) 190–200

Fig. 1. Schematic representation of bio-ethanol production from Scenedesmus obliquus.

UV-1700 Pharma Spec, Japan). The cell dry weight (CDW) was the solvent was evaporated. The total lipid content was determined
achieved by filtering 50 ml from aliquots culture onto pre dried by Bligh and Dyer’s method (Bligh and Dyer, 1959).
and pre-weighed quantitative Sartorius filter paper (Particle reten-
tion 1−2 ␮m; diameter 125 mm) and then the sample was washed
2.5. Physico-chemical characterization
twice to remove adsorbed salts attached on the cell surface with
the help of double distilled water. Then the cells present on the fil-
Calorific value (CV) was done with the help of an automatic
ter paper discs was subjected to drying at 105 ◦ C in an oven until
adiabatic bomb calorimeter (Changsha Kaiyuan Instruments Co.,
the weight was constant. The dry cell weight was then gravimet-
5E-1AC/ML). The sample (SO) was oxidized by the process of com-
rically measured using an electronic balance (Sartorius CPA225D,
bustion in an adiabatic bomb that contains 3.4 Mpsi oxygen under
Germany). The actual cell dry weight of the algae was estimated
pressure. The reporting of mean values was done by performing
as a result of subtracting the dried paper loaded with microalgae
the assays in triplicates. The CHN analysis was done in a CHN ana-
from blank, dried paper. The biomass productivity was calculated
lyzer and the calculation of oxygen content was done by difference.
as follows:
The determination of moisture, volatile matter and ash content of
Productivity(mgCDWL −1 d−1 ) = (CDWL -CDWE )/(TL -TE ) (i) SO were done according to ASTM protocols. Finally the difference
gives the fixed carbon content.
Where; CDWE and CDWL representing the CDW (mgL−1 ) at the start
De-oiled Scenedesmus obliquus residue was subjected to compo-
of the culture (TE ) and late exponential phase (TL ), respectively
sitional analysis following the gravimetric method (Adeeyo et al.,
The growth rate (␮) was calculated using the following equa-
2015). Extractives content was obtained using a Soxhlet apparatus
tion:
and acetone as a solvent (For 2.5 g dried residue 150 mL of acetone
␮ = (LnA1 /LnA0 )/(T1 -T0 ) (ii) was used). The mixture was boiled using the heating mantle at 70
◦ C temperature for time duration of 4 h. After extraction, the dry-
Where, A0 and A1 represent the optical density at times t0 (day 0)
ing of the sample was done in an oven till the weight attained was
and t1 (the last day of the experiment), respectively. The generation
constant. The weight difference between the raw-extractive laden
time (G) calculated as follows equation:
residue and extractive-free biomass was expressed as % (w/w) of
G = ln2/␮(h−1 ) (iii) the extractive content. The hemicellulose was estimated gravimet-
rically after treating with 0.5 M NaOH for 3 h (Adeeyo et al., 2015).
2.4. Lipid extraction Acid-insoluble lignin was obtained similarly by drying the sample
after treating with 72 % H2 SO4 followed by autoclaving at 121 ◦ C
Triplicates of 50 mL samples were harvested by centrifugation for a time duration of 1 h. The absorbance of the acid-hydrolyzed
at 5000 rpm for 10 min. The pellets were rinsed twice with phos- samples was measured at 320 nm for determining the acid soluble
phate buffer then dried in an oven at 80 ◦ C overnight. The dried lignin fraction. The sum of acid-soluble and acid-insoluble lignin
biomass was mixed with 5 mL of 2:1:0.8 methanol: chloroform: gives the total lignin content (Sluiter et al., 2008). The difference
water (v:v:v). The blend was vortexed for 2 min subsequently added gives the cellulose content % (w/w) (Lin et al., 2010).
chloroform and water to adjust the final solvent ratio to 1:1:0.9 Total protein was estimated by Lowry’s method (Lowry et al.,
v/v/v of methanol: chloroform: water and the sample were then 1951). The FT-IR spectrum of algal deoiled residue was recorded
centrifuged at 5000 rpm for 5 min. The lower layer (chloroform on Nicolet IR spectrometer at room temperature. The blending of
layer) was carefully collected in the pre-weighed glass vials, and SO was done with potassium bromide (KBr) powder, and then for
 M. Gohain et al. / Process Safety and Environmental Protection 146 (2021) 190–200 193

measurement these were pressed into tablets. The transmittance

was recorded over a wave number range from 4000–400 cm−1 . The
recording of powder X-ray diffractograms was done on a Rigaku
miniflex diffractometer (CuKa radiation, ␭ =1.5406 Å) in 2␪ range
10−80◦ at a 2◦ scanning rate for crystalline phase determination.
Thermo gravimetric analysis (TGA) was done in order to study
the combustion behaviour of SO de-oiled residue. The thermo-
gravimetric analysis of biomass was done in nitrogen atmosphere
at 10 ◦ C/min heating rate. 10 mg of each sample was heated from
ambient temperature to 800 ◦ C in a Pyris diamond TG/DT analyzer
(PERKIN ELMER). For displacing air in the pyrolytic zone, feeding of
pure nitrogen gas (99.99 %) was done at 100 ml/min constant flow
rate as an inert purge gas, thereby oxidation of the sample which
are unwanted was avoided.

Fig. 2. The time-course of growth for Scenedesmus obliquus.

2.6. Bio-ethanol production

2.6.1. Isolation and molecular identification of yeast the distillation product, is concentrated by using molecular sieve
In this study, Saccharomyces cerevisiae isolate BY01 was isolated (pore size diameter of 3A). The yield of bio-ethanol was found out
from local rice beer starter culture. The isolate was grown on yeast using the Eq. (iv) (Wang et al., 2019b).
and mould agar medium (YMA; HiMedia, India) at 28 ◦ C for 48−72 h Ethanol yield (%)
and preserved as slant culture at 4 ◦ C. The 5.8S internal transcribed
Ethanol mass (g/L)
spacer (ITS) rDNA of yeast isolate was amplified using the primers =   × 100 (v)
ITS-1 and ITS-4. PCR was done in a total reaction volume of 50 ␮l Total carbohydrate mass g/L × 1.11 × 0.511
containing 25 ␮l Dream Taq PCR 2× master-mix, 19 ␮l nuclease
Where,
free water, 2 ␮l each forward and reverse primers; 10 ␮M and 2
0.11 is the coefficient of cellulose being converted to glucose
␮l of template DNA. Amplification reaction was done in a thermo
0.511 is the theoretical conversion coefficient of glucose to
cycler (Eppendorf) using method described elsewhere with slight
ethanol.
modification (Saikia et al., 2018). The amplification parameters are:
initial denaturation at 95 ◦ C for 2 min, followed by 36 cycles of
denaturation at 94 ◦ C for 1 min (36 cycles), annealing at 54 ◦ C for 2.6.3. Analysis of ethanol by HPLC and GC–MS
30 s, elongation at 72 ◦ C for 1 min and then final extension at 72 ◦ C The obtained bio-ethanol was characterized using HPLC
for 10 min. PCR products were separated in 2 % (w/v) agarose gel (Waters) equipped with UV–vis detector and C18 column (5 ␮m
(Sigma) in 1 × TAE buffer. Molecular weight marker Gene Ruler 100- × 250 mm). Sample aliquots were filtered through a 0.2 ␮m nylon
bp DNA Ladder Plus (Fermentas) was applied in order to determine filter. Acetonitrile/water in the ratio of 80:20 (v/v) was used as a
the size of the bands obtained. Amplicons were sequenced by the mobile phase at 1 ml/min flow rate. The temperature of the col-
1st BASE DNA sequencing service. DNA sequences were analysed umn was set at 30 ◦ C. GC–MS analysis was performed in a GC–MS
with basic local alignment search tool (BLAST). 7890A (Agilent) equipped with FID and HP-1MS column (30 m ×
250 ␮m × 0.5 ␮m). 1 ␮l of sample was injected at an injection tem-
perature of 230 ◦ C, while the temperature of the column was held
2.6.2. Saccharification and fermentation
at 80 ◦ C. Helium was used as carrier gas at 0.5 ml/min flow rate.
The alkaline hydrolysis of SO was done using the bio-based
(CBPA, CWH, CCPS, CTGL and K-RAC catalysts (2 wt. %). The solid
3. Results & discussions
phase to the liquid phase ratio is maintained at 1:10. The alkaline
mixture is then autoclaved at 121 ◦ C temperature for 30 min. The
3.1. Microalgae growth
analysis of total carbohydrate in the hydrolysate was done using a
HPLC (Thermo Scientific) [ESI, S1] equipped with a refractive index
Microalgae have gained importance as a potential biomass
(RI) detector according to the protocol described by NREL. Briefly,
source for the generation of renewable energy. Under stress con-
20 ␮l of liquid hydrolysate (neutralized to pH 7) was injected. The
ditions like nitrogen deficient flow of carbon in microalgae is
column (Accucore 150-Amide-Hilic) temperature was set at 60 ◦ C
allocated to energy-rich compounds (carbohydrate, lipids). The
and the mobile phase was 5 mM H2 SO4 at 0.6 ml/min flow rate.
highest carbohydrate production efficiency can be achieved by
The saccharification was calculated using Eq. (iv) (Muthusamy
exploring the optimal time length of cultivating microalgal cells
et al., 2019).
  under nitrogen-deficit conditions (Ho et al., 2013). Hence, the
Total sugar in the hydrolysate g/L time-course of growth for Scenedesmus obliquus under photobiore-
Saccharification (%) = × 100 actor culture mode was studied under nitrogen-deficit conditions
Total sugar in raw sample (g/L)
(iv) to achieve maximum amount of carbohydrate (Fig. 2). There was
steady increase in the test organism’s growth with a lag of one
day followed by the logarithmic phase and the stationary phase
After cooling, the treated biomass sample is then filtered for was attained at 9th days in case of nitrogen-deplete conditions and
separation of solid and the liquid part. Then the hydrolysate was 14th days at 50 % N(-) as well as, normal conditions. The obtained
subjected to fermentation. Herein, Saccharomyces cerevisiae was results revealed that Scenedesmus obliquus cultivated under nitro-
used as the fermentative organism. Yeast inoculum was added to gen deficiency conditions resulted in a reduction in all studied
the hydrolysate and the fermentation process was done at 32 ◦ C growth parameters. On the other hand, the increase in nitrogen
and pH 5 for 7 days at 150 rpm. Later, the fermentation broth was concentration resulted in decreases in lipid content. The most
filtered in vacuum pump filter for the removal of solid and the fil- distinct increase in lipid content was observed under nitrogen
trate is then distilled in the distillation unit. Bio-ethanol, which is depletion conditions, which amounted to 53.38 % compared to con-
194 M. Gohain et al. / Process Safety and Environmental Protection 146 (2021) 190–200

Table 1
Growth parameters and lipid content.

Property Control 50 %N(-) Nitrogen-deficiency

Dry weight (g/L) 1.384 1.352 0.669

Biomass productivity (mg 89.45 87.34 41.79
L−1 day−1 )
Generation time (G) (h−1 ) 6.24 10.77 11.10697
Maximum specific growth 0.11 0.0642 0.06
rate (max ) (h−1 )
Lipid content (mg/L) 315 405 480
Carbohydrate (g/L) 14.84 18.62 21.263

trol (Table 1). These results are in accordance with other published
work, who noticed that the deficiency of nitrogen enriched the lipid
content of Scenedesmus sp. CCNM 1077 by 27.93 % (Pancha et al.,
2014).
The most pronounced decrease in cellular dry weight, biomass
Fig. 3. FTIR spectra of SO.
productivity, and growth rate amounted to 51.6 %, 53.28 %, and
45.45 %, respectively was recorded at a 100 % decrease in NaNO3
(Table 1). These results aligned with the results of earlier published
work, where similarly a severe drop in the biomass of Scenedesmus
acutus was noticed, when grown under nitrogen deplete condi-
tions (Pancha et al., 2014). Likewise, the decreased in the biomass
production of Scenedesmus obliquus was showed under nitrogen-
deficient conditions (Wu and Miao, 2014).

3.2. Molecular identification of the yeast isolate

A 531 bp amplicon was obtained by the PCR amplification of 5.8S

ITS region of the rRNA gene and sequenced. The submission of the
gene sequence has been done to NCBI GenBank [GenBank Accession
no. MN194201]. Based on the nucleotide BLAST sequence similarity,
the yeast strain BY01 that was identified as Saccharomyces cerevisiae
isolate BY01 (Table 2).

Fig. 4. TGA thermogram of SO.

3.3. Physico-chemical characterization of Scenedesmus obliquus
de-oiled residue
3.3.1. FTIR analysis
De-oiled algal biomass residue is generated as a leftover from The FTIR spectrum of SO is shown in (Fig. 3). The correct sen-
biodiesel production process using microalgal lipids (Subhash and tence should be:The 3300–3000 cm−1 region is characteristic for
Mohan, 2014). Moreover, it is considered as a feedstock that is stretching vibrations of C C and Ar-H. The 3000 to 2800 cm−1
economic for biofuel production as it contains more than 70 % region is attributed to stretching vibrations of CH, CH2 and CH3
of carbohydrates and protein by weight (Katiyar et al., 2018). (Velazquez-Lucio et al., 2018; Bardhan et al., 2019b). The 1800 and
Scenedesmus obliquus is a green algae which belongs to the class 1500 cm−1 region signifies characteristic bands of proteins. The
Chlorophyceae and it is used extensively for lipid production absorption at 1650 cm−1 showed the presence of C O of carboxylic
(Duangjan et al., 2016). The physico-chemical property of SO with acid and derivatives. The 1600 and 1500 cm−1 region is charac-
their average characteristic composition has been tabulated in teristic of amide-II bands, which are indicative of NH2 bending
Table 3. vibrations.
Moisture content is considered as an important parame- For bio-ethanol production, the presence of polysaccharide
ter for selecting an appropriate biomass conversion technology peaks is of interest as we are concerned with the conversion of com-
(McKendry, 2002). SO with a low moisture content of 10.91 % seems plex carbohydrate molecules into the simple sugars. The sequence
to be a potential candidate for direct thermo-chemical conversion. of bands in region 1200–900 cm−1 signifies C O, C C, C O C
The cell wall composition of algae typically contains about ∼70 % and C O P stretching vibrations of polysaccharides (Rozenberg
cellulose and ∼43 % hemicelluloses (Welker et al., 2015). In this et al., 2019). The presence of low lipid content has been indicated
study, the cellulose and hemicellulose in SO was found to be 53.08 by the declined intensity of absorption between 3100–2800 cm−1
% and 38.4 % respectively. High content of cellulose in SO makes it (Phukan et al., 2011). Thus, SO is suitable feedstock for bio-ethanol
a suitable feedstock for producing bio-ethanol having only a small production.
amount (∼1 %) of lignin. While lignin is absent in most algae, certain
species of the green algae genus like Coleochaete do contain some 3.3.2. TGA
lignin-like compounds in their cell walls (Domozych et al., 2012). The TGA profile of SO (Fig. 4) reveals an initial loss of weight
De-oiled biomass of other algal strains including Chlorella sp. has between ambient temperature and 160 ◦ C possibly due to elim-
also been reported as a feasible feedstock for bioenergy production ination of physically absorbed water. Subsequently, there was
(Subhash and Mohan, 2014; Phukan et al., 2011). continuous loss of sample weight between 220−490 ◦ C. This is the
 M. Gohain et al. / Process Safety and Environmental Protection 146 (2021) 190–200 195

Table 2
Identification of yeast isolate BY01.

5.8S-ITS region of rRNA gene

Isolate no. Size of PCR product (bp) Homology (%) Closest related Species

BY01 531 100 Saccharomyces cerevisiae strain SE23; MK908003.1

Table 3
Physico-chemical properties of SO.

Properties SO Methods/Ref

Calorific value (MJ/Kg) 15.6

Cellulose 53.08
Hemicellulose 38.4
Structural
Acid-soluble lignin 0.305 (Lin et al., 2010)
carbohydrates (%)
Acid-insoluble lignin 5.03
Extractives 3.212
Moisture 10.91 ASTM D 3173
Volatile matter 69.06 ASTM D 3174
Proximate analysis (%) Fixed carbon 2.04 ASTM D 3175
Ash 17.99

Lipid content (%) <1 % (Bligh and Dyer, 1959)

Total carbohydrate (g/L) 13.68 (Dubois et al., 1956)
Biochemical analysis
Protein content (g/L) 2.548 ± 0.16 (Lowry et al., 1951)
CHN analyzer
Elemental analysis (%) Carbon 38.249
Nitrogen 9.374
Hydrogen 5.326
Oxygen (by difference) 47.05

Table 4
Percentage saccharification using different catalyst.

Catalyst Saccharification (%)

CBPA 50.08
CWH 49.01
CCPS 60.39
CTGL 42
K-RAC 49.24

basic ions by penetrating the small pores present in the lignocel-

luloses (Salehian et al., 2013; Molaverdi et al., 2019). Evidence of
similar results was found in number of studies (Molaverdi et al.,
2019; Noori and Kaimi, 2016; Safari et al., 2017). Hence, the pre-
pared catalyst could be successfully utilized for hydrolysis reaction.

3.4. Bio-ethanol production

Fig. 5. XRD pattern of (a) Raw SO and after utilization of (b) CBPA (c) CWH, (d) CCPS,
(e) CTGL and (f) K-RAC.
3.4.1. Hydrolysis reaction of SO using Bio-based catalyst
The analysis of the total carbohydrate in the samples was done
zone of active pyrolysis (where main degradation occurred). Pas- using NREL protocol and estimated from HPLC. For lignocellulosic
sive hydrolysis occurred in between 500−700 ◦ C. The weight loss biomass dilute acid or bases acts only as a pre-treatment and not as
is very slow until 800 ◦ C signifying some further reaction involving hydrolysis because of their complex structures as lignin is present
char. The thermal degradation profile suggests suitability of SO for (John et al., 2011). However for Scenedesmus obliquus the cellulose
thermo-chemical conversion. present in the inner membrane is free from lignin, hence readily
hydrolysable.
3.3.3. X-ray diffraction (XRD) Availability of the active basic sites may affect the efficiency of
Fig. 5 shows the XRD pattern for SO and SO after hydrolysis base hydrolysis of SO for obtaining the fermentable sugars, due
using calcined Musa balbisiana colla peels (CBPA), calcined water to the specific structure of Scenedesmus obliquus. The conversion
hyacinth (CWH), calcined Carica papaya stem (CCPS), calcined Tec- of carbohydrates of the SO into fermentable sugars was done via
tona grandis leaves (CTGL) and Rhodotorula mucilaginosa deoiled catalytic saccharification for bio-ethanol fermentation. Catalytic
biomass activated carbon impregnated by using potassium hydrox- hydrolysis of the biomass was done at 121 ◦ C, for 30 min. For the
ide (K-RAC) as catalyst. saccharification process, different catalysts were used as shown in
The intense X-ray diffraction peak was detected at 25◦ which the Table 4. The catalysts exhibited high basicity as determined
characteristic of cellulose. From the Fig. 5, it is well disclosed that by Hammet indicators test and CO2 -TPD analysis. K2 O, K2 CO3 , CaO
the peak at 25◦ for the treated samples got reduced compared to and MgO were considered the main factor behind the higher basic-
the raw sample i.e. SO. This portrays release of cellulose and disrup- ity of CBPA. K was found to be the major ingredient and K2 O being
tion of the cellulose lignin structure of SO. Breaking of the linkages a strong base resulted in strong basicity of the bio-based catalysts.
between cellulose, hemicellulose and lignin can be done by the They have also been characterized by XRD, FTIR, SEM TEM and EDX
196 M. Gohain et al. / Process Safety and Environmental Protection 146 (2021) 190–200

Fig. 6. HPLC chromatogram of (a) standard sugar mixture and (b) total carbohydrate in SO.

Table 5 charomyces cerevisiae RL-11 (11.7 g/L) and Pachysolen tannophilus

Ethanol concentration and percentage ethanol yield.
(11.92 g/L) using waste biomass such as giant reed, spent coffee
Catalyst Ethanol concentration (g/L) Ethanol yield (%) grounds and green seaweed Ulva rigida respectively under simi-
CBPA 7.99 66.24 lar fermentation conditions (Scordia et al., 2012; Mussatto et al.,
CWH 5.47 45.03 2012; El Harchi et al., 2018). Furthermore, we performed sepa-
CCPS 8.10 67.16 rate hydrolysis and fermentation (SHF) that resulted in 68 % yield
CTGL 8.24 68.32 (ethanol produced/ g substrate consumed) which was found com-
K-RAC 7.91 64.48
parable to bioethanol yield from empty fruit bunches of palm oil
(Mofijue et al., 2019). The yield obtained in our study was found to
techniques (Gohain et al., 2017; Gohain and Deka, 2018; Gohain be higher compared to simultaneous saccharification and fermen-
et al., 2020a, b; Gohain et al., 2020c). From the table, it is evi- tation (SSF) and continuous fermentation processes using sugars
dent that highest saccharification yield was exhibited by the CCPS derived from cellulosic agro-residues like taro waste (43.55 %),
catalyst while the other catalysts showed lower saccharification wheat straw (24 %) and fruit peels (41 %) (Tomás-Pejó et al., 2008;
yield under the same condition. The hydrolysis gave saccharifica- Choi et al., 2015; Wu et al., 2016). Moreover, SHF provides addi-
tion yield of 60.39 % for CCPS. Lowest saccharification was seen tional advantages like independent temperature optimization for
while using CWH. The reason behind it might be the lower basicity both saccharification and fermentation, use of minimum quantity
of CWH. of hydrolytic enzymes and reduced risk of contaminating microbes
(Tojo and Hirasawa, 2013). Our results indicate that Scenedesmus
3.4.2. Fermentation obliquus deoiled biomass (SO) hydrolysate contains fermentable
The hydrolysate obtained after the hydrolysis SO was subjected sugars that is quite high and has a great potential as a raw material
to fermentation using the fermentative microorganism Saccha- for bio-ethanol production.
romyces cerevisiae. For the fermentation process yeast inoculums
was prepared maintaining a temperature of 32 ◦ C. The fermenta- 3.5. Bio-ethanol characterization
tion was performed using 10 % of yeast innoculum. Yeast should be
capable of utilizing all monosaccharide present while withstand- 3.5.1. High performance liquid chromatography (HPLC)
ing potential inhibitors in the hydrolysate (Sunwoo et al., 2019). In Fig. 6(a), shows a standard sugar mixture for comparison of the
this study, the ethanol concentration obtained was 8.24 g/L which cellulose obtained from SO. The total carbohydrate in the SO was
was comparable to bio-ethanol concentration obtained with other determined by the NREL protocol and estimated 21.263 g/L by HPLC
reported yeast strains like Schefferssomyces stipitis (8.2 g/L), Sac- [Fig. 6(b)]. The hydrolysate obtained after using CBPA, CWH, CCPS,

Table 6
Bio-ethanol production using different algal feedstocks.

Feedstock Hydrolysis Agent Ethanol concentration (C) Or Ref.

Ethanol Yield (Y) (%)

Chlorococcum humicola H2 SO4 7.2 g/L (C); 52(Y) (Harun and Danquah, 2011)
Scenedesmus obliquus H2 SO4 11.7 g/L (C) (Miranda et al., 2012)
Undaria pinnatifida Mixture of enzymes 12.98 g/L (C); 40.12 (Y) (Kim et al., 2013)
Chlorella vulgaris FSP-E H2 SO4 11.7 g/L (C); 87.60 (Y) (Ho et al., 2013)
Arthrospira platensis HNO3 and H2 SO4 16.32 (Y) (HNO3 ) and 16.27 (Y) (H2 SO4 ) (Markou et al., 2013)
Eucheuma cottonii Catalyst Amberlyst (TM) - 15 0.33 g bio-ethanol/g biomass ; 65 (Y) (Tan et al., 2013)
Chlorella vulgaris Enzyme Aspergilluspectinase 89 (Y) (Kim et al., 2014)
Chlorella sp. KR-1 HCl and pectinase enzyme 0.16 g ethanol/g residual biomass (C) (Lee et al., 2015)
Scenedesmus dimorphis Amyloglucosidase enzyme 0.26 g bio-ethanol/g biomass (C) (Cheng et al., 2016)
Mixed Microalgae H2 SO4 and mixture of enzymes 6.41 g/L (enzymatic) (C) and 4.96 g/L (acidic) (Shokrkar et al., 2017)
(C); 92 (Y) (Enzymatic) and 76 (Y) (Acidic)
Porphyridium cruemtum(PC) Pectinase and cellulase enzymes 65.4 (Y) (Seawater PC) and 70.3 (Y) (Kim et al., 2017)
(Freshwater PC)
2 macroalgal biomass HCl and H2 SO4 61 (Y) (Kumar et al., 2018)
Scenedesmus obliquus Green Catalyst (CTGL) 8.24 g/L (C) and 68.32 (Y) This work
 M. Gohain et al. / Process Safety and Environmental Protection 146 (2021) 190–200 197

Fig. 7. HPLC chromatogram of (a) Hydrolysate and (b) bio-ethanol.

Fig. 8. Gas Chromatogram of (a) standard ethanol and ethanol prepared by using (b) CBPA, (c) CWH, (d) CCPS, (e) CTGL and (f) K-RAC.

CTGL and K-RAC was also analyzed to detect the presence of sugar (MSU3) was estimated as a step peak at RT 2.055 min (Muthusamy
with the help of HPLC [Fig. 7(a)]. et al., 2019). Likewise, (Selvan et al., 2019) detected the bioethanol
In this study, the HPLC analysis of bio-ethanol was estimated peak using HPLC (RT of 5.23 min against standard ethanol; RT
as a steep peak at retention time of 2.915, 2.893, 2.865, 2.903 5.538) obtained from fermentation of green microalgal biomass
and 3.024 for CBPA, CWH, CCPS, CTGL and K-RAC hydrolysate Acutodesmus obliquus with S. cerevisiae.
respectively, compared to commercial ethanol standard at reten- It is evident that the highest bio-ethanol yield [Table 5] was
tion time of 2.893 [Fig. 7(b)]. Table 5 shows the bio-ethanol yield obtained from the CTGL treated biomass hydrolysate. The bio-
using different catalysts. Similar HPLC analysis of bioethanol pro- ethanol concentration was found to be 8.24 g/L and yield of 68.32
duced by fermentation of agro-residue using Streptomyces olivaceus %. This indicates that the catalyst CTGL is more efficient for bio-
198 M. Gohain et al. / Process Safety and Environmental Protection 146 (2021) 190–200

Table 7 Acknowledgements
The physico-chemical properties of bio-ethanol.

Property Bio-ethanol Ethanol M. Gohain thanks DBT, New Delhi, India for providing finan-
3
Density (g/cm ) @ 15 C◦
0.805 0.785–0.809 cial support (Grant No- DBT/IC-2/Indo-Brazil/2016-19/04). We
Kinematic Viscosity (cSt) @ 20 ◦ C 1.28 1.2–1.5 acknowledge SAIC Tezpur University, CSIR-NEIST Jorhat, DRL
Flash point (◦ C) 18 16.6 Tezpur and IIT Guwahati for assisting in performing analyses.
Fire point (◦ C) 37 26
Calorific Value (MJ/Kg) 24.82 29.7
Appendix A. Supplementary data

Supplementary material related to this article can be found, in

ethanol production. Also the lowest ethanol concentration of 5.47
the online version, at doi:https://doi.org/10.1016/j.psep.2020.08.
g/L with ethanol yield of 45.03 % was obtained from the fer-
046.
mentation of CWH treated biomass hydrolysate. The results are
comparable with other published articles, wherein researchers
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