In Situ Transesterification of Blue Green Microalgae for Sustainable Biodiesel

Production

A Research Paper

Submitted to the Faculty of the

Chemical Engineering Department

Eulogio “Amang’ Rodriguez Institute of Science and Technology

Nagtahan St. Sampaloc, Manila

In partial fulfillment of

the requirements for

Methods of Research

Submitted by:

Bowman,Jasmine D.

Casono, Aira Mae

Ganaba,Willy Boy E.

Versoza,Sarah pearl C.

Dr. Angelo Edward Jacinto

Research Adviser

Submitted to:

Engr. John Andrew Tria

 2024

Table of Contents

Navigation.................................................................................................................................2

CHAPTER 1

1.1 Background of the Study.....................................................................................................4

1.2 Statement of the Problem....................................................................................................5

1.3 Significance of the Study....................................................................................................6

1.4 Objective of the Study.........................................................................................................7

1.5 Framework of the Study......................................................................................................7

1.6 Research Gap.......................................................................................................................8

1.7 Scope and Delimitations......................................................................................................8

1.8 Definition of Terms..............................................................................................................9

CHAPTER 2

2.0.1 Introduction ...................................................................................................................13

2.0.2 Controversies Surrounding Biodiesel ............................................................................14

2.0.3 Transesterification process…..........................................................................................16

2.0.4 Biodiesel production basics………….………................................................................16

2.1 Feedstock……………………………………....................................................................17

2.2 Algae biomass and its suitability for biodiesel production................................................17

2.3 Catalyst (acid and base catalyst) …..…………………….................................................18

2.4 In situ……………………………………………………................................................19

CHAPTER 3

3.1 Research Design................................................................................................................21

3.2 Data Collection Methods....................................................................................................21

3.3 Materials.............................................................................................................................22

3.4 Procedures ………………………….................................................................................23

3.4.1 Extraction and Characterization of Oil,algae biomass and Biodiesel.................23

3.4.2 Quantification of oil content…………...............................................................25

3.4.3 Protein content of algae biomass. .……..............................................................25

3.4.4 Ash Content of algae biomass………….............................................................26

3.4 Free fatty acid (FFA)..........................................................................................................27

3.5 Acid Value…………..........................................................................................................27

3.6 Properties measurements of Blue Green biomass,oil, and Biodiesel................................28

3.7 Biodiesel Production process.............................................................................................28

3.7.1 Transesterification process……………..............................................................28

3.7.2 Settling,separation,and washing processes..…....................................................30

3.8 Laboratory Experiment Data Analysis...............................................................................32

 CHAPTER 1

Introduction

This chapter shows the Background of the Study, the Statement of the Problem, the

Objectives of the Study, the Significance of the Study,Framework of the

Study,Hypothesis,Research Gap,the Scope and Delimitation,the Definition of Terms and the

Conceptual Framework that gave further details of the research.

1.1 BACKGROUND OF THE STUDY

The Philippines is pretty dependent on fuel imports for its energy needs. This

not only affects the economics of the nation but also increases issues related to the

environment consisting of air pollutants and weather change. In recent years, there has been a

growing interest in locating opportunities and sustainable energy sources, and making biofuel

from algae is a possible remedy. As of today, the primary source of the biodiesel in the

Philippines is coconut oil feedstock, which is 228 000 metric tons expected to be consumed

by 2022.

Microalgae and cyanobacteria can be grown in nutrient-rich wastewater, reducing

nutrient costs for algal growth and bioremediation. With a higher biomass per unit area, they

can produce 10-100% more energy than second-generation biodiesel crops (Morsi et al.,

2023).
 The aquatic eukaryotic group Macroalgae is composed of a variety of non-phylogenetic

aquatic eukaryotes, including Rhodophyta, phaeophyta, and Phaeophyceae. They can be

cultivated in various water types, including wastewater, and have a faster growth rate than

fodder crops. The oil content of algae is 30 times higher than conventional feedstocks, and it

is fully biodegradable and sulfur-free, leading to better quality oil (Godvin et al., 2021).

The aim of the study is to show the efficient way of Situ Transesterification of Blue Green

Microalgae for Sustainable Biodiesel Production.

1.2 STATEMENT OF THE PROBLEM

The ever expanding human population demands for energy security and independence

and can’t rely on the ever depleting, non-renewable resources (i.e. coal, crude oil, natural

gas). The increasing demand and desire for the renewable resources as a substitute for the

non-renewables and the need for reduction of time and energy consumption in algae oil

extraction and the conversion of its lipids to biodiesel necessitate optimization of the

biodiesel production process. In a nutshell there is a need to cut down the cost of production

of biodiesel so that the biofuel manufacturers can meet this expanding market. Microalgae, a

third generation biofuel is being considered as the major raw material in biodiesel production

because of its high oil content. Industry players and researchers have a common knowledge

that the bulk of energy costs during biodiesel production arise from the longer times

experienced during algae dewatering, oil extraction, conversion, and high solvent

consumption during extraction and reaction to final product (biodiesel). This process occurs

in a single stage: there is continuous oil extraction of oil from microalgae 5 biomass and at

the same time this oil is converted to biodiesel. Therefore it would be of great economic
benefit if a means is found of reducing the time of production and thus the energy

consumption. One such method is through the simultaneous algae dewatering, algae oil

extraction and conversion to biodiesel (in situ)

1.3 SIGNIFICANCE OF THE STUDY:

The importance of this study was to address In Situ Transesterification of Blue Green

Microalgae for Sustainable Biodiesel Production

The final result of this study would be beneficial for the following:

TO THE FUTURE RESEARCHERS

This study would become a frame of reference for those who had related topics and investigations.

TO THE ENTREPRENEURS

This product would expand opportunities, particularly for those looking for much safer, affordable

and much sustainable biodiesel.

TO THE PEOPLE

Aside from being more sustainable than other diesel.This biodiesel would be a great help to sustain

the needs of every consumer using algae for production.

TO THE BIODIESEL INDUSTRY IN THE PHILIPPINES

Due to the production of this product, this algae as raw material would be helpful to production of

biodiesel.
1.4 General Objectives

The goal is to establish a sustainable and cost-effective in situ transesterification of Blue

Green microalgae as a renewable feedstock. This approach aims to address the challenges of

greenhouse gas emissions and reliance on non-renewable energy sources.

1.4.2 Specific Objectives

1. To quantify oil content of Blue green microalgae.

2. To characterize Blue green microalgae oil.

3. To study the effect of reaction operating variables: catalyst concentration, methanol

to oil ratio and reaction time on FAME yield, and identify optimal conditions through

the use of Response Surface Methodology.

1.5 FRAMEWORK OF THE STUDY

This framework shows the overall outline of the study. It can be expressed by using

the input-process-output framework (IPO).

Figure 1. To produce biodiesel from algae, the process involves extracting oil, converting it
to biodiesel through transesterification, separating impurities, settling, washing, and drying
before storage.

1.6 RESEARCH GAP

Research on in situ transesterification of blue-green microalgae for sustainable

biodiesel production has gained considerable attention in recent years. However, there are

still some notable research gaps that need to be addressed to optimize the process and

improve its feasibility. A research gap is the limited understanding of the optimal operating

conditions for in situ transesterification of blue-green microalgae. The process involves

converting microalgae lipids into biodiesel in the presence of a catalyst. The selection of

suitable catalysts, for example alkaline or acidic catalysts, as well as their optimal

concentrations, reaction temperatures and reaction times are crucial for achieving high

biodiesel yields. Further research is needed to investigate the effects of these parameters on

the efficiency of the transesterification process and to determine the optimal operating
conditions for different blue-green microalgae species. Another research gap is the

identification and characterization of suitable blue-green microalgae strains with high lipid

contents and growth rates. The lipid content of microalgae is a crucial factor for the

production potential of biodiesel. However, not all blue-green microalgae species have high

lipid content and their lipid productivity can vary significantly under different growth

conditions. Therefore, there is a need for comprehensive screening and characterization of

blue-green microalgae strains to quickly identify those with high lipid content.

1.7 SCOPE AND DELIMITATION

The produced biodiesel was not evaluated in a test engine. This study focused on the

effect/importance on biodiesel production from Blue- Green microalgae on the identified

variables and its future scope as energy security

Oils are valuable, energy-rich compounds that have the potential to replace traditional

fossil fuels through the production of biofuels. Oils have a wide range of industrial and

technical applications in coatings and polymers, printing inks, lubricants, leather processing,

solvents, plastics, pesticides and fuels. In recent years, lipid-based fuel, also called biodiesel,

has been recognized as a suitable energy source for fossil-based fuels because it is renewable,

non-toxic, etc. Several oils, when highly purified and refined to meet pharmaceutical

specifications, are used to act as fillers, binders, solubilizers, emulsifiers, and plasticizers in a

variety of dosage forms. Oil production from biomass continues to represent a significant

bottleneck in biofuel production due to high costs and high energy requirements

1.8 DEFINITION OF TERMS

Agitation: When the reaction mixture is mixed or stirred during transesterification, it is
referred to as agitation. Efficient conversion is promoted by uniformly distributed reactants
and catalysts, which is ensured by proper agitation.

Algae: Algae are simple organisms that resemble plants and range in size from microscopic
unicellular creatures to large forms. Some species of microalgae are widely cultivated
because of their high concentration of lipids, or oil. This lipid is a useful ingredient in the
creation of biodiesel since it can be transformed into that fuel.

Algal Strain: Algal strains are distinct classifications of algae, representing specific varieties
or species. These strains can differ in their lipid content, growth rates, and other
characteristics, which ultimately impact their suitability for the production of biodiesel.

Biodiesel: The term "biodiesel" describes an alternative fuel that burns cleanly and renewable
and is made from biological sources like algae, animal fats, or vegetable oils. Usually, it is
used as a blend or diesel substitute.

Biorefinery: An algal biorefinery is a facility that integrates various processes to convert

algal biomass into a range of valuable products, including biodiesel, bioethanol, and other
bio-based chemicals.

Catalyst Selection: Materials known as catalysts aid or quicken chemical reactions without
getting consumed themselves. Catalysts are employed in transesterification to speed up the
synthesis of biodiesel. Alkaline catalysts, such potassium or sodium methoxide, and acidic
catalysts are common types of catalysts.

Centrifugation: Centrifugation is a method of particle separation from liquids that makes use
of centrifugal force. Centrifugation is frequently used in the production of biodiesel to
separate the fuel from glycerol and other contaminants.

Cultivation: Cultivating algae plays a vital role in the production of biodiesel, as it entails the
deliberate and controlled growth of algae under specific environmental conditions, including
temperature, light, and nutrient availability. Successful algal cultivation directly impacts the
quantity of lipids that can be obtained and processed into biodiesel.

Cyanobacteria: Cyanobacteria, also known as blue-green algae, are a group of

photosynthetic bacteria that can perform photosynthesis similar to plants. They are
characterized by the presence of chlorophyll-a and phycobilins, giving them a blue-green
color. Cyanobacteria are important in the production of oxygen and play a crucial role in
various ecosystems.

Downstream Processing: The processes that come after harvesting, such as lipid extraction,
purification, and transesterification, are referred to as downstream processing. It includes all
steps involved in turning algal biomass into biodiesel.

Ethanol: Ethanol is an alcohol that is frequently used in transesterification reactions to

produce biodiesel.

Flocculation: Flocculation is a technique used in algae harvesting to agglomerate or clump

together the algae cells, making them easier to separate from the growing medium.

Glycerol: Glycerol is a byproduct of the transesterification process used in biodiesel

production. It is separated from biodiesel during the production process and can have various
industrial applications.

Glycerol Layer: Transesterification produces glycerol as a byproduct, which frequently

separates from the reaction mixture due to density differences. The glycerol layer and the
biodiesel are separated once the reaction is finished.

Harvesting: Gathering mature algae from the cultivation system is called harvesting.
Different techniques like flocculation, filtration, and centrifugation are used to separate the
algae from the growing medium.

Large-Scale Algae Cultivation: The term "large-scale algae cultivation" describes the
commercial or industrial growth of algae for a range of uses, such as the production of
biofuel, food additives, and wastewater treatment. There are several options for cultivation
techniques, such as hybrid systems, closed photobioreactors, and open pond systems.

Lipids: Lipids are organic compounds that are insoluble in water but soluble in organic
solvents. In the context of biodiesel production, lipids refer to the fatty acids found in algae,
animal fats, or vegetable oils that can be converted into biodiesel through the process of
transesterification.

Microalgae: Microalgae are microscopic algae that are unicellular or colonial in nature. They
are a diverse group of photosynthetic organisms that can grow in various aquatic
environments. Microalgae are of particular interest in biodiesel production due to their high
lipid content and rapid growth rates.

Open Pond System: An open pond system is a type of algae cultivation system that uses
open-air ponds or raceways to grow algae. These ponds are exposed to sunlight and can cover
a large area. Open pond systems are relatively low-cost but may be susceptible to
contamination and have lower productivity compared to closed systems.

Photobioreactor: A photobioreactor is a closed system used for the cultivation of algae. It

typically consists of a transparent enclosure, such as a glass or plastic tube, in which algae are
grown under controlled conditions. Photobioreactors offer advantages such as higher
productivity, better control over environmental parameters, and reduced contamination risks
compared to open pond systems.

Pyrolysis: Pyrolysis is a thermochemical process that involves heating biomass in the absence
of oxygen to convert it into bio-oil, gas, and char. Pyrolysis can be used as a method for
converting algae biomass into biofuels, including bio-oil that can be further processed into
biodiesel.

Strain Selection: Strain selection refers to the process of choosing specific strains or species
of algae for cultivation based on their desirable traits, such as high lipid content, fast growth,
and tolerance to environmental conditions. Selecting the right algal strains is crucial for
maximizing biodiesel production efficiency.

Transesterification: Transesterification is the chemical reaction that converts triglycerides,

found in algae oil or other vegetable oils, into biodiesel. It involves reacting the triglycerides
with an alcohol, such as methanol or ethanol, in the presence of a catalyst to produce
biodiesel and glycerol as a byproduct.

Ultrasonication: Ultrasonication is a technique that uses high-frequency sound waves to

break down cell walls and improve the extraction of lipids from algae. It is often used as a
pre-treatment method in biodiesel production to enhance the efficiency of lipid extraction.

Upstream Processing: Upstream processing refers to the initial steps in biodiesel production,
including algae cultivation, harvesting, and biomass preparation. It involves the preparation
and optimization of the algal biomass for downstream processing.
 Chapter 2

REVIEW OF RELATED LITERATURE

This chapter presented the relevant literature and studies that the researchers considered in

strengthening the claims and importance of the present study.

2.0.1 Introduction

The world is rapidly transitioning from non-biodegradable and non-renewable forms of

energy to pollution-free alternatives. The continuous exploration of crude oil, combined with

high consumption rates, is gradually depleting this valuable resource. It is crucial, therefore,

for the world to explore other energy sources, particularly third-generation biofuels, in order

to meet current and future energy demands. Unlike first and second generation biofuels,

third-generation biofuels, such as biodiesel from algae, have the potential to fulfill the

growing need for biodiesel (Jones & Mayfieldt, 2012).

The invention of the diesel engine by Rudolf Diesel in the 1890s allowed for the use of

various fuels, including vegetable oil. In fact, one of Diesel's new engines, showcased at the

Paris Exposition in 1900, was powered by peanut oil. However, due to the availability and

affordability of cheap petroleum fuels, alternative options received little attention (Gerpen,

2019).

As early as the 1930s, there was interest in separating fatty acids from glycerin in vegetable

oil to create a thinner product similar to petroleum diesel. In 1937, G. Chavanne obtained a

Belgian patent for an ethyl ester of palm oil, which is essentially biodiesel as we know it
today. In 1938, a passenger bus fueled by palm oil ethyl ester successfully operated between

Brussels and Louvain (Knothe, 2005). During World War II (1939-1945), several countries,

including Brazil, Argentina, China, India, and Japan, utilized vegetable oil as a fuel when

petroleum supplies were disrupted. However, once the war ended and petroleum became

readily available again, these vegetable oil fuels were largely forgotten (Boakye, 2013).

The petroleum oil embargo in the 1970s prompted many governments to reconsider vegetable

oil as a viable fuel source. Scientists from Austria, the United States, South Africa, and other

countries confirmed that straight vegetable oil could once again be used to power diesel

engines. However, the high viscosity of vegetable oil resulted in poor fuel spray quality,

posing challenges (Boakye, 2013).

2.0.2 Controversies Surrounding Biodiesel

The rapid expansion of the biodiesel industry has raised concerns that farmers may be

incentivized to grow more and more fuel crops, reducing the amount of land available for

food production. The "food versus fuel" debate emerged as global food prices rose in 2008,

sparking serious discussions. While it is possible to grow feedstock crops for biofuels (such

as sunflower oil, sugarcane, corn, wheat, and barley), there are doubts about the potential

impacts on food supply and security. The debate between fuel and food is exacerbated by the

extensive cultivation requirements and the significant resource levels needed. Therefore,

there is a pressing need for alternative, sustainable fuels and feedstocks to replace food-based

sources of biofuels. Scientists are exploring the production of fuel from non-oilseed

feedstocks like inexpensive, non-edible biomass sources (such as agricultural residue, wood
industry waste, switchgrass, and other grasses) that can be converted into a diesel substitute

known as second-generation biofuel. While converting vegetable oil or animal fat into

biodiesel is relatively straightforward, the process of converting cellulosic feedstocks into

fuel is more complex and costly. Typically, biomass is first converted into a synthetic gas

using high heat to produce a hydrocarbon fuel, which can then be turned into a liquid diesel

fuel. Due to the drawbacks associated with first and second-generation biofuels, a

third-generation biofuel was developed, commonly produced from algae. Algae, which are

single-cell or multicellular organisms, are usually classified based on their habitat, such as

freshwater algae, marine algae, or wastewater algae. A specific type of algae is selected for

biodiesel production based on its lipid content.

2.0.3 Transesterification process

Most of the biodiesel used in commercial applications is produced through a chemical

process known as transesterification. This process entails combining the feedstock oil with an

alcohol, usually methanol or ethanol, along with a catalyst, either an acid like 𝐻2𝑆𝑂4 or a

base like 𝑁𝑎𝑂𝐻. The outcome of the reaction is the formation of methyl esters (when

methanol is utilized) or ethyl esters (when ethanol is utilized) - these components make up

the biodiesel (fuel) and glycerin by-product (Murphy et al, 1995). Methanol is commonly

chosen for its cost-effectiveness, as per a study conducted by the University of Idaho, which

states that the physical and chemical properties of methyl esters and ethyl esters are

"comparable" (Idaho, 2003).

2.0.4 Biodiesel production basics

Biodiesel as a fuel is made up of mono-alkyl esters of long chain carboxylic acids derived

from vegetable oils, and or animal fats. The diesel obtained can be used in diesel engines and

for heating purposes (Marchetti et al, 2007).

Biodiesel is commonly produced through the process of transesterification. This method

involves the reaction between triglyceride, the main component of oil/fats, and alcohol in the

presence of a catalyst. The result of this reaction is the production of biodiesel and glycerol as

a byproduct. Transesterification is a reversible reaction that occurs in three steps. The first

step involves the conversion of long-chain triglycerides to diglycerides, followed by the

conversion of diglycerides to monoglycerides. Finally, the monoglycerides are converted to

glycerol. Each step of the reaction produces one molecule of an alkyl ester, as shown in the

reaction below.
 In the above reactions, 𝑅’ is the alkyl group for the alcohol whereas 𝑅1, 𝑅2 and 𝑅3 are

carbon chains of the fatty acids(Kumar A. , Chirchir, Namango, & Kiriamiti, 2016).

2.1 Feedstock

Biodiesel is a promising alternative fuel for diesel engines, derived from various sources

such as vegetable oils, animal fats, and microalgae oils. The ideal feedstock for biofuel

production should have a carbon chain length ranging from 𝐶14 to 𝐶22, with lower levels of

unsaturation for optimal conversion efficiency. The chain length is a crucial factor to

consider, with high unsaturation levels in algae biodiesel negatively impacting oxidative

stability, cetane number, and heat of combustion.

2.2 Algae biomass and its suitability for biodiesel production

2.3 Catalyst (acid and base catalyst)

According to Rahman et al. (2017), researchers encountered several challenges when using

conventional methods for biodiesel production. These challenges included low biodiesel

production percentage and the lengthy duration required to achieve the desired objective.

When transesterification is carried out in the presence of an alkali catalyst, algae, which

contains a higher percentage of free fatty acids (FFA), may lead to soap formation, increased

catalyst consumption, catalyst fouling, and decreased biodiesel yield (El-Shimi et al., 2013;

Chiou et al., 2008).

Rahman et al. (2017) also mentioned that these issues may result in biodiesel separation

problems from glycerol. Additionally, in alkali reactions, using an excess of alcohol may lead
to better conversion of triglycerides, but it makes glycerol recovery more challenging.

Therefore, the optimal ratio between alcohol and raw material should be determined

experimentally for each process (Marchetti et al., 2007). On the other hand, acid catalysts

have slower reaction rates compared to base catalysts (Wang et al., 2007). Hence, it is

necessary to determine the acid value for esterification or the excess amount of base to be

added before carrying out the transesterification process with a base catalyst.

The use of alkaline catalyzed transesterification technology is not suitable for biodiesel

production from microalgae oil due to the high FFA content in microalgae lipids. This is

because using alkaline catalysts with high FFA-containing oils would result in soap

production (Ehimen et al., 2010; Al-Zuhair, 2007), leading to difficulties in biodiesel

separation and downstream purification. Instead, the use of sulphuric acid as the reaction

catalyst has been considered for microalgae lipid transesterification. This is because the

transesterification and esterification reactions in biodiesel production can be facilitated

through acidic catalysis, and sulphuric acid is insensitive to the FFA content of the

microalgae feedstock (Nagle & Lemk, 2015).

2.4 In situ

The process of in situ biofuel production involves combining lipid extraction and

transesterification steps to produce biodiesel. Hincapie & Lopez(2011) proposed that

integrating these stages reduces production costs by minimizing the use of reagents and

solvents, making analysis simpler and more cost-effective. Deepalakshimi et al., (2014)

optimized biodiesel production from waste avocado seeds using the in-situ method, achieving

a yield of 94.4 wt% relative to the total oil content of the seeds. This highlights the
effectiveness of the in-situ method in enhancing biodiesel production. ElShimi et al., (2013)

conducted a study on producing biodiesel from Blue Green Microalgae through the in-situ

method, determining that 8 hours was the optimal time for the reaction when utilizing direct

heat from a hot surface. Microwave energy was not utilized in this research, and the longer

reaction time of 8 hours was necessary to ensure the progression of reactants in the desired

direction.
 CHAPTER 3

MATERIALS AND METHODS

This chapter presents the explanation about the procedures that are taken in this study

to find out the answers to the research question.

3.1 Research Design

Experiments were conducted in this study to assess the efficiency of simultaneous

extraction and transesterification (in situ) in determining the yield of FAME. The FAME

yield was evaluated by considering the catalyst concentration, the volume ratio of methanol

to oil, and the reaction time. Furthermore, additional experiments were carried out to

determine the oil yield in algal biomass, as well as to characterize the Blue Green microalgae

oil and the biodiesel produced. It is worth noting that most biodiesel production processes

typically operate at temperatures below 70 °C (Fajardo, Leon, & Vargas, 2010). To optimize

and maximize the production of the three variables under investigation, Response Surface

Methodology (RSM) and Central Composite Design (CCD) were employed.

3.2 Data Collection Methods

The data will be gathered through laboratory experiments, the researcher will assess

the mechanical properties. The experiment included the Situ Transesterification of “Blue

Green Microalgae”.
3.3 Materials

The materials used in this study are summarized in Table 3.3.1 below.

Table 3.3.1: The Materials Used and their Sources.

Item Chemicals & Solvents (analytical grade)

NO

1. Blue Green microalgae biomass

2. Methanol Gels Up

3. Sodium Hydroxide Gels Up

4. Hexane Gels Up

5. Sulphuric Acid (ρ=1.01) Gels up

6. Hydrochloric acid Gels up

7. Biodiesel standard (Methyl Heptadecanoate)

8. Phenolphthalein Indicator

9. Ethanol Gels up

10. Isopropyl Alcohol

11. Fatty acid

12. Potassium hydroxide

13. Distilled water

14. Phenolphthalein indicator

15. Sodium hydroxide

3.3.2 Equipment The equipment used in this study to produce and analyze biodiesel from

Blue Green microalgae by in-situ is summarized in Table 3.3.2

Table 3.3.2: The Apparatus/Equipment Used

 Item Name of equipment/Apparatus
No.

1. Gas Chromatograph Uni

2. Rotary Vacuum Evaporator

3. Muffle Furnace

4. Hot Plates

5. pH/ORP meter

6. Analytical Balance

7. Stirrer

8. 3-Necked Round Bottom Flasks

9. Condensers

10. Soxhlet Apparatus

11. Centrifuge

12. Desiccator

3.4 Procedure

3.4.1 Blue Green microalgae Extraction and characterization of oil, algae biomass and

biodiesel

The extraction process using a Soxhlet apparatus involves the use of a condenser and a

round bottom flask. In this particular case, a 250ml round bottom flask was used to hold a

solvent mixture of hexane and isopropyl alcohol in a ratio of 3:2, totaling 120ml. The solvent

was heated inside the flask using a hot plate with temperature control to maintain the boiling

point temperature. The vapors of the solvent are then condensed and collected in the Soxhlet

chamber, where it extracts the oil from the algae biomass contained in a porous thimble.
Figure 3.4: Soxhlet Extraction Set-up for the Blue Green Algae Oil Extraction Process.

3.4.2 Quantification of oil content

The quantification of the extracted oil yield of microalgae biomass (%) was calculated

according to the following equation:(Elshimi, Attia, & Allah, 2015).

3.4.3 Protein content of algae biomass

The protein content of Blue green microalgae was analyzed in the laboratories of

Chemical Engineering (CHE). Following the extraction of oil/lipids from the algae biomass
(Index 3.2.1), the defatted powder was air-dried and stored at 4°C. The defatted algae

biomass was mixed with distilled water (8% w/v), stirred for 60 minutes, and then filtered

using Whatman filter paper number 1. The crude extract filtrate was subsequently incubated

in ice. An optimal concentration of 40% (w/v) ammonium sulfate (NH4)2SO4 was utilized to

gradually increase the salt concentration in the algae biomass filtrate to 30% (w/v) for the

removal of proteins and other materials with minimal or no coagulation activity through a

process known as salting out. The precipitates were separated by centrifugation at 3000 RPM

for 10 minutes, and the supernatant was collected for further cautious addition of 40%

(NH4)2SO4 (w/v) (Parimi, Singh, Kastner, C., & Fusberg, 2015). The precipitated proteins

were then centrifuged at 3000 RPM for 10 minutes. The resulting pellets were dissolved in

distilled water and dialyzed overnight (12 hours) against 2L of distilled water at 4°C in a

beaker. Fresh distilled water was substituted after the initial 4 hours of dialysis to maintain

the concentration gradient. The total protein content as a percentage (%) of the total algae

biomass was subsequently determined.

3.4.4 Ash Content of algae biomass

A precise measurement of two grams of microalgae biomass in powdered form (with a

particle size of 160µm) was carefully weighed and transferred into an open crucible. The

crucible was then placed inside a muffle furnace, which was maintained at a temperature of

550°C for a duration of 6 hours. Afterward, the sample was allowed to cool down to room

temperature within a desiccator. Once cooled, the sample's weight was measured again. The

difference in weight between the initial biomass and the residue left in the crucible represents

the ash content. Typically, this ash content is expressed as a percentage (Liu, 2019).
3.4 Free fatty acid (FFA)

The acid value and free fatty acid content of both microalgae oil and biodiesel were

analyzed at the Chemical and Process Engineering laboratories (CPE Labs). The acid value

represents the amount of potassium hydroxide required to neutralize the free fatty acid

present in one gram of oil or fat. The free fatty acid content in the Blue green oil/biodiesel

was determined through a titration method. A mixture of 1g of filtered oil/biodiesel, 25ml of

ethanol, and 2-3 drops of phenolphthalein indicator was prepared. The sample was dissolved

in warm 95% ethanol with stirring, followed by titration using a standard solution of 0.1M

sodium hydroxide and phenolphthalein indicator. The percentage of free fatty acid

wascalculated using Eq.3. (Sameer Mohammed., 2016).

3.5 Acid value

Acid value (𝐴. 𝑉. ) is given by the number of milligrams of potassium hydroxide needed

to neutralize the free fatty acid in one gram of oil/fat.

3.6 Properties measurement of Blue Green algae biomass, oil, and biodiesel

The specific gravity, kinematic viscosity, calorific value, acid number, flash point, ash

content, and moisture content properties of Blue Green biomass and biodiesel were analyzed

through different techniques in accordance with the American Standard Test Methods (ASTM

D 6751), which are also applicable for algae biodiesel determination.

3.7Biodiesel production process

3.7.1 Transesterification reaction

Biodiesel production and analysis were conducted at the CHE laboratories. The production of

biodiesel was done in batches, with the process staggered over time through

transesterification. This study aimed to optimize process variables such as algae to methanol

ratio (ranging from 1:4 to 1:14 g/ml), algae biomass to catalyst concentration (ranging from
1:0.0032 to 1:0.036818 wt. %), and reaction time (ranging from 1 to 11 min) for biodiesel

production using an in-situ method with the aid of microwave irradiation. The ideal

conditions for biodiesel production were determined based on experiments in a controlled

setting and the resulting data. Prior extraction of oil from microalgae was not performed

before the transesterification process to convert it into biodiesel. To ensure complete

dissolution, the catalyst and methanol were mixed using a stirrer (BibbySterilin ltd, UK -

CAT NO. SS10). A total of 15 grams of bluish-green, fine uniform dry powder (160μm mesh)

was mixed with the catalyst and methanol for analysis. The mixture was blended for

uniformity before being transferred to a reaction vessel (a 250ml, 3-necked round bottom

flask) equipped with a quick-fit condenser and an external stirrer. The reaction to produce

biodiesel took place in a 1300-watt microwave (Shivaki, Japan), which provided the

necessary energy for the process. The extraction and transesterification of bluish-green

microalgae biomass, along with the conversion of its lipids to biodiesel and glycerol,

occurred simultaneously within the reaction vessel. The reactants were heated and maintained

under microwave energy for a specific duration. After the conversion process, the resulting

product underwent a series of steps before obtaining the pure final product.

Figure 3.2: Schematic Diagram of a Microwave Synthesis Unit

3.7.2 Settling, separation and washing processes

Once the reaction had occurred, the mixture underwent a cooling process using running

cold water in order to halt the reaction. Subsequently, the mixture was decanted to separate

the solid residue from the liquid phase. To ensure thorough removal of any remaining liquid

phase (methyl esters) in the residue, the solid cake was washed with 30ml of methanol.

Filtration was then performed to separate the solid cake from the flowing liquid phase. The

Rotary Vacuum Evaporator (RVE) from HAHNVAPOR, Hahnshin Scientific Co. ltd, Korea,

was utilized, rotating at 20 RPM, to separate the solvent from the biodiesel and glycerol

formed. The RVE maintained the internal temperature of the rotating flask at 22°C, while the

external temperature of the water in the RVE water bath was set at 50°C. The RVE pump

effectively removed internal heat, ensuring that the separation occurred at a lower

temperature (22°C) and preventing any further reaction. To guarantee the separation of

biodiesel from the heavier glycerol and suspended solids, a centrifuge from Ittettich,

ZENTRIFUGEN D-7200 Tuttlingen, was employed, rotating at 6,000 RPM for a duration of

30 minutes. The centrifuge resulted in the formation of two distinct layers: a dark-brown,

more viscous substance known as glycerol settled in the collecting vessel, while the biodiesel

(light brown) could be observed on top of the glycerol layer. Decantation was then used to

separate the biodiesel from the glycerol. The pH of the biodiesel was subsequently

determined. The remaining layer, primarily consisting of biodiesel, underwent multiple

washes using warm distilled water to eliminate any soap, unreacted catalyst, and solid

particles. The mixture was gently heated to about 100°C to remove excess solvent and water.

The resulting substance is commonly referred to as biodiesel.

Figure 3.3: Process Flow Diagram of in situ Process of Biodiesel Production.

3.8 Laboratory Experiment Data Analysis

The analysis of fatty acid methyl esters (FAME) from Biodiesel was conducted using a

modified gas chromatography system (MRC Scientific Instrument: MRC/GC/39621381)

equipped with a flame ionization detector. The detector was maintained at 260°C, while the

injector port was set at 240°C. The temperature program for the oven was as follows: initially

held at 100°C for 2 minutes, then increased to 140°C at a rate of 10°C/min, further increased

to 190°C at 3°C/min, then to 260°C at 30°C/min, and finally maintained at 260°C for 2

minutes. Nitrogen gas served as both the carrier and make-up gas, flowing through a 30m x

0.25mm x 0.25µm Zebron ZB-FAME column installed in the injector and detector ports of

the Gas Chromatography system. Prior to elution through the capillary column, the Biodiesel

sample (50mg) was mixed with 5 ml of Methyl Heptadecanoate (C17:0, purity 99%) to create

a solution of 10mg/ml. The standard solution (Methyl heptadecanoate, 2mg/ml) was prepared

by dissolving in GC grade Hexane (purity 98.9%). An injection volume of 1µL was used with

a split ratio of 1:50 (EN-14214:2003).

 Gantt Chart

In Situ Transesterification of Blue Green Microalgae for Sustainable Biodiesel Production

Bowman, Jasmine , Ganaba, Willy Boy ,Versoza, Sarah Pearl

October November December January February March April May

Task W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W
1 W2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

Proposal of Topic and Title

Consultation

Approval of Topic

Gathering RRLs

Approval of Advisory

Writing of Chapter

Approval of Advisory

Revising of Chapter 1

Finalizing of RRLs

Writing for Chapter 2 :

Related Literature

Writing of Chapter 3:
Methodology

Proposal Defense

Loofah Fiber Collection and

Preparation

Alkali Treatment

Paper Bag Production

Tensile testing test

Collecting and interpretation

of the data

Writing of Chapter 4: Results

and Discussion
Writing Chapter 5:
Conclusions and
Recommendation

Executive Summary

Bibliography

Final Defense
Line Budget Distribution

Materials/ Tools quantity total cost

Methanol or
Ethanol 1000 ml 249
Hexane 500 ml 750
Isopropyl
Alcohol 500 ml 175
Ethanol 500 ml 334
Biodiesel
standard (Methyl
Heptadecanoate) 1g >99.0% 11,500
Potassium
hydroxide 500 grams 1,500
Phenolphthalein
indicator 250 ml 3,031
Total 17, 539