COMPARATIVE STUDY FOR BIOMASS IMPROVEMENT

STRATEGY OF MICROALGAE USING DIFFERENT

WASTEWATER AND SUBSEQUENT BIOETHANOL
PRODUCTION
submitted in partial fulfilment of the requirements

for the award of the degree of

Masters of Science

In

FOOD PROCESSING AND NUTRITION SCIENCE

By

ARGHYA MUKHERJEE

ENROLLMENT NO. -2022CTM005

SESSION-2023-2024

Under the Guidance of

DR. SHANTONU ROY

SCHOOL OF COMMUNITY SCIENCE AND TECHNOLOGY

INDIAN INSTITUTE OF ENGINEERING SCIENCE AND TECHNOLOGY,

SHIBPUR, HOWRAH -711103, WEST BENGAL

JULY 2024

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 DECLARATION

I hereby declare that the research work embodied in the thesis entitled " Comparative study
for biomass improvement strategy of microalgae using different wastewater and
subsequent bioethanol production" is my own work conducted between March 2023 to
June 2024 under the supervision of Dr. Shantonu Roy, Assistant Professor, School of
Community Science and Technology, IIEST Shibpur (711103).

I further declare that to the best of my knowledge, neither this thesis nor any part of it has
been submitted for any academic award anywhere before.

Signature of supervisor

Dr. Shantonu Roy

Assistant Professor

School of Community Science and Technology

IIEST, Shibpur

Howrah (711103)

Signature of the candidate

Arghya Mukherjee

Enrollment no.- 2022CTM005

IIEST, Shibpur
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INDIAN INSTITUTE OF ENGINEERING SCIENCE AND TECHNOLOGY
SHIBPUR, HOWRAH (711103)

FORWARD
We hereby forward the thesis entitled " Comparative study for biomass improvement
strategy of microalgae using different wastewater and subsequent bioethanol
production" prepared by Arghya Mukherjee(2022CTM005) under our guidance and
supervision in partial fulfillment of the requirements for the degree of Master of Science in
Food Processing and Nutrition Science at IIEST, Shibpur, Howrah (711103).

Dr. Shantonu Roy

Assistant Professor

School of Community Science and Technology IIEST, Shibpur

Howrah (711103)

Prof. Ajit Kumar Mahapatra

HOD SOCSAT

IIEST, Shibpur Howrah (71110)

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INDIAN INSTITUTE OF ENGINEERING SCIENCE AND TECHNOLOGY
SHIBPUR, HOWRAH (711103)

CERTIFICATE OF APPROVAL
The thesis entitled " Comparative study for biomass improvement strategy of microalgae
using different wastewater and subsequent bioethanol production" is hereby approved as
a creditable study of an applied science subject carried out and presented in a satisfactory to
warrant its acceptance as a prerequisite to the degree of Master of Science in Food Processing
and Nutrition Science. It is understood that by this approval the undersigned do not
necessarily approve any statement made, opinion expressed and conclusion drawn therein but
approved the thesis report only for the purpose of which it is submitted.

Board of examiners:

1.

2.

3.

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INDIAN INSTITUTE OF ENGINEERING SCIENCE AND TECHNOLOGY
SHIBPUR, HOWRAH (711103)

ACKNOWLEDGEMENT
At the onset, I would like to express my dense of respect and gratitude to Dr. Shantonu Roy
(Assistant Professor, SOCSAT, IIEST Shibpur) for his valuable advice, resourceful guidance,
active supervision and constant encouragement without which it would not have possible to
submit the thesis in a shape in time. I am also thankful to Dr. Jayati Bhowal (Asst. Prof,
SOCSAT, IIEST, Shibpur) and Dr. Dipsikha Kalita (Asst. Prof, SOCSAT, IIEST,Shibpur)
for the constant encouragement and co-operation. I am also thankful to research scholars, my
friends, who helped me to prepare this work.

Date- 15/07/2024
School of Community Science and Technology Arghya Mukherjee

Indian Institute of Engineering Science and Technology 2022CTM005

Shibpur, Howrah -711103, West Bengal

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TABLE OF CONTENTS PAGE NO.
ABSTRACT……………………………………………………………………………. 10
1. INTRODUCTION…………………………………………………........................... 11-12

2. LITERATURE REVIEW………………………………………….......................... 13-39

2.1Wastewater and Environmental pollution
2.2 Microalgae as a medium for wastewater treatment
2.3 Modes of cultivation of microalgae
2.3.1 Photoautotrophic
2.3.2 Heterotrophic
2.3.3 Mixotrophic
2.4. Biochemical composition of microalgae
2.4.1 Proteins
2.4.2 Carbohydrates
2.4.3 Lipids
2.5 Wastewater for microalgae cultivation
2.5.1 Characterization of wastewater
2.5.2 Nutrients from wastewater for microalgae cultivation
2.5.2.1 Municipal wastewater
2.5.2.2 Agricultural wastewater
2.5.2.3 Industrial wastewater
2.6 Efficiency of Microalgal growth in wastewater
2.6.1 Microalgal growth in municipal wastewater
2.6.2 Microalgal growth in agricultural wastewater
2.6.3 Microalgal growth in industrial wastewater
2.7. Biofuel production from microalgae
2.8. Bioethanol production from microalgae
2.8.1 Accumulation of carbohydrate during microalgae cultivation
2.8.2 Fermentation and hydrolysis process of microalgal biomass
2.8.2.1 Dark Fermentation for Bioethanol Production
2.8.2.2 Photo fermentation for Bioethanol Production
2.9 Yeast in Ethanol Production
2.9.1 Diversity of Yeast
2.9.2 Genetics of Yeast
2.9.3 Yeast in the Production of Bioethanol
2.10 Application of bioethanol
2.11 Bioethanol purchase policy in India

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3. MATERIALS AND METHODS………………………………………………… 40-46
3.1 Wastewater collection
3.2 Characterization of wastewater
3.2.1 Estimation of total nitrate content of wastewater samples
3.2.2 pH determination of different wastewater samples
3.2.3 Determination of Biological Oxygen Demand (BOD) of different wastewater
Samples
3.2.4 Determination of Chemical Oxygen Demand (C.O.D.) of different wastewater
Samples
3.2.5 Estimation of the carbohydrate content of different wastewater samples
3.2.6 Protein estimation by Lowery method
3.2.7 Estimation of total reducing Sugar of the wastewater samples
3.3 Cultivation of Micractinium sp. in different wastewater
3.3.1 Microalgae seed culture
3.3.2 Optimization of different wastewater concentrations for biomass estimation
3.3.3 Biochemical profile analysis of microalgae cultivated in different wastewater
3.3.3.1 Estimation of carbohydrate content of microalgae
3.3.3.2 Estimation of reducing sugar content of microalgae
3.3.3.3 Estimation of total starch content of microalgae
3.3.3.4 Estimation of cellulose content of microalgae
3.3.3.5 Determination of amylase activity of microalgae
3.4 Nutrient removal efficiency study of algae
3.5 Ethanol Production from algal biomass
3.5.1 Optimization of acid concentration for pre-treatment of algal biomass
3.5.2 Effect of Microwave heating time exposure
3.5.3 Optimization of saccharification by enzyme
3.5.4 optimization of overall pre-treatment and saccharification process
3.5.5 Optimization of yeast concentration
3.5.6 Production of bioethanol under optimized treatment
3.5.7 Estimation of ethanol yield

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4. RESULTS AND DISCUSSIONS………………………………………………… 47-55
4.1 Characterization of wastewater
4.2 Characterization of algal biomass
4.3 Estimation of nitrate and phosphate removal efficiency
4.4 Effect of sulphuric acid concentration on acid hydrolysis
4.5 Effect of microwave heating on algal biomass hydrolysate
4.6 Optimization of the overall pre-treatment and saccharification process
4.7 Effect of Yeast Inoculum on Bioethanol yield
4.8 Estimation of bio-ethanol yield

5. CONCLUSIONS………………………………………………………………….. 56
REFERENCES……………………………………………………………………… 57-66

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 List of Diagrams
Fig.1- Phosphate and nitrate removal on a daily basis………………………………….. 50

Fig 2- Effect of sulphuric acid concentration on reducing sugar content………………. 51

Fig 3- Effect of microwave heating on reducing sugar content………………………… 52

Fig 4- Influence of overall optimization process on reducing sugar content…………………... 53

Fig 5- Effect of yeast inoculum on bioethanol yield……………………………………. 54

Fig 6- Overall Bioethanol yield…………………………………………………………. 55

Fig 7- Ethanol content vs. reducing sugar content of three hydrolysate………………… 55

List of tables
Table 1- Biochemical composition of different microalgal species…………………….. 18

Table 2- Characterization of wastewater……………………………………………….. 47

Table 3- Characterization of algal biomass…………………………………………….. 48-49

Table 4- Phosphate and nitrate removal efficiencies of microalgae (Micractinium sp.) in

different wastewater…………………………………………………………………….. 50

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 Abstract

Microalgae biomass can produce high quantities of biochemicals used in various applications
such as bioethanol, biogas, and aquaculture feed. The potential of sterilizing wastewater for
microalgae-based wastewater treatment on a lab scale is well-introduced. The present study
aimed to evaluate the growth of Micractinium sp. in three different types of wastewater
(artificial, slaughterhouse, and starchy) to compare the improvement in the biomass
concentration and to check the nutrient removal efficiency of the microalgae in three different
types of wastewater and subsequent production of bioethanol from that cultivated algal
biomass hydrolysate. Microalgae was cultivated in different concentrations of wastewater
collected from the local market. Under optimized conditions, 7.34±0.15 gL-1 maximum
biomass yield was observed in slaughterhouse wastewater. Micractinium sp. in crude
slaughterhouse wastewater achieved 97.36±2.41% and 98.86±3.91% of phosphate and nitrate
removal efficiencies, respectively, which was 9.27% (Phosphate) and 9.18%( nitrate) higher
than the control artificial wastewater. The results also showed that the microalgae cultivated
in slaughterhouse wastewater contained the highest amount of reducing sugar (58±0.6%)
after different treatment was done to the algal biomass hydrolysate. It was observed that the
pretreatment and saccharification gave the maximum ethanol percentage of 22% using the
yeast volume of 10%. This study demonstrated that Micractinium sp can grow well in three
different wastewaters while effectively removing phosphates and nitrates. also, cheap and
cost-effective bioethanol can be produced from microalgae.

Keywords: microalgal biomass; nitrate removal; reducing sugar; ethanol yield; removal
efficiency; Micractinium sp; phosphosphate removal

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 1. Introduction

With the increasing global population, rapid urbanization, and, eventually, economic
advancements, the energy demand has increased worldwide. However, it is essential to note
that fossil fuels are not sustainable long-term energy resources. Burning fossil fuels can
increase greenhouse gas (GHG) emissions, contributing to the environmental effects
associated with global warming (Hill et al., 2006). Renewable energy sources provide
environmentally friendly alternatives to traditional fossil fuels. Presently, the primary forms
of renewable energy widely available in the market are bioethanol and biodiesel. These
biofuels have gained popularity due to their environmentally friendly nature and sustainable
production methods. Bioethanol is derived from crops such as corn, sugarcane, or wheat,
while biodiesel is typically made from vegetable oils or animal fats. These alternative fuels
offer a promising solution to reducing reliance on fossil fuels and combating climate change.
Crop-based biofuels are in direct economic competition with the production and pricing of
food (Hill et al., 2006). Microalgae-based biofuels have emerged as a promising alternative
that avoids any negative impact on agriculture. Microalgae are believed to have a higher
capacity for producing biomass than traditional plant crops, requiring less land for
cultivation. Additionally, they are expected to have a lower cost per yield and offer the
potential to decrease greenhouse gas emissions by substituting fossil fuels. Microalgae
growth relies on several key elements, including sunlight, water, carbon dioxide (CO2), and
essential nutrients. These inputs are crucial for the development and sustenance of
microalgae. Water and inorganic nutrients are recognized as crucial limiting factors for the
cultivation of microalgae. The essential elements needed for microalgae growth (primarily
nitrogen and phosphorus) can be acquired from liquid effluent wastewater. Improper
wastewater management, produced from domestic, industrial, and agricultural activities,
poses a substantial environmental risk. Wastewater frequently contains elevated levels of
nutrients like nitrogen and phosphorus, which are crucial for the growth of plants.
Nevertheless, excessive quantities can result in a process called eutrophication. Therefore, in
addition to creating an optimal environment for microalgae growth, there is also the
opportunity to utilize waste effluents for treatment.

Using algae, microalgae, and cyanobacteria to create third-generation biofuels offers

numerous benefits compared to using higher plants for producing first and second-generation
biofuels. Due to their rapid growth and ability to thrive in various environments, including
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wastewater, these organisms exhibit accelerated development. Under optimal growth
conditions, microalgae have a diverse biochemical composition consisting mainly of proteins
(30-50%), carbohydrates (20-40%), and lipids (8-15%), without any nutrient restrictions. (Hu
et al.,2013, Cardoso et al.,2011, Ho et al.,2013).

Three potential pathways exist for utilizing microalgal and cyanobacterial biomass in
bioethanol production. The initial method follows the conventional procedure where the
biomass goes through pre-treatment stages, enzymatic breakdown, and fermentation by yeast.
In dark conditions, the second approach involves utilizing metabolic pathways to shift the
focus of photosynthesis toward generating hydrogen, acids, and alcohols (like ethanol). The
third method involves "photo fermentation," which is not practical. The final approach
involves genetic manipulation to redirect the existing biochemical pathways of microalgae to
achieve a more personalized and effective bioethanol synthesis.

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 2. LITERATURE REVIEW

2.1 Wastewater and Environmental Pollution

Wastewater pollution poses an increasing danger to both humans and marine life, and it
constitutes the highest proportion of coastal pollution on a global scale. According to reports,
approximately 80 percent of wastewater, including human sewage, is released into the
environment without treatment. This releases harmful pollutants into the ocean, directly
harming individuals and coral reefs. Shockingly, more than 40 percent of the world's
population, equivalent to 3.46 billion people, do not have access to sanitation services that are
managed safely.

Wastewater contamination arises from various origins, such as industries, farming, and urban
areas. This pollution enters the ocean through different pathways, including runoff from land
surfaces, direct and treated discharge, and infiltration into the groundwater.

Dealing wastewater into the ocean and combined with seawater leads to the dispersion of
pollutants. The severity of the impacts of wastewater pollution is influenced by geography,
population size, infrastructure type, and climate change. These impacts include:

• Coral reefs, seagrasses, and salt marshes suffer physical and biological damage due to
the increased presence of nutrients, pathogens, plastics, and pharmaceuticals.
• Harmful algal blooms that harm marine life result in beach closure and contribute to
human diseases.
• Pathogens, heavy metals, and toxic chemicals cause the transmission of human and
animal diseases.

2.2 Microalgae as a medium for wastewater treatment

In wastewater treatment, removing inorganic nitrogen and phosphorus poses a significant

challenge. However, microalgae offer a promising solution as they can use these pollutants
for their growth. By harnessing the power of microalgae, it becomes possible to effectively
reduce the concentration of inorganic nitrogen and phosphorus in wastewater (Ahluwalia et
al.,2007). Numerous types of microalgae can thrive in wastewater environments by
efficiently utilizing the ample inorganic nitrogen and phosphorus in the wastewater.

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Consequently, large-scale microalgae cultivation holds excellent potential for implementing
wastewater treatment as a tertiary process (Martin et al.,1985).

The cost of implementing a comprehensive tertiary process in wastewater treatment, which

focuses on removing nitrogen and phosphorus, is approximately four times higher than that of
primary therapy (de la Noüe et al.,1992). Microalgal cultures provide a sophisticated answer
to tertiary treatment because they can utilize inorganic nitrogen and phosphorus for their
growth. This makes them an excellent option for addressing the needs of tertiary treatment.
Furthermore, their ability to eliminate heavy metals and certain harmful organic substances
does not create additional pollution. To summarize, utilizing microalgae cultures in
wastewater treatment can significantly aid in maintaining water ecosystems by offering a
cost-effective and environmentally friendly solution.

Mass-cultured microalgae offer several significant benefits compared to traditional aerobic

wastewater treatment systems. These advantages include cost reduction through decreased
energy consumption, lower initial capital investment, and reduced operational expenses
(Simpson et al.,1997). On the other hand, there are numerous drawbacks to consider. One
such disadvantage is the space needed for cultivating microalgae. Due to their reliance on
photosynthesis, sunlight must reach the microalgae. As a result, microalgae-based wastewater
treatment systems should be implemented in areas with low land costs where ample sunlight
and warm temperatures are available.

The standard wastewater treatment process can be divided into three main stages: separating
solid and liquid components, conducting anaerobic fermentation horizontally, and utilizing an
activated sludge method in aerobic treatment. Following this three-step approach, the levels
of biochemical oxygen demand (BOD) and suspended solids (SS) in the treated wastewater
are significantly decreased (Hongyang et al.,2011). The wastewater discharged contains
nitrogen, phosphorus, and other essential nutrients, while the flue gas contains CO2. The
effluent and flue gas serve as sources of nutrition for microalgae growth. By combining the
treatment of wastewater with the utilization of flue gas, we have developed an integrated
system that is both environmentally friendly and sustainable. This system effectively treats
wastewater and mitigates CO2 emissions (Rawat et al.,2011).

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2.3 Modes of cultivation of microalgae

The type of microalgal cultivation greatly influences the pattern of microalgal growth. It also
influences the quality and quantity of biodiesel products. Three modes of cultivation have
been observed in microalgae- photoautotrophic, heterotrophic, and mixotrophic.

2.3.1 Photoautotrophic

The most common and energy-saving microalgae cultivation type is a photoautotrophic

cultivation mode. The whole cultivation process is carried out inside a photobioreactor (PBR)
or in an open pond. Light is the most crucial factor in microalgal cultivation. ATP is
generated from absorbed light energy, and NADPH is also generated. Photosynthetic
microalgae utilize light as an energy source, whereas inorganic carbon serves the purpose of
the carbon source. CO2 is an essential factor in photoautotrophic cultivation since the high
concentration of CO2 can enhance biomass productivity. Although photoautotrophic mode is
an established method of microalgal cultivation, there is a significant disadvantage of this
type of cultivation process; as the reaction progresses, increases in broth turbidity decrease
the light penetration exponentially, which inhibits achieving high biomass productivity. The
selection of strain is an essential factor in photoautotrophic cultivation, as the amount of lipid
accumulation by microalgae depends on the strain type used in the cultivation process. The
percentage of lipid accumulation varies between 5% to 68% (Chen et al., 2011). Studies show
that Chlorella sp. achieves the highest lipid content under the photoautotrophic cultivation
method provided with 2% carbon-di-oxide and 0.25 vvm aeration of 179mg/L/d (Yen et
al.,2016). Generally, a limiting or nutrient-limiting environment could be favorable for
obtaining higher lipid content; however, the amount of biomass obtained in this condition is
much lower than under normal circumstances, sometimes resulting in much lower lipid
production. This photoautotrophic mode of cultivation can be done in an open-scale platform
(like a pond). The cultivation cost of microalgae is very high, which is one of the significant
drawbacks of this procedure.

The photo-bioreactors used for microalgal cultivation should be exposed

to light energy with optimum photons for photosynthesis. One of the significant
disadvantages of using a photobioreactor is that higher microalgal density inhibits light
penetration. Because of that, less light reaches the bioreactor's inner part, resulting in less
biomass productivity (Chen et al., 2011).

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 2.3.2 Heterotrophic

In the heterotrophic mode of microalgal cultivation, microalgae consume organic compounds

as both carbon and energy sources. This process is independent of light; thus, it can avoid the
limitations associated with light in photoautotrophic cultivation. The lipid content is also
higher in heterotrophic cultivation. About 40% of higher lipid contained was obtained by
Chlorella protothecoides when shifting the cultivation mode from photoautotrophic to
heterotrophic (Xu et al., 2006). Glucose, lactose, galactose, and fructose are commonly used
as organic carbon sources for microalgae growth (Liang et al., 2009). High lipid and biomass
productivity of 932mg/L/d and 2g/L/d were obtained by using the strain Chlorella
protothecoides, and as an organic carbon source, corn powder hydrolysate was used (Ananthi
et al., 2021). Again, using the same strain under the fed-batch mode of cultivation, the highest
amount of lipid productivity obtained was 3700 mg/L/d (Ananthi et al., 2021; Liang et
al.,2009). The heterotrophic cultivation mode uses sugar as an organic carbon source, which
sometimes can lead to contamination by some microorganisms, affecting the product's
quantity and quality. Therefore, obtaining axenic monoalgal culture is very important in this
cultivation process. In heterotrophic cultivation, organic carbon sources are used as both
carbon and energy sources; thus, it increases the cost of the substrate, which is required for
microalgal growth.

2.3.3 Mixotrophic

Mixotrophic cultivation is how microalgae can operate on photoautotrophic and heterotrophic

conditions. Mixotrophic microalgae perform photosynthesis and use it as an energy source. It
also utilizes both inorganic and organic carbon sources. Those microalgal strains assimilate
organic compounds and CO2, which is released due to the respiration by microalgae; it is also
trapped and later utilized under light energy sources in photoautotrophic conditions (Ananthi
et al., 2021). This process also decreases the amount of CO2 in the environment; thus, it is
also environmentally friendly.

There are particular controversies regarding the growth rate of the mixotrophic mode of
cultivation. According to a group of scientists, it is approximately the sum of
photoautotrophic and heterotrophic modes of microalgal growth. In contrast, some believe it
is not the simple combination of two other modes of microalgal growth. However,

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photosynthesis of photo autotrophy and respiration of heterotrophy affect each other and add
a synergistic effect that increases biomass productivity.

Since mixotrophic cultivation can drive both photoautotrophic and as well heterotrophic, it
does not depend solely on photosynthesis, which also means that light energy is not an
essential factor for mixotrophic growth; therefore, chances of photo-inhibition or photo
limitation becomes very much less in mixotrophic mode (Molina et al.,2017). Since organic
compounds are being utilized in a mixotrophic mode, the chance of contamination is also
high. To prevent contamination, a closed bioreactor is recommended for this type of
microalgal growth, in which an artificial light source will be provided (Shu et al.,2016).

Mixotrophic cultivation has several advantages: a) the growth rate of mixotrophic cultivation
is higher than the other two modes; thus, the production of biomass is also higher within a
shorter growth cycle b) the more extended exponential phase, c) biomass loss during the dark
reaction also becomes less as the photoinhibition effect also declined in this process d)
interconversion between heterotrophic to photoautotrophic mode is easy ( Ananthi et
al.,2021; Wang et al.,2014; Kröger et al., 2011; Chojnacka et al.,2004 )

2.4 Biochemical composition of microalgae

Microalgae produces many biochemical compounds, including proteins, carbohydrates, fats,

nucleic acids, vitamins and minerals. The amount of these biochemical compounds inside the
cell depends on the different biochemical strains of microalgae depending on their responses
to abiotic and biotic factors, e.g., photoperiod, light intensity, nutrients, and growth phase
(Barkia et al., 2019). The biochemical composition of some selected species of micro-algae is
shown in the table below:

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 Table 1: Biochemical composition of different microalgal species:

Species % Protein(w/w) %Fat(w/w) %Carbohydrate(w/w) References

Chlamydomonas 48 21 17 Tibbetts et al., 2015
reinhardtii
Nitzschia 26 13 9.8 Barkia et al., 2019
Closterium
Dunaliella 11 - - Barkia et al., 2019
tertiolecta
Dunaliella salina 57 6 32 Becker et al., 2007
Chaetoceros 40 23 37 Velasco et al., 2016
calcitrans
Chaetoceros 59 31 10 Velasco et al., 2016
muelleri
Spirulina maxima 60-71 6-7 13-16 Becker et al., 2007
Pavlova sp. 24-29 9-14 6-9 Brown et al.,1991
Thalassiosira 34 19 8.8 Brown et al.,1991
pseudonana
Nannochloropsis 30 22 10 Kent et al.,2015
sp.
Porphyridium 28-39 9-14 40-57 Becker et al., 2007
cruentum
Prymnesium sp. 28-45 22-31 25-33 Ricketts et al.,1966
Isochrysis galbana 27 11 34 Aranda et al.,2013
Chlorella vulgaris 51-58 14-22 12-17 Becker et al., 2007
Scenedesmus sp. 31 15 28 Kent et al.,2015

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 2.4.1 Proteins

Proteins influence the structure and metabolism of microalgal cells. Proteins are the essential
constituents of cell membranes and the numerous catalytic enzymes involved in
photosynthesis (Williams et al.,2010). The amount of proteins in different microalgal cells
varies quantitatively and qualitatively (Batista et al.,2013; Graziani et al.,2013). The report
shows that several microalgae species contain a very high protein content, starting from 42%
and going up to 72% in certain cyanobacterial species (Millovanovic et al., 2012; Plaza et
al.,2009). Microalgal protein contains all the essential amino acids that mammalian cells
cannot synthesize; thus, in terms of quality, microalgal protein is much richer. Furthermore, a
well-balanced amino acid profile is observed in microalgae, and protein sources are
comparable with some high-quality animal and plant protein sources like egg albumin, soy,
and lactoglobulin (Williams et al.,2010). The presence of non-protein components (e.g.,
chlorophyll) in microalgal-based products affects the taste and color of the products (Becker
et al.,2007). Thus, it limits the application of microalgal proteins in foods.

2.4.2 Carbohydrates

Carbohydrates (mono, poly, and oligosaccharides) play an essential role in the metabolic and
structural functions of the cell. Glycolipids, glycoproteins, and other complex
polysaccharides are the major constituents of the cell wall (Arad et al.,2010). Moreover,
microalgae synthesize glucose- or starch-based energy-rich products through photosynthesis
(Williams et al.,2010).

Different strains of microalgae produce various forms of accumulation s of polysaccharides.

Cyanobacteria are known for glycogen production, although some specific microalgal strains
synthesize semi-amylopectin (α-poly glucans) (Nakamura et al.,2005). Amylose and
amylopectin, the two building blocks of starch, are synthesized by Chlorophyta (Busi et
al.,2014), while Floridian starch (a type of carbohydrate polymer produced by Rhodophyta

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(Gügi et al., 2015). Chrysolaminarin (a storage polysaccharide) is produced by Diatoms
(Heterokontophyta, Bacillariophycae). (1,3)-β-D-glucan can be stored up to 30% of its dry
weight by some diatoms species during the log phase of the growth cycle, and it can go up to
80% under nutrient stress (Dismukes et al., 2008).

Even though microalgae are a valuable source of carbohydrates, their use in the food industry
is minimal. In contrast, microalgal polysaccharides are becoming increasingly crucial in the
cosmetic industry as hygroscopic agents (de Jesus Raposo et al., 2013).

2.4.3 Lipids

Among all the biomolecules, microalgal lipids have received the most attention recently.
Microalgal lipids mainly consist of polar lipids such as glycolipids and phospholipids,
unsaturated fatty acids (UFAs), and triacylglycerol (TAG). Among these, UFAs and glycerol
are utilized to obtain energy. Polar lipids in cell membranes and other organelles contain
UFAs (Lupette et al., 2020). Polar lipids are the most abundant compound during the
exponential phase of the microalgal growth cycle. Under nutrient-limited and stress
conditions, TAGs are accumulated (Rodolfi et al., 2009). Microalgal fatty acids contain both
saturated and unsaturated long-chain fatty acids (C16 and C18), which include w-3 and w-6
fatty acids. Saturated fats are found in neutral lipid bodies. The polar lipids are predominantly
associated with unsaturated fatty acids that help maintain the membrane fluidity (Williams et
al.,2010). Numerous parameters, viz., light intensity, growth environment, strain type,
temperature, and pH, affect the microalgal lipid content.

Microalgal lipids generally account for up to 20% to 50% of the dry biomass (w/w).
However, it has been reported that the amount of intracellular lipid in a nitrogen-limiting
environment increases significantly. It was also reported that microalgal cultures during the
stationary phase increase their total neutral lipid content by a factor of two. (Robert et al.,
2003, Thompson et al., 1993).

2.5 Wastewater for microalgae cultivation

Over the last twenty years, significant research has been dedicated to studying the cultivation
of microalgae with the use of wastewater. Several studies have shown that the application of
microalgae can effectively eliminate nitrogen, phosphorus, and heavy metal components from
wastewater (Wang et al., 2010). Furthermore, the presence of various nutrients in wastewater

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substantially impacts the growth of microalgae and their overall biomass and lipid production
(Cai et al., 2013). The discussion will focus on the nutrient composition of various
wastewater streams that cultivate microalgae.

2.5.1 Characterization of wastewater

The utilization of wastewater in microalgal cultivations can be categorized into three primary
types based on its source: municipal, agricultural, and industrial liquid waste products. The
process of treating wastewater typically involves two or three phases. Firstly, a primary
treatment phase separates solid materials through sedimentation and floatation. Secondly, a
secondary treatment phase employs physical, chemical, and biological methods to eliminate
suspended and dissolved organic substances. Lastly, a tertiary treatment phase, which
includes disinfection and filtration, ensures the final purification of the water before it is
released into the environment (Ramalho et al., 1977). The effluent refers to the discharged
water undergoing a wastewater treatment process.

In the effluents of wastewater, various types of waste are commonly found, such as sludge
and scum, organic waste, inorganic waste, nutrients, toxins, and pathogenic organisms. This
review article focuses on the importance of nutrients in wastewater for microalgal growth.
The presence of excessive nutrients, like nitrogen and phosphorus, in sewage can lead to
eutrophication in lakes and disrupt the balance of the ecosystem (Cai et al., 2013). Nutrients
are essential for the development of microalgae. The critical nutrients of concern include
nitrogen and phosphorous. Nitrogen can be found in effluent as ammonia, nitrogen bound to
organic compounds, or nitrite and nitrate. Phosphorous is predominantly present in influent
and effluent as phosphates.

2.5.2. Nutrients from wastewater for microalgae cultivation

The nitrogen and phosphorus concentrations found in wastewater can vary greatly depending
on the type of wastewater. Nitrogen is often present in wastewater in the form of ammonia
and nitrates, which are commonly found chemicals containing nitrogen. Among the various
chemical forms of nitrogen, ammonium is one of the most frequently encountered types that a
wide range of microalgal species and strains can easily absorb. A cost-effective nitrogen
source found in wastewater or effluent can be utilized to cultivate microalgae (Razzak et al.,

21 | P a g e
2013). High levels of total nitrogen, particularly ammonium, can hinder the growth of
microalgal cultures in wastewater, as indicated by specific studies. However, it has been
observed that when the culture's pH and other growth conditions are carefully regulated,
ammonium can serve as a dependable nitrogen source (De-Bashan et al.,2010). To
summarize, despite the adverse impact on microalgae growth when supplemented with
ammonium, it remains the favored nitrogen source as long as the environmental conditions
necessary for the culture's optimal growth are carefully regulated (Razzak et al., 2013).
Phosphorus is a vital element needed for the growth and metabolism of microalgae. It plays a
crucial role as ATP in the cells of these microorganisms. Consequently, the availability of
phosphorus dramatically influences the growth of microalgae, particularly during the process
of photosynthesis (Razzak et al., 2013). Phosphorus is typically present in wastewater as an
inorganic anion species, such as H2PO4 - and HPO42- (Martinez et al., 1999).

The levels of TN and TP in domestic secondary effluent are relatively low, with total nitrogen
ranging from approximately 15-90 mg L-1 and total phosphate ranging from about 5-20 mg
L-1. These concentrations are typical for domestic wastewater. On the other hand, the
concentrations of total nitrogen and total phosphate in wastewater from livestock breeding
and agriculture are much higher, usually ranging from 185-3,213 mg L-1 for TN and 30-987
mg L-1 for total phosphate. Such wastewater includes anaerobic digested poultry litter
effluent, swine, or dairy manure. However, these types of wastewater contain incredibly high
levels of nutrients and must be diluted before being used for microalgal cultivation.

2.5.2.1 Municipal Wastewater

The expansion of urban populations and the increasing urbanization have led to a rise in
municipal wastewater, also known as domestic wastewater. The composition of municipal
wastewater varies significantly from one location to another. Typically, it contains human
and other organic waste, nutrients, microorganisms, household chemicals, and industrial
chemicals. Municipal wastewater has lower nitrogen and phosphorus levels than industrial
and agricultural wastewater. However, due to the activities of localized, small-scale factories,
raw municipal sewage may also contain significant amounts of heavy metals such as lead,
zinc, and copper. The cultivation of microalgae using municipal wastewater as a nutrient
source has been extensively studied, with a focus on removing nitrogen and phosphorus (Li et
al., 2011; Ruiz-Marin et al., 2010). While the nitrogen and phosphorus found in municipal
wastewater can serve as nutrient sources, it is crucial to consider the wastewater's complexity,
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diversity, and toxicity at each treatment stage to meet the wastewater discharge standards.
Therefore, it is essential to study microalgae cultivation using wastewater and consider the
effectiveness of wastewater treatment in removing the most harmful components (Cabanelas
et al., 2013). Typical examples of municipal wastewater include rice gruel water,
slaughterhouse wastewater, etc.

2.5.2.2 Agricultural wastewater

In most cases, agriculture accounts for the highest water consumption globally, with only a
few regions being exceptions. The wastewater generated from agricultural activities, which
includes animal manure, plant stalks, hulls, and leaves, among others, carries water that is
contaminated with waste materials. The point source wastewater, originating mainly from
large-scale livestock and poultry operations, is a significant contributor. Over the past few
decades, there has been a notable shift in livestock operations from small-scale to large-scale,
resulting in intensifying levels of nitrogen and phosphorus as the primary components in the
wastewater produced by animal farms (Zhu et al., 2013). Ammonium and organic nitrogen
are the primary constituents found in agricultural wastewater. The predominant form of
nitrogen waste in animal waste is ammonium, accounting for nearly half of the total nitrogen
content. The nutrient composition of animal wastewater is greatly influenced by various
factors, including animal diet, usage, productivity, and location (An et al., 2003; de Godos et
al., 2009; Wang et al., 2010; Wilkie & Mulbry, 2002; Zhu et al., 2013)

2.5.2.3 Industrial Wastewater

There are numerous categories of industrial wastewater, which are determined by the specific
industry and the contaminants involved. Each sector generates its unique blend of pollutants.
In contrast to agricultural wastewater, the composition of industrial wastewater varies
depending on the activities carried out. Heavy metals are commonly found in industrial
wastewater, while nitrogen or phosphorus levels are typically lower than in municipal or
agricultural wastewater (Chinnasamy et al., 2010). The quantity of wastewater produced
varies based on the technological advancements in different industry sectors. As industrial
technologies continue to evolve, there is a gradual reduction in sewage generated. Developing
countries are believed to experience higher rates of industrial wastewater than developed
countries.

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Screening and isolating microalgae species and strains with a high tolerance for metals and
organic materials is essential for achieving optimal growth efficiency in industrial
wastewater. However, more research is needed on cultivating microalgae with high metal
tolerance and their ability to remove nitrogen and phosphorus (Ahluwalia & Goyal, 2007).

2.6 Efficiency of microalgal growth in wastewater

Numerous factors influence the growth of microalgae in wastewater. These factors can vary
depending on the type and source of the wastewater. Additionally, different microalgae
species have varying tolerance levels to specific wastewater conditions. Chlorophytes, a large
group of microalgae, have a diverse range of species and can be found in various parts of the
world. Chlorophyte microalgae have demonstrated potential for use in different wastewater
conditions and high efficiency in removing nutrients from wastewater. Chlorella, a strain of
Chlorophytes, has been extensively studied and has proven effective in removing nitrogen
and phosphorus from sewage, even at different initial concentrations. Many studies have
examined the growth of Chlorella species cultured in wastewater (Wu et al., 2014).
2.6.1 Microalgal growth in municipal wastewater
Many studies in published literature have focused on using municipal wastewater for
cultivating microalgae. This is primarily because municipal wastewater treatment methods
are well-established in developed countries. However, the chemical composition of municipal
wastewater can be complex due to the mixing of low concentrations of industrial wastewater
from local small factories. Some municipal wastewater may also contain trace amounts of
heavy metals or chemicals, which are diluted by domestic wastewater to meet biological
treatment and discharge standards more easily (Gizgis et al.,2006). Therefore, the impact on
the development of microalgal culture would differ. In this analysis, we have compiled a
comparison of microalgal biomass generation using various types of municipal wastewater,
primarily focusing on the levels of phosphorus and nitrogen.
Microalgae Chlorella efficiently removes nutrient pollution and utilizes it when the algal
cells are cultivated in domestic wastewater. An investigation was conducted on the capability
of C. vulgaris to remove nutrients, which resulted in an 86% removal efficiency for inorganic
nitrogen and 78% for inorganic phosphorus. Recent findings indicate that cultivating C.
vulgaris using various streams of municipal wastewater yields microalgal biomass ranging
from 39 to 195 mg L-1 d-1 (Cabanelas et al., 2013). According to Cho et al. (2013), Chlorella
24 | P a g e
sp. was found to have the highest biomass production of approximately 3.0 g L-1 when using
10% anaerobic digestion tanks. They also used a combination of 90% wastewater and a
conflux line as nutrients for microalgal cultivation. Li et al. (2011) demonstrated that
Chlorella sp. had a biomass productivity of 0.9 g L-1 d-1 when grown in the center, a
concentrated municipal wastewater stream produced from the activated sludge thickening
process. Therefore, utilizing municipal wastewater for microalgal culture could be an
effective and practical approach as an environmentally friendly treatment method.

2.6.2 Microalgal growth in agricultural wastewater

In contrast to municipal wastewater, agricultural wastewater derived from manure or animal
waste can contain significantly higher levels of nitrogen, phosphorus, and organic matter.
These substances exist in both soluble and solid particle forms within the effluent. Despite
the elevated nutrient concentration, research has shown that microalgae could be suitable for
effectively utilizing agricultural waste. Additionally, microalgae have demonstrated their
ability to efficiently remove nitrogen and phosphorus from manure-based wastewater (An et
al., 2003; Wilkie & Mulbry, 2002). Nitrogen and phosphorus in agricultural wastewater, such
as piggery wastewater, can be a valuable nutrient for microalgae growth. The abundance of
carbon and nitrogen in piggery wastewater, favorable temperatures, and solar radiation
contribute to the increased productivity of microalgal biomass. However, the excessively
high concentration of NH4+ in livestock wastewater may hinder growth. Additionally,
volatile fatty acids, such as acetic acid, propanoic acid, and butyric acid, in piggery
wastewater can act as a carbon source and help regulate pH levels. Huo et al. (2012)
employed acetic acid to regulate pH to achieve increased oil content and specific growth rate
in C. zofingiensis culture when utilizing dairy wastewater. Interestingly, the organic carbon
present in piggery wastewater can create a mixotrophic environment favorable for
microalgae. Numerous studies have demonstrated that biomass and lipid production are
greatly enhanced through heterotrophic or mixotrophic cultivation conditions (Perez-Garcia
et al., 2011).

6.3 Microalgal growth in industrial wastewater

Industrial wastewater can be categorized into various types, depending on the industry and
the contaminants involved. Each sector has its unique blend of pollutants. Unlike agricultural
wastewater, the composition of industrial wastewater differs based on the specific operations

25 | P a g e
of the source. Heavy metals are commonly found in industrial wastewater, while nitrogen and
phosphorus levels are generally lower than in municipal or agricultural wastewater
(Chinnasamy et al., 2010). The volume of wastewater produced is influenced by the level of
technological advancement within each industry sector and will decrease over time as
industrial technologies improve. Developing countries are believed to experience higher rates
of industrial wastewater generation than developed countries.
Achieving high growth efficiency in industrial wastewater requires the screening and
isolating of microalgae species and strains that exhibit high tolerance to metal and organic
materials. However, a limited amount of literature is available on microalgae cultivation with
high metal tolerance and the ability to remove nitrogen and phosphorus.

2.7 Biofuel production from microalgae

Photosynthesis is an essential procedure that propels the creation of all biofuels, transforming
light energy into biomass, carbon storage substances (like carbohydrates and lipids), and a
minor volume of hydrogen. In green algae, the light-capturing compound (LHC) (comprising
chlorophylls and carotenoids) takes in photons from solar light as chemical energy. This
energy is utilized by the photosystem II (PS II) to carry out water oxidation, forming protons,
electrons, and molecular oxygen. The electron transport chain receives electrons with low
potential, contributing to the reduction of ferredoxin and, subsequently, the creation of
nicotinamide adenine dinucleotide phosphate (NADPH). After the water is oxidized within
the thylakoid lumen, an electrochemical gradient is established, and a discharge happens.
This process is leveraged to generate adenosine triphosphate (ATP) through ATP synthase.
The Calvin-Benson cycle utilizes the products of photosynthesis (NADPH and ATP) as raw
materials. CO2 is transformed into C3 molecules, which are then incorporated to create
sugars, lipids, and other crucial biomolecules that facilitate cell growth (Beer et al.,2009).
Microalgae biofuels have been at the heart of rigorous study, primarily centered on producing
biodiesel and biogas. However, bioethanol and biohydrogen are also being looked at with
interest. The production methods and operational prerequisites differ for every biofuel.
Numerous research efforts have already proven the feasibility of manufacturing biodiesel
through industrial procedures, with some proposing the use of anaerobic digestion following
the extraction of lipids from algal biomass (Santander et al.,2014; Sawaengsak et al.,2014;
Tercero et al.,2014)

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The possibility of producing bioethanol from microalgae is a sustainable technological
advancement. These sources have demonstrated greater yields than some crops like sugarcane
and corn, already established as raw materials for bioethanol production. Microalgae have the
potential to comprise up to 50% of their dry mass in the form of carbohydrates. These
carbohydrates can subsequently be broken down and fermented, with a high degree of
productivity.

Bioethanol can be produced from microalgae through three distinct methods: (i) biomass
hydrolysis followed by fermentation, (ii) a process known as dark fermentation, and (iii) a
method called "photo fermentation."

2.8 Bioethanol production from microalgae

The process involves creating microalgae biomass in photobioreactors, followed by

preliminary stages such as the disintegration of cell structures and biomass hydrolysis. Often,
this is accompanied by the introduction of enzymes. The biomass, once processed, is
subjected to fermentation using yeasts or bacteria to produce ethanol. The primary
disadvantages of this path include the necessity for multiple stages, which increases energy
consumption, and the employment of enzymes and yeasts, which constitute a significant part
of the expenses. Contrarily, hydrolysis and fermentation are the most efficient methods to
convert biomass due to the effectiveness of enzymes and yeasts in transforming biomass into
end products.

2.8.1 Accumulation of carbohydrate during microalgae cultivation

Primarily, microorganisms are selected for bioethanol production based on their carbohydrate
accumulation ability. Environmental and nutritional factors influence this. Salinity,
temperature, pH, and light intensity are a few environmental factors, while the availability of
carbon, nitrogen, phosphorus, and sulfur are nutritional factors (Chen et al.,2013; Markou et
al.,2012). Rigorous studies have been done on several Chlorella, Chlorococcum, and
Scenedesmus species to study their bioethanol production capacity. Typically, growing under
a high light intensity that varies between 150 to 450 μm−2s−1, utilizing a blend of CO2 in air
from 2% to 5%, and sustaining mesophilic temperatures (20–30°C) results in achieving
roughly 50% carbohydrate content primarily due to nitrogen deficiency. There is an
advantageous impact when light intensity heightens, leading to starch and lipids build-up.
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However, this advantage is only achievable to a specific limit, typically equal to the
saturation point of photosynthesis under certain conditions of a particular species (Vitova et
al.,2015). Nutritional factors influence the photosynthesis rate and biochemical composition
of microalgae. Restricting the amount of macro elements like nitrogen, sulfur, or
phosphorous has proven to be the most effective method to boost starch accumulation and is
currently the most commonly used (Vitova et al.,2015). Synthesis of DNA, pigment, and
proteins in microalgae is enhanced in the presence of nitrogen; Iron levels influence the
transportation of electrons during photosynthesis, reduction of nitrite/nitrate and sulfate,
nitrogen fixation, and neutralization of reactive oxygen species (ROS) (Sunda et al.,1997).
Sulfolipids, polysaccharides, and proteins are formed with the involvement of sulfur, which
also plays a role in the electron transport chain. In circumstances with a limited amount of
sulfur, it hampers the process of cell division. On the other hand, high sulfur levels hinder the
photosynthetic absorption of carbon-rich substances, such as carbohydrates (Markou G et
al.,2012). Carbon dioxide (CO2) is the primary carbon source in autotrophic conditions.
When there is a lack of nitrogen (N), supplementing CO2 and increased light intensity
enhances the absorption and conversion of carbon into carbohydrates, resulting in more
efficient utilization (Chen et al.,2013). After depleting the nitrogen source, Scenedesmus
obliquus CNW-N was found to store approximately 50% of its dry weight as carbohydrates in
a regular-growth medium. The researchers noticed that when the protein levels were reduced,
there was a significant increase in the amount of carbohydrates in the S. obliquus cells (Ho et
al.,2013). The researchers examined the growth of Synechococcus sp. PCC 7002 under
different nitrate concentrations. They discovered that once the nitrate was depleted during the
initial stages of cultivation, there was a notable build-up of carbohydrates in the microalgae
cells, accounting for approximately 60% of the dry weight (Möllers et al.,2014). When
Tetraselmis subcordiformis cells were grown under conditions of phosphorus depletion (0.5–
6.0 mM), there were no significant differences in the accumulation of carbohydrates, which
accounted for approximately 45% of the cell content (4.6–5.3 g L−1 DW). However, cellular
productivity was lower under phosphorus depletion than nitrogen (nitrate 3–11 mM) or sulfur
(sulfate 0.4–0.8 mM) depletions despite the similar carbohydrate content. Under phosphorus
depletion, cellular productivity ranged from 45% to 50%, with dry weight ranging from 5–6 g
L−1. (Yao et al.,2012; Yao et al.,2013). When subjected to different salt concentration levels
(ranging from 5.4 to 67.5 grams per liter of NaCl), the same microalgae achieved a dry
weight of 2.7 to 4.2 grams per liter. Furthermore, the carbohydrate content accounted for

28 | P a g e
approximately 30 to 40 percent, predominantly observed during nitrogen deprivation (Yao et
al.,2013). When Chlorella vulgaris FSP-E and ESP-6 are grown without sufficient nitrogen,
the amount of carbohydrates in their composition rises significantly, from 15-20% to 49-54%.
Similarly, Chlamydomonas orbicularis Tai-04 experiences an increase in carbohydrate
content from 34% to 47% under similar conditions of nitrogen depletion (Ho et al.,2013). C.
vulgaris strain CCALA 924, known for its ability to adapt to various growth conditions, was
subjected to different nutrient depletions, including phosphorus, nitrogen, and sulfur.
Interestingly, it was observed that sulfur limitation resulted in the highest percentage of starch
accumulation in the microalgae. In fact, under sulfur starvation, the microalgae accumulated
approximately 60% of their dry weight as starch, which is 50% higher compared to
conditions without sulfur depletion (Brányiková et al.,2011). One downside of employing
tactics to deplete nitrogen, phosphorus, and sulfur is that it significantly reduces the
effectiveness of the process by decreasing biomass yield. However, it does lead to a greater
accumulation of carbohydrates (Brányiková et al.,2011). Examining microalgae cultivation
requires a thorough investigation into the interaction between nutritional factors. A single
factor was previously believed to increase biomass yield and promote high carbohydrate
accumulation. For example, the strain C. vulgaris P12 was grown using a restricted amount
of iron (FeNa-EDTA) and urea. It was observed that the removal of the nitrogen source (urea)
had a notable impact on the starch levels within the cells, leading to an increase of
approximately 40% (Dragone et al.,2011). T. subcordiformis was grown by investigating the
correlation between nitrogen (nitrate 0–11 mM) and sulfur (sulfate 0–0.8 mM) limitations.
The research revealed that the limitation of nitrogen had a greater impact on the accumulation
of starch than the limitation of sulfur or the combination of both (Yao et al.,2013). The
identical strain of microalgae was also grown under conditions of osmotic pressure (NaCl -
salinity), and it was observed that the impact on starch accumulation was even greater than
under nitrogen scarcity conditions (Yao et al.,2013). Research revealed that nitrogen
deficiency was a major factor affecting the buildup of carbohydrates in nearly all scenarios.
This is likely due to its tendency to inhibit the production of nitrogen-containing compounds,
specifically proteins.

2.8.2 Fermentation and hydrolysis process of microalgal biomass

The primary types of carbohydrates found in microalgae and cyanobacteria, which are
utilized in bioethanol production, include starch, glycogen, and cellulose. Starch plays a
29 | P a g e
significant role as a carbon source in microalgae, making it a valuable feedstock for
bioethanol production. It is considered one of the primary carbon sources in microalgae,
contributing to bioethanol production. The cellulose found within the cell wall of microalgae
can also serve as a viable source for producing bioethanol (Ho et al.,2012). The primary
microorganisms employed in ethanolic fermentation are yeasts from the Saccharomyces
genus or bacteria from the Zymomonas genus. Cyanobacteria produce a glucose polymer
called glycogen, which serves as an energy reserve and is called cyanophycean starch. The
properties of cyanophycean starch l are pretty intriguing. They exhibit more excellent
solubility in water and have shorter polymer chains. Additionally, cyanobacteria can be
readily hydrolyzed to produce bioethanol (Möllers et al.,2014). When it comes to the
operational conditions of the process, it seems that the microalgae biomass necessitates gentle
conditions for hydrolysis and fermentation. Furthermore, breaking down microalgal biomass
through acid and enzyme hydrolysis necessitates only a tiny quantity of reactants. This is
especially true for enzymatic hydrolysis, where high conversion yields can be achieved with
minimal input.

The complete breakdown of carbohydrates in S. obliquus was achieved by subjecting it to

acid hydrolysis at a temperature of 120°C, using sulfuric acid with a concentration of 2-3 N
for 30 minutes. This process resulted in nearly total hydrolysis of the carbohydrate content,
comprising 71-97% of the total carbohydrates. Amongst the carbohydrates, glucose
accounted for approximately 65%, and the solid concentration ranged from 20 to 500 g L−1
(Miranda et al.,2012). Scenedesmus bijugatus, which contains 26% carbohydrates after
removing lipids, was subjected to acid hydrolysis using H2SO4 at temperatures of 130°C for
45 minutes and a solid concentration of 20 g L−1. This process resulted in the
saccharification of 84% of the sugars in the biomass and led to a conversion rate of 70% for
ethanol production (Ashokkumar et al.,2015). The process of enzymatic hydrolysis and
fermentation was conducted on Chlamydomonas reinhardtii, with a biomass concentration of
50 g L−1 and a carbohydrate content of 59.7%. Separated hydrolysis and fermentation were
employed, utilizing amylases to break down the biomass. Liquefaction was achieved through
the use of 0.005% α-amylase from Bacillus licheniformis at 90°C for 30 minutes, followed by
saccharification using 0.2% glucoamylase from Aspergillus niger at 55°C for 30 minutes,
with a pH of 4.5. This process resulted in a 94% hydrolysis of the carbohydrates in the
microalgae. Subsequently, fermentation was carried out using Saccharomyces cerevisiae
S288C, yielding 60% (Choi et al.,2010). Chlamydomonas fasciata Ettl 437 effectively
30 | P a g e
extracted 98% of the carbohydrates (specifically starch) using an ultrasonic homogenizer (30
W and 20 kHz, for 40 minutes). The extracted carbohydrates were then hydrolyzed using
glucose-AN derived from A. niger, followed by fermentation with S. cerevisiae AM12. This
fermentation process yielded an impressive yield of 80% (Asada et al.,2012). Treating C.
vulgaris FSP-E with sulfuric acid is more effective hydrolysis than an enzymatic treatment
involving a combination of amylases and cellulases. According to a study conducted by Ho et
al. in 2013, they found that when hydrolysis was carried out using H2SO4 (at concentrations
ranging from 0.036 to 1.8 N) at a temperature of 121°C for 20 minutes and with biomass
concentration ranging from 10 to 80 g L−1, it resulted in a 95% conversion of glucose content
in the biomass. They also observed that approximately 90% of the expected fermentation
yield was achieved within 12 hours when further fermentation was performed using
Zymomonas mobilis ATCC 29191 (Ho et al.,2013). C. vulgaris was also subjected to various
cell disruption methods, including autoclaving, bead beating, and sonication. The research
revealed that combining bead beating with pectinase treatment (derived from Aspergillus
aculeatus) proved more effective in extracting sugars than cellulases, amylases, and
xylanases. This combination increased the sugar extraction rate from 45% to 70%, leading to
a fermentation yield of 89% after 12 hours using S. cerevisiae KCTC 7906. The results of this
study also revealed the presence of substantial levels of pectin in the cell wall of C. vulgaris
(Kim et al.,2014)—Chlorella sp. KR-1, with a carbohydrate content of 49.7%, achieved
saccharification of over 98% using 0.3 N HCl at 121°C within 15 minutes. Additionally, it
obtained an 80% fermentation yield when combined with S. cerevisiae (Lee et al.,2015).
Dunaliella tertiolecta, initially containing 37.8% carbohydrates and 51.9% of dry weight
after removing lipids, underwent acid hydrolysis using HCl and H2SO4 (0.1–1 N at 121°C
for 15 minutes) and enzyme hydrolysis (such as amyloglucosidase, cellulase, and Viscozyme
L) with a biomass concentration of 50 g biomass/L. The chemo-enzymatic treatment,
precisely the combination of amyloglucosidase and HCl 0.5 N, exhibited the highest
efficiency. This treatment resulted in a biomass hydrolysis level of 80% based on the
theoretical sugar content and a fermentation yield of 82% using S. cerevisiae YPH500 (Lee et
al.,2013).

Extensive research has been conducted to explore the viability of utilizing Chlorococcum
humicola biomass as a promising source for bioethanol production. In their research, Harun
et al. (2011) investigated the acid hydrolysis process on microalgae. They experimented with
different acid concentrations ranging from 0.36 to 3.6 N and conducted the hydrolysis at
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temperatures between 120°C and 160°C. As a result of their study, they achieved a final
ethanol concentration of 7.2 g L−1 using S. cerevisiae. The microalgae were subjected to
alkali hydrolysis using NaOH (0.2–0.5 N) at temperatures ranging from 60 to 120°C,
resulting in an ethanol yield of 26% (based on ethanol produced per gram of biomass) (Harun
et al.,2011). Different research revealed that Trichoderma reesei ATCC 26921's cellulases
can hydrolyze around 70% of the sugars (specifically glucose-based) found in C. humicola
biomass. This finding highlights the potential of enzymatic treatment in converting the
microalga into bioethanol inputs (Harun et al.,2011).

Previous investigations have revealed a wide range of diversity in the cellular structure of
microalgae. Additionally, it has been observed that each species necessitates a unique enzyme
in order to undergo efficient saccharification. Typically, using amylase plays a significant
role in breaking down microalgae cultivated under limited nutrients. To date, glucose has
emerged as the most commonly detected sugar following the saccharification process of
biomass. Another crucial factor to consider is that applying chemical hydrolysis techniques
can aid in extracting lipids from microalgae biomass. This method enables the recovery of
fermentable sugars and facilitates the retrieval of lipids through solvent extraction.
Nannochloropsis gaditana, C. sorokiniana, and Phaeodactylum tricornutum underwent a
steam explosion treatment with sulfuric acid (H2SO4) at temperatures ranging from 120 to
150°C for 5 minutes. This process resulted in hydrolyzation of approximately 96% of the
sugar content using 0.6 N of the acid at a temperature of 150°C. The lipid extraction
efficiency was enhanced by the acid hydrolysis of these microalgae biomasses (Lorente et
al.,2015). Wang et al. (2014) discovered a 25% increase in lipid content when comparing the
amounts obtained before and after hydrolyzing Tribonema sp. microalgae with 1N H2SO4.
The carbohydrate content was reduced by 80% through hydrolysis, using a biomass
concentration of 50 g L−1 of solution at 121°C for 45 minutes. After fermentation with S.
cerevisiae, they achieved 70% of the expected yield (Wang et al.,2014).

To summarize, acid hydrolysis, using sulfuric, nitric, or chloridric acid at temperatures

ranging from 120 to 140°C for 15 to 30 minutes, leads to over 80% saccharification and
fermentation. However, employing large amounts of catalysts (both acids and alkalis) may
impede the fermentation process due to the creation of salts following the neutralization of
the solution. Enzymatic hydrolysis is achieved using enzymes such as amylases, cellulases,

32 | P a g e
and pectinases. The specific choice of enzyme depends on factors like the cultivated species,
the biochemical composition, and the type of carbohydrates involved.

2.8.2.1 Dark Fermentation for Bioethanol Production

Dark fermentation is commonly known as transforming organic substances into biohydrogen.

Fermentative and hydrolytic microorganisms break down complex organic polymers into
smaller units known as monomers. These monomers are then transformed into a combination
of low molecular weight organic acids and alcohols, with acetic acid and ethanol being the
primary ones. Numerous microalgae and cyanobacteria can release ethanol outside of their
cells through an intracellular process, even without light (Ueno et al.,1998). Nevertheless,
dark fermentation has a drawback when it comes to hydrogen production. This is because
around 80-90% of the original chemical oxygen demand (COD) persists as acids and alcohols
after the procedure. Even when operating conditions are at their best, the usual yields range
from 1 to 2 moles of H2 per mole of glucose. The synthesis of ethanol is facilitated by the
build-up of carbohydrates within the microalgae cells through photosynthesis. Subsequently,
when the microalgae transition to dark conditions, they are compelled to directly convert their
carbohydrate and lipid reserves into ethanol through fermentative metabolism (Beer et
al.,2009; Abo-Hashesh et al.,2011). Based on the available information, it can be inferred that
the dark fermentation of microalgae is not an effective method for producing bioethanol.

2.8.2.2 Photo fermentation for Bioethanol Production

The process of photofermentation has gained significant attention, particularly since the
unveiling of industrial facilities that utilize genetically modified cyanobacteria to produce
bioethanol (Piven et al.,2014) directly. The "homofermentative" pathway, also known as
Photanol, is a natural process that efficiently converts sunlight into fermentation products
through a metabolic pathway (Hellingwerf et al.,2009). Photanol is not only restricted to the
production of ethanol, but it also finds applications in a wide range of naturally derived
products that are formed through fermentation based on glycolysis.

Creating ethanol through metabolic pathways can be summarized as follows: First, inorganic
carbon is converted through the Calvin cycle, forming phosphoglycerate. This
phosphoglycerate is then transformed into pyruvate by two enzymes, namely pyruvate

33 | P a g e
decarboxylase (PDC) and alcohol dehydrogenase (ADH). Finally, pyruvate is converted into
ethanol.

Hence, the process of "photofermentation" to acquire ethanol consists of two phases:

photosynthesis and fermentation. Each phase has crucial elements that dictate the process's
effectiveness and the cyanobacteria's metabolic requirements. In any scenario, this method
necessitates the utilization of genetically altered microorganisms.

2.9 Yeast in Ethanol Production

Yeasts are classified as fungi belonging to the ascomycetes or basidiomycetes groups. They
can reproduce through budding or fission and produce spores not contained within a fruiting
body (Wolf et al.,1996). The initial classification of fungi is based on their sexuality, either
belonging to Ascomycotina or Basidiomycotina or lacking a sexual phase in their life cycle,
known as Deuteromycotina. Further taxonomic divisions such as families, subfamilies,
genera, species, and strains are determined by their morphological, physiological, and genetic
characteristics, including their ability to undergo sexual reproduction (Kurtzman et al.,2011).

2.9.1 Diversity of Yeast

The amount of identified yeasts has been on the rise each year. Over 2500 species of yeast
were documented by 2005. It is believed that only 1% of yeast species are currently
recognized, which is approximately 1500 species. The total count of yeast species on Earth is
projected to reach 150,000 (Barriga et al.,2011). The yeast species found in specific
environments varies based on their ability to use various carbon sources and their preferences
for specific nutrients. This specialization is a critical factor in determining the diversity of
yeast species within different niches. Yeasts can be found in various environments, including
land, water, and air. Plants are particularly favorable for yeast communities to thrive. Some
species of yeasts have formed commensal or parasitic relationships with animals. Yeasts can
also survive in extreme conditions such as low water availability (high sugar or salt
concentration) and low temperatures. Yeast cells exhibit a wide range of size, shape, and
color diversity. The size of yeast cells is determined by their species and the conditions in
34 | P a g e
which they grow. While some yeast cells can be as small as 2-3 μm, others can reach 20-50
μm lengths. Most yeast cells have a width within the 1 to 10 μm range. Typically, brewing
strains of S. cerevisiae are more significant compared to laboratory strains (Hough et
al.,1982). Numerous yeast varieties, such as Saccharomyces spp., exhibit an ellipsoidal or
ovoid morphology characterized by their creamy-colored colonies.

2.9.2 Genetics of Yeast

Bioethanol production relies on yeasts' capability to break down six-carbon molecules like
glucose into two-carbon components, such as ethanol, without converting them into the end
product of oxidation, CO2. Crabtree-positive yeasts, like S. cerevisiae, produce ethanol when
exposed to oxygen. In contrast, the crabtree-negative yeast Candia albicans metabolizes
sugars into CO2 in the presence of oxygen (De Deken et al.,1966). The occurrence of
carbohydrates with six carbon atoms inhibits the oxidative respiration pathway in yeasts that
exhibit the Crabtree effect, and energy for growth is produced through glycolysis. When the
supply of six carbon molecules is depleted, the catabolism switches to the oxidation of two
carbon molecules, producing CO2 (Postma et al.,1989). This phenomenon is commonly
called the occurrence referred to as the 'diauxic shift. ' The bioethanol production process,
which involves fermentative metabolism and the diauxic shift, relies on the enzyme Alcohol
Dehydrogenase (EC 1.1.1.1), which is encoded on the ADH1 locus. ADH1 facilitates the
conversion of acetaldehyde into ethanol during glucose fermentation. Additionally, it can
catalyze the reverse reaction, where ethanol is converted back into acetaldehyde. However, it
should be noted that this reverse reaction occurs with a lower efficiency level than the
forward reaction. The yeast S. cerevisiae possesses two genes responsible for encoding ADH.
ADH1 is expressed continuously, whereas ADH2 expression is triggered by decreased
internal glucose concentration. Ethanol serves as the substrate for the ADH2 enzyme (Alper
et al.,2006). Transcription factors control the regulation of the ADH2 gene's expression.
Genome sequencing and transcriptome analysis have uncovered these regulatory proteins'
structure and DNA binding elements (Alper et al.,2006). Advancements in synthetic biology
have recently been centered around modifying the ADH gene to enhance its ability to process
specific substrates and improve catalytic activity. Additionally, researchers have been
working on manipulating the yeast genome by introducing protein-coding genes that increase
ethanol tolerance and enhance the ability to catalyze various carbon sources (Matsushika et
al.,2009). Molecular biologists are searching for new genes that code for ADHs using

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metagenomic methods. This endeavor has discovered several distinct variations (Wexler et
al.,2005).

2.9.3 Yeast in the Production of Bioethanol

For centuries, yeasts like S. cerevisiae have played a crucial role in alcohol production,
particularly in the brewing and winemaking industries. Throughout thousands of years, these
yeasts have been utilized to create various alcoholic beverages. Maintaining a high ethanol
yield, productivity, and the ability to withstand high ethanol concentration effectively reduces
the distillation cost. This allows for a more efficient production process while keeping
expenses low (Kasavi et al.,2012). Currently, yeasts are being utilized to produce fuel ethanol
from sustainable energy sources (Kosaric et al.,1995). Numerous yeast strains, including
Pichia stipitis (NRRL-Y-7124), S. cerevisiae (RL-11), and Kluyveromyces fagilis (Kf1), have
been identified as proficient ethanol producers when exposed to various sugar sources.
(Mussatto et al.,2012).
S. cerevisiae is widely used in industrial ethanol production due to its ability to withstand
various pH levels (Lin et al.,2012). Baker's yeast has historically been employed as a primary
culture in ethanol production because it is inexpensive and readily accessible. Nonetheless,
baker's yeast and other strains of S. cerevisiae have struggled to compete with wild-type
yeast, resulting in contamination during industrial operations. The yeast's inability to survive
during fermentation can be attributed to stressful conditions such as higher ethanol levels,
elevated temperatures, osmotic stress, and bacterial contamination (Basso et al.,2008).
Flocculent yeasts were employed in ethanol production through biological fermentation due
to their ability to assist downstream processing, operate at high cell density, and enhance
overall productivity (Domingues et al.,2000; Jin et al.,1998). One benefit of this process is
that it lowers the expense of recovering cells. It allows the cells to separate from the
fermentation medium without centrifugation (Choi et al.,2010). During sugar fermentation,
yeasts commonly encounter two main challenges: an increase in temperature, ranging from
35 to 45 degrees Celsius, and a rise in ethanol concentration exceeding 20% (Tofighi et
al.,2014). Yeasts' growth rate and metabolism escalate as the temperature rises until it reaches
optimal. An elevation in ethanol concentration during the fermentation process can hinder the
growth and viability of microorganisms (Alexandre et al.,1998; Attfield et al.,1997). The
inability of S. cerevisiae to thrive in media with a high alcohol concentration hinders ethanol
production (Fiedurek et al.,2011). One challenge in yeast-based bioethanol fermentation is

36 | P a g e
the yeast's limited ability to ferment pentose sugars. S. cerevisiae is widely utilized in
bioethanol production; nevertheless, it can only ferment hexoses and not pentoses (Kumar et
al.,2009). Certain Pichia, Candida, Schizosaccharomyces, and Pachysolen yeasts can convert
pentoses into ethanol through fermentation (Mussatto et al.,2012). The challenges yeasts face
can be effectively addressed by utilizing yeast strains resistant to ethanol and high
temperatures. These resilient strains can be obtained from various natural sources such as
soil, water, plants, and animals. This is because cells gradually adapt to their surroundings
through natural selection. Conducting ethanol fermentation at elevated temperatures is
advantageous because it allows for the selection of microorganisms that can tolerate high
temperatures, eliminating the need for cooling expenses and the use of cellulose (Fonseca et
al.,2008). K. marxianus is a type of yeast that can tolerate high temperatures and ferment
hexose and pentose sugars. It can survive at 42–45 °C (Yanase et al.,2010). To address the
challenges associated with pentose fermentation, a potential solution is the utilization of
hybrid yeast strains, genetically engineered strains, or co-cultures involving two different
yeast strains. Hybrid yeast strains can ferment both pentose and hexose sugars into ethanol
simultaneously. These hybrid strains are developed by combining the protoplast of S.
cerevisiae with xylose-fermenting yeasts such as P. tannophilus, C. shehatae, and P. stipites
(Kumari et al.,2013). Researchers have successfully created genetically modified S.
cerevisiae and implemented a co-culture system involving two strains to produce bioethanol
from xylose efficiently. By utilizing recombinant DNA technology, genetic engineering has
allowed the up-regulation of stress tolerance genes, enabling the organisms to overcome
inhibitory environments (Doğan et al.,2014). The genes for xylose reductase and xylitol
dehydrogenase from S. stipitis were inserted into S. cerevisiae to create a strain to ferment
xylose. Modified yeast strains can convert cellulose into ethanol faster than unaltered yeast
strains. In a co-culture process, two different yeasts are cultured and grown in a single reactor
(Tanimura et al.,2012). To efficiently utilize both hexose and pentose sugars, a combination
of pentose-utilizing yeasts such as Pichia fermentans and Pichia stipitis with S. cerevisiae is
used in co-culture. This allows for effectively utilizing both types of sugars (Singh et
al.,2014; Karagöz et al.,2014). S. cerevisiae has been extensively researched as one of the
most commonly studied yeasts. Various types of feedstock have been employed to produce
bioethanol. The yeast strain S. cerevisiae has been extensively researched, making it one of
the most studied yeasts. Various feedstocks have been employed to manufacture bioethanol
(Kim et al.,2014). The wild-type yeast strain, S. cerevisiae KL17, can utilize glucose and

37 | P a g e
galactose simultaneously. This indicates that wild-type yeasts have a significant capacity for
fermenting sugars into ethanol. Furthermore, Silva Filho et al. discovered that wild-type
strains might outperform commercial strains in terms of industrial efficiency. When
fermenting giant reed with S. stipitis CBS 6054, the resulting ethanol concentration was
recorded at 8.2g/L, with a productivity rate of 0.17g/L/h (Scordia et al.,2012). When the
sugars are released under optimal conditions, the toxic degradation byproducts surpass the
threshold level, rendering the conditions unfavorable for yeast fermentation.

2.10 Application of bioethanol

Bioethanol can be used in gasoline engines as an alternative to traditional fuel. It can be
mixed with gasoline in varying proportions. Most gasoline engines currently in use can run
on bioethanol/gasoline blends of up to 15%. Bioethanol has a higher octane rating than
ethanol-free gasoline, which increases the compression ratio of an engine and improves
thermal efficiency. It is also utilized as a fuel for bioethanol fireplaces, making it an ideal
choice for home use as it is flueless and does not require a chimney. Bioethanol is a thermal
combustion fuel for generating electricity and can be used as an energy source in
cogeneration units. In the chemical industry, it acts as a feedstock for various processes.
Additionally, thermochemical reactions involving bioethanol can provide fuel for fuel cells.

2.11 Bioethanol purchase policy in India

The Ethanol Blended Petrol (EBP) Programme has a range of objectives, including
addressing environmental concerns, reducing reliance on imports, and supporting the
agriculture sector. The government has implemented various measures since 2014 to boost
domestic ethanol production. These include implementing an administered price mechanism,
creating alternative avenues for ethanol production, amending the Industries (Development &
Regulation) Act of 1951 to grant exclusive control of denatured ethanol to the Central
Government, reducing the Goods & Service Tax (GST) from 18% to 5%, enacting the
National Policy on Biofuels in 2018, expanding the range of raw materials for ethanol
procurement, offering interest subsidies to enhance ethanol production capacity, and
extending the EBP Programme to cover the entire country except for the Andaman Nicobar &
Lakshadweep islands starting from April 1st, 2019.
• The 2018 National Policy on Biofuel (NPB) aims to achieve 20% ethanol blending in
petrol by 2030. To move towards this objective, oil marketing companies (OMCs)

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 will obtain ethanol from various sources such as C heavy molasses, B heavy
molasses, sugarcane juice, sugar, sugar syrup, damaged food grains unsuitable for
human consumption, surplus food grains determined by the National Biofuel
Coordination Committee (NBCC) under the NPB-2018, and even fruit and vegetable
waste. Under the Ethanol Blended Petrol (EBP) Programme, OMCs procure and
blend up to 10% ethanol in petrol.
• Since 2014, the government has decided to regulate ethanol prices for the EBP
Programme. The government has set fixed prices for the procurement of ethanol from
various sugarcane-based raw materials, such as C heavy molasses, B heavy molasses,
sugarcane juice, sugar, and sugar syrup. These prices are applicable for an Ethanol
Supply Year (ESY), which runs from December to November. However, the pricing
of ethanol derived from damaged and surplus food grains is determined by OMCs.
OMCs estimate the demand for ethanol based on the estimated petrol demand at their
locations and the fixed ethanol prices for the ESY. They then float a tender or
Expression of Interest (EOI) to procure the required ethanol.
In summary, if applicable, the OMCs determine the annual amount of ethanol to be
procured (off-take assurance) and the price of ethanol based on damaged and surplus
food grains. On the other hand, the government sets the price of ethanol derived from
sugarcane-based raw materials, considering the state of the sugar sector. The
government provides directives to the OMCs regarding the prioritization of raw
materials for ethanol procurement, guidance on transportation rates (which are
determined by the OMCs), payment of GST, and other administrative requirements to
advance the EBP Programme.

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 3. MATERIALS AND METHODS

3.1 Wastewater collection

Starchy and slaughterhouse wastewater was collected from the local market. The wastewater
was brought to the laboratory, and it was stored in the refrigerator to prevent any further
degradation of the water.

3.2 Characterization of wastewater

Starchy and slaughterhouse wastewater was collected from the local market, and further
characterization was done. An artificial wastewater was also prepared and used as a control
for this study.

3.2.1 Estimation of total nitrate content of wastewater samples

Total nitrate content was estimated using the Brucine-Sulfanilic acid reagent method. Briefly,
5 ml of sample was taken. An aliquot of sodium chloride (NaCl) solution was added to it,
followed by the required amount of sulphuric acid (4:1) solution was added to that mixture,
along with a specific volume of brucine-sulphanilic acid solution added to it. The reaction
mixture was shaken well for proper mixing, and the absorbance of the reaction mixture was
recorded at 410 nm using a UV-Vis spectrophotometer. The absorbance value was then
compared with the standard curve of sodium nitrate solution.

3.2.3 pH determination of different wastewater samples

The pH of different wastewater samples was measured using a digital pH meter. An aliquot
of the sample was taken, the probe of the pH meter was dipped into it, and the pH of different
samples was recorded.

3.2.4 Determination of Biological Oxygen Demand (BOD) of different wastewater

samples

The B.O.D. bottle was filled with a specific volume of wastewater sample without making
any air bubbles. Then, 2ml of manganese sulfate solution was added carefully to the
wastewater sample, followed by an aliquot of the alkali-iodide-azide solution was added to
the solution in the same manner. The solution was appropriately mixed by inverting the bottle

40 | P a g e
once or twice, and the appearance of a brownish cloud indicated the presence of oxygen. The
brown precipitate was allowed to settle down to the bottom. After that, 2 ml of concentrated
sulfuric solution was added carefully to the reaction mixture. The solution was appropriately
mixed to dissolve the precipitate. The bottle was kept inside of a B.O.D. incubator for five
days. After five days of incubation, 50 ml of that solution was titrated with 0.025N sodium
thiosulfate to pale yellow. Then, 2 ml of 1% starch solution was added, and the titrated
solution turned blue. The titration was continued until the blue color disappeared, and a clear
solution was observed. The reading was noted down. The amount of B.O.D. was calculated.
Using the following equation-

3.2.5 Determination of Chemical Oxygen Demand (C.O.D.) of different wastewater

samples

10 ml of sample was taken into a round bottom flask. Some glass beads were added to
prevent the bumping of the solution when heated. An aliquot of Mercury sulfate (HgSO4)
solution was added to the flask, followed by a certain volume of potassium
dichromate(K2Cr2O7) added to the mixture. Then, 15 ml of silver sulfate-sulfuric acid
solution was added to the mixture. A reflex condenser was set up, and the solution was
digested using a hot plate for 2 hours. After digestion, the solution was collated, and a certain
volume of distilled water was added. A few drops of ferroin indicator were added to the
mixture and titrated with ferrous ammonium sulfate solution to the endpoint. The C.O.D. of
the samples was calculated using the following equation-

3.2.6 Estimation of the carbohydrate content of different wastewater samples

Total carbohydrate content was estimated using the phenol-sulphuric acid method. 0.2 mL of
the sample prepared was taken, and the volume was adjusted up to 1.0 mL using distilled
water. A 1 mL of 5% (w/v) phenol solution was added to the sample, followed by 96 wt%
sulphuric acids. The reaction mixture was shaken well for proper mixing and incubated for 10
min. After incubation, the mixture was shaken and kept in a water bath at 30˚C for 20 min.
The absorbance of the mixture was recorded at 490 nm using a UV-Vis spectrophotometer.
The absorbance value was then compared with the standard curve of glucose. Total
carbohydrate content was estimated using Eq.

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 3.2.7 Protein estimation by Lowery method
The protein content of the sample was determined using the Lowery method. A 1 mL sample
was mixed with reagent 1 (a mixture of 1% CuSO4, 2% Na-K tartrate, and alkaline Na2CO3).
Thereafter, the mixture was incubated for 10 minutes. After incubation, 0.5 mL of folin
reagent was added to the reaction mixture and incubated at room temperature in the dark for
30 min. The absorbance of the reaction mixture was recorded at 660 nm. The protein content
was estimated using a standard Bovine Serum Albumin (BSA) curve.
3.2.8 Estimation of total reducing Sugar of the wastewater samples
Reducing sugar was estimated using the 3,5-dinitrosalicylic acid reagent (DNS) method. An
aliquot of sample was taken, and 1 ml of DNS reagent was added. After that, the reaction
mixture was incubated at 95˚C for 5 minutes. Then, 8 ml of distilled water was added to that
mixture, and the absorbance of the reaction mixture was recorded at 540nm using a UV-Vis
spectrophotometer. The absorbance value was then compared with the standard curve of
glucose. The reducing sugar content of the samples was determined by using the following
Eq.
3.3 Cultivation of Micractinium sp. in different wastewater

3.3.1 Microalgae seed culture

Micractinium sp. (Gen Bank accession no. PP033763) was isolated from freshwater used in
this study. Starchy and slaughterhouse wastewater was collected from the local market, and
the artificial wastewater was prepared in the laboratory.
The microalgae were cultivated using an artificial wastewater medium containing the
following components (per liter): 1.5 g NaNO3, 60 mg MgSO4, 7H2O, 60 mg KH2PO4, 0.075
g, 1.0 mg FeSO4.7H2O and Urea (NH2CONH2). The cells were incubated at 25±1˚C under
constant illumination with 150 µmol m-2 s-1 light intensity using high CRI cool day white
LED 6500K tubes. The light intensity was measured using a lux meter (HTC instrument LX-
103, China), and the values were converted into µmol m-2 s-1 by the following conversion
factor. 70 lux = 1µmol m-2 s-1 (white light -CRI 80) for as a generalization (Sharakshane, A.
2018).
3.3.2 Optimization of different wastewater concentrations for biomass estimation
Microalgae were cultivated in different wastewater concentrations, i.e., crude, 1:1, and 1:9.
Among them, the best-suited concentration was used for further analysis.

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 3.3.3 Biochemical profile analysis of microalgae cultivated in different wastewater
The biochemical composition of microalgae cultivated in three wastewaters was analyzed
using standard protocols, including total carbohydrate, total reducing sugar, total starch
content, cellulose content, etc.
3.3.3.1 Estimation of carbohydrate content of microalgae
Total carbohydrate content was estimated using the phenol-sulphuric acid method. Briefly, 2
mL of culture broth was centrifuged, the pellet was collected, and the weight of the pellet was
recorded. The sample was prepared by dissolving the pellet in 1 mL of distilled water. After
that, 0.2 mL of the sample prepared was taken, and the volume was adjusted up to 1.0 mL
using distilled water. A 1 mL of 5% (w/v) phenol solution was added to the sample, followed
by 96 wt% sulphuric acid. The reaction mixture was shaken well for proper mixing and
incubated for 10 min. After incubation, the mixture was shaken again and allowed to be kept
in a water bath at 30˚C for 20 min. The absorbance of the mixture was recorded at 490 nm
using a UV-Vis spectrophotometer. The absorbance value was then compared with the
standard curve of glucose. Total carbohydrate content was estimated using Eq.
3.3.3.2 Estimation of reducing sugar content of microalgae
Reducing sugar was estimated using the 3,5-dinitro salicylic acid reagent (DNS) method. 2
mL of culture broth was centrifuged, the pellet was collected, and the weight of the pellet was
recorded. The sample was prepared by dissolving the pellet in a specific volume of distilled
water. An aliquot of sample was taken, and 1 ml of DNS reagent was added. After that, the
reaction mixture was incubated at 95˚C for 5 minutes. Then, 8 ml of distilled water was
added to that mixture, and the absorbance of the reaction mixture was recorded at 540nm
using a UV-Vis spectrophotometer. The absorbance value was then compared with the
standard curve of glucose. The reducing sugar content of the samples was determined by
using the following Eq.

3.3.3.3 Estimation of total starch content of microalgae

2 mL of culture broth was centrifuged, the pellet was collected, and the weight of the pellet
was recorded. The sample was prepared by dissolving the pellet in a specific volume of
distilled water. One drop of iodine solution was added to it and was appropriately mixed. The
absorbance of the samples was recorded at 610nm using a UV-Vis spectrophotometer. The

43 | P a g e
absorbance value was then compared with the standard curve of starch. The starch content of
the sample was determined by using the following Eq.

3.3.3.4 Estimation of cellulose content of microalgae

To estimate the total starch content of microalgae, an aliquot of sample was taken, and 200ml
of 0.1M HCL was added to it. After that, the mixture was heated at 100˚C for a few hours.
Then, the mixture was filtered thoroughly. The residue was cleaned and left to dry at 40˚C
overnight. After that, it was treated with 0.1N NaOH, and the mixture was heated at a specific
temperature for 2 hours. Then, the mixture was filtered, and the residue was cleaned with
0.1N NaOH and was left to dry overnight at a particular temperature. After that, it was treated
with 15% acetic acid, 20% H2SO4 and 10% H2SO4. Then, the whole mixture was heated at
75˚C for bleaching. The dry weight of the residue was measured. The total cellulose content
was measured using the following Eq.

3.3.3.5 Determination of amylase activity of microalgae

To determine the amylase activity of microalgae, an aliquot of sample was taken, and 1 ml of
DNS reagent was added to it. After that, the reaction mixture was incubated at 95˚C for 5
minutes. Then, 8 ml of distilled water was added to that mixture, and the absorbance of the
reaction mixture was recorded at 540nm using a UV-Vis spectrophotometer. The absorbance
value was then compared with the standard curve of glucose. The enzymatic activity was
determined using the following Eq.

3.4 Nutrient removal efficiency study of algae

. 2 mL of culture broth was centrifuged, and the supernatant was collected; an aliquot of
sample was taken from that supernatant. Using that sample, different nutrient estimation
assays were performed. Based on the results of those assays, the nutrient removal efficiency
was calculated using the following equations.

3.5 Ethanol Production from algal biomass

3.5.1 Optimization of acid concentration for pre-treatment of algal biomass

For pre-treatment of algal biomass with dilute acid, 5%(w/v) of the remaining biomass was
autoclaved at 121˚C for 15 minutes in the presence of sulphuric acid (H2SO4) (0.5, 1,1.5., 2,

44 | P a g e
2.5, 3 N). After hydrolysis, the samples were cooled to room temperature and centrifuged at
10000g for 5 min. The supernatant containing the released reducing sugar was collected as
the acid hydrolysate. The reducing sugar content of the samples were estimated using the
DNS method.

3.5.2 Effect of Microwave heating time exposure

For pre-treatment of algal biomass with microwave heating, 5% (w/v) of the residual biomass
was subjected to different heat exposures (1,3,5,7,9 min). The reducing sugar content of the
samples were estimated using the DNS method.

3.5.3 Optimization of saccharification by enzyme

The enzymatic saccharification was done using two types of enzymes: amylase and cellulase.
1% of both enzymes were used in the saccharification process, and a mixture of 1% cellulase
and amylase was used in the saccharification process. The reducing sugar content of the
samples were estimated using the DNS method.

3.5.4 optimization of overall pre-treatment and saccharification process

The best results from the above three experiments were chosen to optimize the overall pre-
treatment and saccharification process. Those results were paired together, and three new
experimental set-ups were formed. Among them, one pre-treatment was carried out with acid
treatment only, and the other two were microwave heating with enzyme treatment and acid
plus enzyme treatment, respectively. The reducing sugar content of the samples were
estimated using the DNS method.

3.5.5 Optimization of yeast concentration

For this experiment, commercial yeast (S. cerevisiae) powder was used. The inoculum was
prepared by transferring yeast cells into Yeast Peptone Dextrose (YPD) broth media.
Different concentrations of yeast were used in this experiment (5%, 10%, 15%, 20%) (w/v).
The cultures were incubated at 37˚C at 48h.

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 3.5.6 Production of bioethanol under optimized treatment
The algal biomass hydrolysate was fermented using different concentrations of S. cerevisiae.
The fermentation process was performed under complete anaerobic conditions at 30 ± 2 °C.
The fermentation was carried out in a fermentation medium containing the following
components (per liter): ammonium sulfate 2g, K2HPO4 1g, KH2PO4 1g, ZnSO4 0.2g, MgSO4
0.2g, yeast extract 2g, yeast 10%(w/v) and pH was adjusted to 4.5. Bioethanol was separated
from the aqueous solution by evaporation at 70˚C using a rotary evaporator (Khalil et
al.,2015).

3.5.7 Estimation of ethanol yield

The ethanol was estimated using the colorimetric potassium dichromate method (Crowell et
al.,1979). 2ml of the sample was mixed with 10ml of potassium dichromate, and the mixture
was heated in a water bath at 60˚C for 20 minutes and then cooled to room temperature. The
absorbance was measured using a UV-Vis spectrophotometer at 600nm. The ethanol yield
was estimated using the following Eq.

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 Results and Discussions

4.1 Characterization of wastewater

The physical and chemical characteristics of the three types of wastewater, viz. artificial
wastewater, slaughterhouse wastewater, and starchy wastewater used in this study, were
analyzed, and the results are shown in Table 1. The properties showed that the pH was
within the permissible range of the Central Pollution Control Board of India. The BOD5 of
both the slaughterhouse and starchy wastewater was pretty high. The starchy wastewater was
found to have a BOD of 567.93±3.48 mg L-1, while slaughterhouse and artificial wastewater
had a BOD of 215.67±1.52 mg L-1 and 77±4.58 mg L-1, respectively. The starchy and
slaughterhouse wastewater also showed a high amount of COD. The starchy wastewater
showed a COD of 3076.66±89.05 mg L-1, while in slaughterhouse and artificial wastewater,
the amount of COD was 4253.511±73.51 mg L-1 and 1682.6±67.48 mg L-1, respectively.
Phosphate in artificial, slaughterhouse, and starchy wastewater were 61.16±2.73 mg L-1,
2±0.80 mg L-1, and 19.23±3.66 mg L-1, respectively. Similarly, other characteristics like
total carbohydrate and total reducing sugar were also measured for the three wastewater
types, shown in Table 1.

Table 2: Characterization of wastewater.

Parameters AWW(Artificial) SWW(Slaughterhouse) STWW(Starchy)

pH 7.16 7.38 5.52

BOD 77±4.58 mg L-1 215.67±1.52 mg L-1 567.93±3.48 mg L-1

COD 1682.6±67.48 mg L- 4253.511±73.51 mg L-1 3076.66±89.05 mg

1
L-1

Phosphate 61.16±2.73 mg L-1 2±0.80 mg L-1 19.23±3.66 mg L-1

Nitrate 252.4±2.14 mg L-1 1.34±0.575mg L-1 0.503±0.63 mg L-1

Total carbohydrate NA 413±2.38 mg L-1 502±3.16 mg L-1

Total Red sugar NA 312±1.96 mg L-1 320±3.72 mg L-1

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 4.2 Characterization of algal biomass

The algal cells in different wastewater were harvested during the stationary phase to
determine their biochemical composition. The distribution of biochemical compositions is
summarized in Table 2. Using different wastewater for microalgae cultivation was observed
to lead to varying percentages of biochemical compositions in the algal cells. Microalgae
cultivated in Starchy wastewater were observed to contain the highest amount of
carbohydrates (53.17±3.76%), which was reasonably expected as microalgae utilized the
significant amount of carbohydrate present in the starchy wastewater and accumulated it in
their cells. The slaughterhouse and artificial wastewater algae showed 46.18±1.34% and
41.92±2.03% of carbohydrates, respectively. Similarly, the total amount of reducing sugar
observed in microalgae cells cultivated in a synthetic, slaughterhouse, and starchy wastewater
was 10±2.33%, 21±2.88%, and 14±1.55%, respectively. Similarly, a few other
characteristics, like total starch content, cellulose content, and amylase activity of microalgae
cultivated in three types of wastewaters, were observed, which are mentioned in Table 2. The
difference in biomass concentration and biochemical composition observed under different
treatment strategy could be due to the variation in the composition of wastewaters. The
nitrogen limitation can shift the carbon assimilation towards the production of total
carbohydrate. This finding is associated with the result obtained in this present study, where,
STWW cultivated microalgae showed higher percentage of total carbohydrate compared to
SWW and AWW. Similarly, the cellulose content was also increased compared to the control
biomass (AWWAB), possibly due to the nitrogen starvation. However, improvement of
cultivation condition i.e., cultivation of the microalgae under different light regime, different
stress conditions (salinity, pH stress etc.) may improve the total carbohydrate content and cell
wall polysaccharide content.

Table3: Characterization of algal biomass

Parameters AWW (Artificial) SWW(Slaughterhouse) STWW(Starchy)

Total Carbohydrate 41.92±2.03% 46.18±1.34% 53.17±3.76%

Total Reducing Sugar 10±2.33% 21±2.88% 14±1.55%

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Total Starch content 19±3.54% 18±1.76% 16.12±3.45%

Cellulose content 21±3.29% 28±1.58% 25.34±2.14%

Amylase activity 1.14±2.45 µmol min- 2.07±0.7 µmol min-1 2.11±3.5 µmol min-1
1

4.3 Estimation of nitrate and phosphate removal efficiency

To assess the potential of nutrient removal by microalgae as a solution to wastewater

treatment, the nutrient removal efficiencies in three types of wastewater were checked daily
during cultivation. As illustrated in Table 3, Microalgae Micractinium sp. achieved 97% and
98% phosphate and nitrate removal efficiency in slaughterhouse wastewater, while in starchy
wastewater, this efficiency was 87% and 91%. 88% and 89% phosphate and nitrate removal
efficiencies were observed in artificial wastewater. A study reported that by cultivating
microalgae Spirulina platensis and Chlorella vulgaris in wastewater, 89.97% and 89.90% of
nitrate removal was achieved (Sayadi et al.,2016), which is very close to the present study.
Phosphate is essential for nucleic acid synthesis and energy storage in microalgae; therefore,
microalgae can remove phosphate efficiently from wastewater (Su et al.,2021). Previous
studies reported that although during the initial phase of growth, microalgae need a small
quantity of phosphorus, they absorb a high amount of phosphorus during the early stage of
growth and accumulate phosphorus in the intracellular polyphosphate forms (Kulkarni et
al.,2017; Jiang et al.,2016). Another study reported that by cultivating microalgae Spirulina
platensis and Chlorella vulgaris in wastewater, 82% and 88% of phosphate removal was
achieved (Sayadi et al.,2016), which is very similar to the present study.

The variation in removal efficiency between the nitrate and phosphate was observed. The
lower removal efficiency of phosphate by the microalgae could be due to the microbial
physiology. Higher rate of nitrate removal by microalgae causes the change in pH to the
alkaline range, this suggests better microalgal growth (Yirgu et al., 2021). This finding
correlates with the result observed in the present study.

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Table 4: Phosphate and nitrate removal efficiencies of microalgae (Micractinium sp.) in
different wastewater

Biomass Phosphate removal (%) Nitrate removal (%)

AWWAB 88.45±3.69 89.2±4.87

SWWAB 97.36±2.41 98.86±3.91

StWWAB 87.4±4.11 91.75±4.01

a) b)

c)

Figure 1(a,b,c): Phosphate and nitrate removal on daily basis

50 | P a g e
4.4 Effect of sulphuric acid concentration on acid hydrolysis

The reducing sugar content is the primary indicator for assessing quality and quantity control
for the hydrolysis process. The influence of different acid concentrations on reducing the
sugar content of Micractinium sp. is shown in Figure 2.

Figure 2:Effect of sulphuric acid concentration on reducing sugar content

The reducing sugar content of the different hydrolysates was increased along with the
increasing concentration of sulphuric acid up to a certain point. After that, with the rising acid
concentration, the reducing sugar content decreased gradually. It was observed that on 1N
sulphuric acid concentration, the highest amount of reducing sugar was achieved for all three
hydrolysates; among them, the microalgae cultivated in slaughterhouse wastewater showed
the highest amount of reducing sugar (56%). A study reported that saccharification yield
increases with the low acid concentration. After that, with the increasing acid concentration,
the yield gradually decreases (Lee et al.,2015).

The concentration of acid affects the effectiveness of treatment for the production of reducing
sugar for ethanol fermentation. Higher amount or higher concentration of acid could lead to
excessive degradation of algal biomass and total carbohydrate content. This probably results
in the loss of fermentable sugars (Elshobary et al., 2024), which could affect the overall
production of ethanol. Treatment with higher acid concentration produces several compounds
such as hydroxymethyl furfural, furfural, and levulenic acid, which ultimately inhibits the
fermentation process (Singh et al., 2020). The result observed in the present study, is
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consistent with the facts observed tin the reported literature. The decrease in reducing sugar
content when treated with 3N suphuric acid could be due to the excessive degradation of
carbohydrates and production of several compounds unsuitable for fermentation.

4.5 Effect of microwave heating on algal biomass hydrolysate

The microwave irradiation was carried out at 100˚C under time exposures (1,3,5,7,9 mins). It
was observed that the highest amount of reducing sugar was achieved for all three
hydrolysates under 5 mins of heating time exposure. Among them, algal biomass hydrolysate
of slaughterhouse wastewater showed 43% of reducing sugar content, which was the best
among them. The influence of different microwave heating times on lowering the sugar
content of algal biomass hydrolysate is shown in Figure 3. According to a study, more
extended time of heating exposure can degrade glucose and levulinic acid, and formic acid
formation takes place; thus, reducing sugar decreases (Kumar et al.,2016).

The microwave exposure for 5 min produced higher amount of reducing sugar compared to
other exposure times. The higher yield of reducing sugar in 5 min exposure time could be to
the improved permeability of cell membrane. It disturbs the cell membrane protein and
ultimately can’t control the entry or exit molecules through the membrane (Bchir et al.,
2016). MW exposure for 5 min time with interval instead of continuous exposure may
increase the reducing sugar yield further.

Figure 3: Effect of microwave heating on reducing sugar content

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4.6 optimization of the overall pre-treatment and saccharification process

For the overall optimization process, it was found that algal biomass hydrolysate of
slaughterhouse wastewater treated with sulphuric acid ( 1N) and a cocktail of the enzyme(
1% cellulase+ 1% amylase) gave the best possible result with 58% reducing sugar content.
This result was expected to be the best possible combination from the above three
experiments, which was chosen for this optimization process. The influence of this overall
optimization process on reducing sugar content is shown in Figure 4.

The improved result in enzyme treatment could be possibly due to the availability of the
substrate for sufficient reducing sugar production. The parallel results obtained from different
studies with fungal enzyme treatment was observed. The better hydrolysis process lead to
increase in the total reducing sugar content.

Figure 4 Influence of overall optimization process on reducing sugar content

4.7 Effect of Yeast Inoculum on Bioethanol yield

It was found that by using 10% of yeast (v/v), the highest amount of ethanol (22%) was
obtained. 10% yeast is the optimal amount for bioethanol production. A study reported that a
bioethanol yield of 18.7gL-1 was achieved by fermenting 98.7 gL-1 algae using 15.09% yeast
(El-Mekkawi et al.,2019)The amount of bioethanol significantly decreased with the
increasing amount of yeast concentration. The probable reason is that excess yeast
concentration caused nutrient depletion for the yeast cells in the fermentation media, thus

53 | P a g e
hindering the entire process. The lower bioethanol yield in 5% yeast set, could be due to the
lower yeast volume. At higher yeast concentration (15% and 20%) lowers the ethanol yield
could be due to the hindrance of yeast activity by the excess amount of ethanol which
ultimately affect the overall bioethanol yield (Mekkawi et al., 2019). The overall effect of
yeast inoculum on bioethanol yield is shown in Figure 5.

Figure 5:Effect of yeast inoculum on bioethanol yield

4.8 Estimation of ethanol yield

Ethanol estimation was done daily, and it was found that a maximum ethanol of 22±0.5%
was obtained within 72 hours of the fermentation process. According to a study, 11.46 gL-1
bioethanol was obtained by fermenting 100 gL-1 algal biomass with a fermentation time of
72hr. (El-Mekkawi et al.,2019). Another study reported that fermentation of microalgal
biomass Dunaliella tertiolecta resulted in 7.26 gL-1 of bioethanol at pH 6 (Karatay, et al
2016). Both the data are close to the result of our present study. Figure 6 represents the
overall bioethanol yield, while Figure 7 represents ethanol production daily and the
simultaneous depletion of reducing sugar.

54 | P a g e
 The higher amount of ethanol production at 72 hours allows for the sufficient time for the
yeast to metabolize available sugars and convert them to ethanol (Elshobary et al., 2024).

The result also suggested that combination of acid +enzyme treatment improved the ethanol
content as well as the overall fermentation efficiency. However, optimization of several other
factors related to ethanol fermentation needs to be addressed and thereby improving the
ethanol content further.

Figure 6: Overall ethanol yield

Figure 7: Ethanol content vs. reducing sugar content of
three hydrolysate

55 | P a g e
 5. Conclusions

This study explored the potential of using algal biomass, especially Micractinium sp., as
sustainable source for ethanol production. The eco-friendly treatment of microalgal biomass
with cheap cultivation media (different wastewater) used in this study to improve microalgal
biomass profile. The higher carbohydrate content in the microalgae made it a promising
source for fermentation feedstock. Different pretreatment and scarification strategy increases
reducing sugar content upto 55% (w/w). This result (31%, w/w ethanol) indicated that the
microalgae grown under optimized condition could be used as an alternative feedstock for
ethanol production.

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