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Microalgae biomass as a sustainable source for biofuel, biochemical and

biobased value-added products: An integrated biorefinery concept

Article  in  Fuel · January 2022

DOI: 10.1016/j.fuel.2021.121782

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 Microalgae biomass as a sustainable source for biofuel, biochemical and
biobased value-added products: An integrated biorefinery concept

Sk. Yasir Arafat Siddiki1, M. Mofijur2,3*, Senthil Kumar4, *, Shams Forruque Ahmed5,*, A. Inayat6, F.
Kusumo2, P. Irfan Anjum Badruddin7, T. M. Yunus Khan7, L. D. Nghiem8, Hwai Chyuan Ong2, T. M. I.
Mahlia2

1
Department of Chemical Engineering, Khulna University of Engineering and Technology, Khulna, 9203,
Bangladesh
2
Centre for Green Technology, School of Civil and Environmental Engineering, University of Technology,
Sydney, NSW 2007, Australia
3
Mechanical Engineering Department, Prince Mohammad Bin Fahd University, Al Khobar, 31952, Saudi
Arabia
4
Department of Chemical Engineering, Sri Sivasubramaniya Nadar College of Engineering, Chennai 603110,
India
5
Science and Math Program, Asian University for Women, Chattogram 4000, Bangladesh
6
Department of Sustainable and Renewable Energy Engineering, University of Sharjah, Sharjah, 27272,
United Arab Emirates
7
Mechanical Engineering Department, College of Engineering, King Khalid University, Abha 61421, Saudi
Arabia
8
School of Civil and Environmental Engineering, University of Technology Sydney, Ultimo, NSW 2007,
Australia

*Corresponding author: [email protected] (P. Senthil Kumar);

[email protected] (M. Mofijur); [email protected] (Shams Forruque Ahmed)

Abstract
Microalgal biomass has been proved to be a sustainable source for biofuels including bio-oil,
biodiesel, bioethanol, biomethane, etc. One of the collateral benefits of integrating the use of
microalgal technologies in the industry is microalgae’s ability to capture carbon dioxide during the
application and biomass production process and consequently reducing carbon dioxide emissions.
Although microalgae are a feasible source of biofuel, industrial microalgae applications face energy
and cost challenges. To overcome these challenges, researchers have been interested in applying the
bio-refinery approach to extract the important components encapsulated in microalgae. This review
discusses the key steps of microalgae-based biorefinery including cultivation and harvesting, cell
disruption, biofuel and value-added compound extraction along with the detailed technologies
associated with each step of biorefinery. This review found that suitable microalgae species are
selected based on their carbohydrate, lipid and protein contents and selecting the suitable species are
crucial for high-quality biofuel and value-added products production. Microalgae species contain
carbohydrates, proteins and lipids in the range of 8% to 69.7%, 5% to 74% and 7% to 65%
respectively which proved their ability to be used as a source of value-added commodities in multiple

1
industries including agriculture, animal husbandry, medicine, culinary, and cosmetics. This review
suggests that lipid and value-added products from microalgae can be made more economically viable
by integrating upstream and downstream processes. Therefore, a systematically integrated genome
sequencing and process-scale engineering approach for improving the extraction of lipids and co-
products is critical in the development of future microalgal biorefineries.

Keywords: Third generation biodiesel; fourth-generation biofuel; biohydrogen; photobioreactor;

medicine, animal feed, pigment.

1. Introduction
Microalgae species are photosynthetic, can grow rapidly and generate large amounts of biomass on
land not achievable via traditional agriculture [1, 2]. The medical crises in the Second World War
initiated the application of microalgae for medical reasons as a potential source of protein and
antibiotics. In the 1970s, during the energy crisis, the production of renewable energy using
microalgae became a subject of growing interest. There were a few project proposals on the
application of microalgae and research commenced on the production of hydrogen and methane [3].
Since then, there has been ongoing research to develop the technology related to the treatment of
wastewater and sequestration of carbon using microalgae [4]. Extensive research has also been going
on since the 1970s on the development of microalgae and their contribution to the production of
important economical products. The first large-scale culture of Chlorella species was initiated in the
1960s by a Japanese man named Nihon Chlorella [5]. However, the first-ever research and
development (R&D) program on microalgal biodiesel production, the Aquatic Species Program
(ASP), was commenced in 1978 by the US National Renewable Energy Laboratory (NREL) and
persisted until 1996 [6]. In 1987 in New Mexico, a large-scale microalgae culture began in two high-
rate 1000 m2 ponds and continued until 1990 [7]. Currently, microalgae of various species are used
as a source of biofuel which can be used in different applications.
Microalgae is termed as a feedstock of third-generation biofuel which does not compete with
foods and land crops. The cultivation can be undertaken in close, open, or undeveloped land [8].
Edible oil including palm, soybean, corn, wheat, moringa, and vegetable oil is the feedstock for first-
generation biofuels [9]. It has been reported that first-generation biofuel has detrimental consequences
in the areas of food security, water availability, global food markets, and deforestation [10].
Consequently, these forms of biofuels have sparked a lot of debate [11]. Furthermore, second-
generation biofuels produced from non-edible oils (Jatropha curcas, Simarouba glauca, Pongamia
pinnata, and others), forest residues, and lignocellulose biomass necessitate vast swaths of land
coverage that could otherwise be utilized for food production [12-14]. In current times, technologies
2
that are effective for the commercial processing of waste products into a source for generating biofuel
are also lacking in second-generation biofuel production. Microalgae biofuel tends to be a viable
alternative energy source to substitute or complement fossil fuels [15], based on the disadvantages
mentioned above related to first- and second-generation biofuels. However, by genetically modifying
microalgae, a new generation of biofuel termed fourth-generation biofuels can be produced. The
distinctive features of first. second, third and fourth generation biofuel has been presented in Figure
1.

Figure 1: Distinctive features of first, second, third and fourth generations of biofuels. Third-
generation biofuel which is produced from microalgae does not create food versus fuel conflict.

Fuel derived from microalgae has benefits which include biodegradability, being environmentally
friendly and consisting of higher energy. This fuel can be blended with diesel, jet fuel, and gasoline
and can be used as transportation fuel [16]. As a feedstock, microalgae yield a much larger amount
of oil in comparison to any other feedstock. Harvesting microalgae is very convenient as it can be
performed at any time of year [17]. Microalgal growth can occur in systems that are both open and
closed. For microalgal culture, various nutrients and CO 2 are prerequisites, the supply of which can
be acquired from wastewater streams and combustion of fossil fuel, respectively [18]. Furthermore,
3
using this method to convert CO2 into chemical and fuel products without polluting the atmosphere
is a lucrative way to minimize emissions of greenhouse gases while still boosting financial benefits
[19].
Botryococcus braunii, Dunaliella primolecta, Nannochloropsis sp., Crypthecodinium cohnii, and
Chlorella sp. are just a few existing microalgae that contain lipids and hydrocarbons in large amounts.
Botryococcus braunii, green microalgae that are colonial, can produce a lot of hydrocarbons relative
to their biomass [20]. Additionally, they can create other compounds that are commercially valuable
including polysaccharides and carotenoids [21]. Microalgae species have oil content that can reach
80%, with levels ranging from 20% to 50% being very typical [22]. There is up to 50% lipids in the
microalga Chlorella, with Botryococcus braunii having the highest oil content at around 80% [7].
Microalgae, in addition to producing biofuel, produce various types of bioactive compounds that have
a broad range of uses in nutraceuticals, the chemical and food industries, and pharmaceuticals [23,
24], as shown in Figure 2. Therefore, this review attempts to focus on the biorefinery concept of
microalgae to highlights its ability to be converted into commercial products and energy. In the first
part of this review, we comprehensively discuss the microalgae species, culture and harvesting
systems; in the second part, the production of diverse types of biofuel from microalgae using
thermochemical and biochemical technologies, and in the third part, different biobased products and
fine chemical production from microalgae. Finally, a guideline for further research have been
provided.

Figure 2: Potential application of microalgae in producing biofuel. Different biofuels can be

produced from microalgae through anaerobic digestion, fermentation, transesterification process.
4
2. Biorefinery concept for sustainable processing of microalgae biomass
Biorefineries combine various process schemes and equipment to produce biofuels, electricity, and
high-value chemicals from biomass. The “Biorefinery” concept is based on petroleum refineries,
which produce a variety of fuels and products from crude oil. This concept aims to convert biomass
into commercial products and energy. The biorefinery's ability to produce multiple commodities
increases the value of biomass feedstock (Figure 3). The cultivation and harvesting of microalgae,
cell disruption of biomass and compound extraction are among the steps involved in microalgae-
based biorefineries [25]. The fundamental objective of biorefinery operations is to separate and
recover the needed components from the same batch of biomass, in a manner of generation of intact
commercial products. In the biorefinery concept, fractionation is cited as a key bottleneck, because it
is difficult to separate the various compounds.

Figure 3: Schematic of microalgae biorefinery steps along with the list of involved technologies in
each step. The ‘biorefinery’ concept is started with microalgae cultivation, harvesting, drying, cell
disruption, fractionate extraction and conversion into biofuel and products.

5
2.1. Species of microalgae and cultivation
Microalgal growth takes place in ponds, agricultural land, photobioreactors, and wastewater with
the provision of essential requirements like CO2 and sunlight [26]. So far, over 15.7 × 104 microalgae
species have been identified and many other species remain unidentified. Maintaining minimal
expenses for sources and manufacture is necessary to produce sustainable biofuel and other value-
added products. Therefore, the selection of the microalgae strain is crucial for producing biofuel and
high-value co-products to maximize revenue. Suitable microalgae species are selected based on their
carbohydrate, lipid and protein contents which are important for producing high-quality biofuel. The
average carbohydrate, lipid and protein contents of different microalgae species have been shown in
Figure 4.

6
 80
Average carbohydrate, lipid and protein (% dry wt.) Carbohydrate Lipid Protein 74
70
70 66
63 63
60
60 57 57 56 57 56
55 55 55 55
52
50 49 50
48 49
50 45
41 40
40 35 35
37 37
35 34
32 32 32 33
32
30
30 28 27 28 28
26 26 25
23 24 21 24 23
21 22 22
20 21 20
18 18 19 18
20 16 17 16
15 15 15 15 15
13 12 12 11
11 12 11
8 9 9 8
10 6 5 6 7 7
2 2 2 3
0
0

Microalgae species

Figure 4: Average carbohydrate, lipid and protein contents of terrestrial, marine and freshwater microalgae. Microalgal strains contain carbohydrates,
proteins and lipids in the range of 8% to 69.7%, 5% to 74% and 7% to 65% respectively [27-35]

7
2.2. Microalgae culture systems
Cultivation of microalgae can take place in two types of systems, open culture systems like ponds
and lakes, and closed culture systems like photobioreactors (PBRs). Each of these systems has several
benefits and drawbacks. One of the oldest and easiest methods of large-scale microalgae cultivation
is open pond cultivation [36]. Open culture systems typically require less cost to install and maintain
than closed systems and are more robust than reactors that are large and closed, relatively easy to
scale up, consume less energy, and have a higher production capacity [37]. The comparison between
open and closed culture systems has been presented in Table 1. Nonetheless, more energy is needed
by ponds to homogenize nutrients and a minimum water level (15 cm or 150 L/m2) must be regulated
for the microalgae to obtain sufficient solar energy for development.

2.2.1 Open culture system

Because of the open nature of the system, ponds are more vulnerable to meteorological conditions as
they lack control over water temperature, light intensity, and evaporation, which can influence the
growth rate of microalgae. Furthermore, some conditions can have a major impact on microalgae
productivity in open ponds, e.g.: rainwater runoff, which has an effect on microalgae growth
conditions including salinity and pH; erosion of banks, which increases water turbidity and leakage;
and contamination from protozoa and bacteria due to the large cultivation area [38]. Additionally,
since there is only 0.03–0.06% CO2 in the atmosphere, microalgae cell growth is predicted to be
slowed by mass transfer restrictions. These systems work best in areas with a lot of sunlight and free
access to water, which are mostly found in coastal areas. The most common microalgae cultivated in
these systems are Scenedesmus, Spirulina, and Chlorella [39].
Examples of open ponds include natural water sources like rivers and lakes, as well as artificial
or man-made water bodies like raceways and circular ponds. Circular ponds, named after the circular
shape of the culture tank, were the first type of artificial pond used for microalgae cultivation on a
large scale [38]. The raceway pond is regarded as the best available design for open pond cultivation
because of its energy efficiency [40]. In this design, a single paddlewheel has enough strength to
appropriately agitate a raceway pond as large as five hectares [40].

2.2.2 Closed culture system

Closed photobioreactors, in contrast to open-air systems, are used primarily for the cultivation of
axenic single-species microalgae. Photobioreactors have been used successfully for the production of
large quantities of microalgal biomass in a controlled environment. A photobioreactor (PBR) is a type
of reactor that carries out photobiological reactions by growing or using phototrophs (microbial, plant
cells, or algal). PBRs are adaptable systems that can be tailored to the physiological and biological

8
characteristics of the algae species being grown, allowing for the cultivation of species that would
otherwise be impossible to grow in open ponds.
The walls of a PBR limit or prohibit cultivated cells from being exposed to light, contaminants,
and gases (e.g. microorganisms and dust), thus preventing any direct exchange of these entities
between the cultivated cells and the atmosphere. PBRs have numerous advantages over the open pond
due to their shape or design. Tubular, column, membrane, and flat plate photobioreactors (PBRs) are
different types of closed systems [41]. The design features provide good control over growth and
culture parameters (mixing, pH, temperature, nutrients), higher volumetric outputs, and increased
nutrient and metabolic efficiency. They allow an increased density of microalgae or cell
concentrations, require less land, and provide a safer and highly controlled environment, resulting in
an uncontaminated solitary strain microalgae culture.
The type of bioreactor used should be determined by the microalgae strain, space availability and
location, and the nature of the targeted product [42]. Despite their benefits, PBRs have several flaws
that must be considered and addressed. A few of their major drawbacks include the high cost of
construction and operation, the production of algal biomass, overheating, biofilm formation resulting
in oxygen accumulation in the culture, cell damage from extreme stress, and disintegration of photo-
stage material [41, 42]. Though PBRs are more expensive than ponds to produce biomass, the cost
can be low enough for some species of microalgae and applications to be tempting for aquaculture.
Nevertheless, PBRs might not be capable of compensating the high capital and operating costs with
their high cell concentration and productivity.

Table 1: Comparison between open and closed culture systems [7, 38, 39, 43, 44]
Factors Open culture system Closed culture system
Process technology Relatively simple Complex
Controllability Poor Easy
Contamination risk High Very low
Mixing Usually poor Uniform
Sterile condition Not achievable Achievable
Algal cell density Low High
Energy demand Low High
Capital and operational cost Low High
Maintenance Relatively easy Difficult
Land requirement More Less
Environmental factors Uncontrolled Controlled
Evaporation losses High Low

9
 Productivity Low 3~5 times more
Stress damage on algal cells Low High
Scalability Possible Extremely difficult

2.3 Reactor technologies for culturing microalgae

Various types of photobioreactors including tubular, flat plate, column, and biofilm photobioreactors,
are used to grow microalgae [45]. Among these, tubular photobioreactors are the most common.
Airlift, flat inclined, bubble column, column aeration, solar penthouse-roof, and multistage
continuous flow are all possible configurations of these photobioreactors. Figure 5 shows the
classification of different photobioreactors used in microalgae production. The advantages and
disadvantages of different tubular, flat plate and fermenter type photobioreactors have been presented
in Table 2.

Figure 5: Classification of photobioreactors that are used to produce microalgae [45]

2.3.1 Tubular photobioreactor

Tubular type photobioreactors are the most prevalent and regarded as the most appealing since they
produce a high amount of biomass in a short amount of time and have a short harvesting timeframe.
In order to enhance sun exposure, they are constructed of transparent parallel tubes made of plastic
or glass (diameter no greater than 0.2 m). A pump or airlift technology circulates microalgae through
the tubes [41–43]. The configuration of tubular photobioreactors includes a simple vertical column,
10
horizontal and helical. Vertical tubular reactors such as airlift or bubble columns are constructed as
vertical transparent tubes through which light enters the reactors, ensuring adequate light penetration
into the cultivation. However, the inclined-pipe photobioreactors and reactors that form α-shape, as
well as polythene bags and sleeves installed on supporting structures, are also the example of tubular
bioreactors.
Horizontal tubular reactors come in a variety of shapes (loop shape, α shape) and sizes. In outdoor
culture, its shape leads to higher light energy conversion due to its positioning towards the sun [46].
In these reactors, a dedicated gas exchange system is used to introduce a CO2 gas mixture into the
tube connection. However, photobleaching occurs when excess oxygen accumulates during
photosynthesis and thus lowers the photosynthetic efficiency [47]. In addition, another drawback of
horizontal tubular reactors is much higher energy consumption (2000 W/m3) than vertical tubular
reactors (50 W/m3). Additionally, tubular photobioreactors can be designed helically to increase the
surface area to volume ratio. This design maximises light penetration, minimises contamination,
enables simple temperature control, and maximises CO2 transfer in the culture medium. Helical
photobioreactors are made up of transparent and flexible coiled tubes with a small diameter and a
degassing unit that is either separate or attached [48].

2.3.2 Flat plate bioreactors

The flat plate reactor is constructed of transparent materials such as glass, plexiglass, polycarbonate,
and other similar materials. Because of its cuboidal shape and minimal light path, it has a high surface
area to volume ratio and open gas disengagement systems, which distinguishes it from the
competition [49]. It is either bubbled with air from one side or rotated mechanically by a motor to
increase biomass productivity as the mixing rate influences the production rate. For illumination, they
have a high surface-area-to-volume ratio and are simple to construct [50]. It has a higher illumination
surface area and is easier to scale up than horizontal tubular photobioreactors. Aeration rates,
temperature control during cultivation, and biofouling are all disadvantages of flat plate bioreactors
[51].

2.3.3 Fermenter type photobioreactors

The most common reactor is the fermenter, which uses mechanical agitation via impellers of various
sizes and shapes. The bottom of the tank is bubbled with CO2-enriched air, which serves as a source
of carbon for algae growth [47]. Aside from natural light, it is also possible to use artificial lighting
in it from the outside with fluorescent lamps or optical fibres to convert this type of bioreactor into a
photobioreactor. However, the main limitation of this method is the low surface area to volume ratio,
which reduces the light absorption and efficiency of harvesting. Cultivation can be done periodically,

11
semi-continuously, or continuously. Extensive experience in biotechnology processes and running
them on various scales in the pharmaceutical and food industries may provide the operation realised
in an axenix culture [51]. The bacteria from annex culture can be kept in a reactor for a long time,
which is important for the generation of high-value metabolites. The biomass productivity in
fermenter reactor systems is low, approximately 30–50 mgL-1d-1, hence the reactor design is
constantly improved to enhance algal biomass growth efficiency [9].

2.3.4. Hybrid type photobioreactor

Microalgae growth phases are combined in a two-stage system known as a hybrid system. The first
stage of algae growth occurs in a closed system (photobioreactor), while the second stage occurs in
an open system [47]. In hybrid photobioreactors, the advantages of both types of reactors are
combined to overcome the disadvantages of the other. It allows for better control of culture variables,
higher productivity, and consume less power [47]. Thus, hybrid systems contain components and
design solutions from open and closed cultivation systems. It is done in a photobioreactor to reduce
the risk of contamination by alien organisms while maintaining favourable conditions for cell
proliferation. The second stage of production involves exposing cells to nutritional stress, which
should stimulate the synthesis of desirable metabolites and lipid products.

12
 Table 2: Comparative advantages and challenges of different tubular, flat plate and fermenter types photobioreactor used for producing microalgae [45,
47, 50, 51]
Photobioreactors Schematic diagram Advantages Challenges
Tubular  Excellent biomass yield  Cells’ susceptibility to shear stress
 High efficiency of photosynthesis  Low illumination area due to vertical
 Satisfactory mass transfer configuration
 Limited photo-inhibition and photo-oxidation  Light exposure is restricted by allowable
 Exposure to alternate L/D cycle column diameter and height
 Relatively small land footprint  Possibility of biofouling on reactor walls
 Suitable for outdoor cultivation
 Low risk of contamination
 Low energy demand
 Suited for algae immobilization
 Economical, compact and easy to maintain
 Maximum S/V ratio  Uneven mass transfer
 Maximum exposure to light  Risk of dissolved oxygen accumulation
 Suitable for outdoor cultures  Some degree of biofouling
 Good biomass output  Large land footprint
 Adequate angle towards sunlight  Photo-inhibition
 Optimum use of light  Cells’ susceptibility to shear stress
 Relatively cheap  Separate gas exchange unit required
 Reasonable scalability
13
  Rapid unidirectional flow with a minimal air flow  Foam formation as a result of the high cell
rate concentration
 High surface area

 Expanding the number of units makes it much  Oxygen build-up

easier to scale up.  Separate gas exchange
 High surface area  More shear resulted from pump use
 Cell debris pile up inside
 High heating and illumination costs
Flat plate  Maximum possible exposure to solar energy  Scalability is not easy; any expansion of
 High surface to volume ratio production scale necessitates the use of
 Well-suited for outdoor farming additional modules and support structure
 High yield of biomass  Complexities in regulating cultivation
 Uniform access to light across total cultivation temperature
volume  Likelihood of biofouling
 Relatively inexpensive  Probability of hydrodynamic stress in
 Simple to construct, retain, clean and operate some algae species leading to cell damage

 High efficacy of photosynthesis

 Suitability for algae immobilization
 Low dissolved oxygen concentration

14
Fermenter  Possibility of running axenic cultivation  Low surface to volume ratio
 Open gas exchange possible  Poor light absorption and conversion
 Used for optimization analysis  Low output
 Precise control of all process parameters  High operation cost

15
2.4 Factors affecting the microalgae culture system
2.4.1 Light intensity
Light intensity has been considered an important limiting factor in the cultivation of microalgae.
Microalgae photosynthesis is directly affected by light span and intensity, which also influences the
biochemical composition and microalgal biomass production yield [52-54]. Higher light intensities
help to boost the rate of photosynthesis until it reaches a point where a balance is achieved between
photorespiration and photoinhibition, as shown in a study on Scenedesmus abundans microalgae [55].
While light is required for photosynthesis in microalgae, some results [56-58] indicate that light and
dark periods should be alternated. Differences in biomass yield and rate of growth have been proven
experimentally by observing the growth of the same strain of algae under two variable factors: light
intensity and time duration [59]. According to reports, the light intensity required to achieve optimal
growth varies depending on the type of algae species being used. The majority of studies have
concluded that 16 hours of light followed by 8 hours of darkness is the best environment for
microalgae development [60].

2.4.2 Temperature
Regulation of temperature plays a critical role in the processes of biological and microalgae growth
[52, 61, 62]. Different types of microalgae species have different optimal growth temperatures and
any fluctuation of the temperature from the temperature of optimal growth, whether higher or lower,
may stifle or even stop microalgae operation and growth and reduce the biomass output [63]. pH
value and solubility of CO2 in the medium are parameters that are related to microalgal growth and
are also affected by temperature [64]. Most microalgae organisms grow best at temperatures between
20° and 30° C [65]. Thermophile algae, on the other hand, can withstand higher (as high as 80° C)
temperatures [53]. The temperature may also be used as a stress treatment to trigger the development
of useful metabolites [66]. Production of carbohydrates and lipids was found to be high in growing
Chlorella Vulgaris at 25° C in comparison to 30° C [67].

2.4.3. pH and salinity

Photosynthesis is affected by variations in the medium’s pH which causes stress on the growth rate
of microalgae. Microalgal organisms have a distinct optimum pH and salinity [68]. Biomass growth
is higher when microalgae are grown in alkaline environments as it allows CO2 to be easily captured
from the atmosphere [69]. Most microalgae were found to grow well in pH ranges of 6 to 9 [66],
however, it was observed that the amount of chlorophyll in microalgae decreased when the pH was
increased to 9.5 [70]. Chlorella Vulgaris is an exception as it can grow in a pH range of 4 to 10 and
can produce the highest amount of biomass at a pH of 9 to 10 [57, 71]. Microalgae culture under
16
acidic conditions (pH range of 3 to 4) is also advised to avoid the contamination of the cultivation
system with lethal fungus [72]. It is very harmful to algal cells if the pH rises as it causes an increase
in the salinity of the culture media [73].

2.4.4. Mixing
In microalgae culture, mixing ensures that nutrients, pH, CO2, and temperature are distributed evenly
throughout the culture system [74]. Mixing also allows light to penetrate the culture and distribute
evenly, preventing the biomass from settling down and aggregating in an area [75]. The algal cells
go through a rotation where they are exposed to an alternating light and dark cycle (L/D cycle) [58].
This mixing causes a shift between the dark and photic zone (flashing light effect) that promotes
microalgae photosynthesis reactions [58]. The flashing light effect was found to increase microalgal
photosynthesis while also improving the quantity and quality of the algal biomass [76]. Another study
identified that setting the L/D cycle frequency at 60 Hz increased biomass production by more than
55 percent [77]. However, the sheer force of excessive mixing on microalgae can cause cell damage
and lysis [46]. Microalgae cultures must therefore be constantly blended to keep all cells suspended
and exposed to light. An appropriate mixing device in a photobioreactor not only allows the proper
dissolution of nutrients and penetration of light into the culture, but it also allows the effective
exchange of gases [78].

2.4.5. Nutrients
Different microalgae organisms have different nutritional requirements [62], but the basic
requirements are the same for all. Microalgae's backbone is made up of nitrogen, phosphorus, and
carbon, which are all macronutrients needed for growth [66]. The nutrient that plays the most essential
role in contributing to biomass production is nitrogen. The nitrogen content in biomass varies based
on the microalgae species and the nitrogen amount supplied, but it usually ranges from 1% to more
than 10% [79]. Microalgae growth and biomass yield can be hampered by a lack of nitrogen in the
culture [66]. The sources of nitrogen that are most commonly consumed are nitrate (NO 3-), urea, and
ammonia (NH4+) [80]. Examples of nitrogen compounds include potassium nitrate, peptone,
ammonium nitrate, and ammonium sulphate. Chlorella Vulgaris produces 3.43 g/l biomass and the
optimum nitrogen concentration has been determined to be 0.5 g/l [57].
Phosphorus concentration affects biomass composition, especially lipid and carbohydrate
percentage [66]. The capacity of green microalgae to amass lipid under a variety of phosphorus
conditions (control, phosphate-starvation (PS), sequential phosphate addition (SPA), and biphasic
phosphate starvation (BPS)) has been recently studied [81]. When compared to the amount obtained
from SPA, BPS has been proven to provide more lipid recovery. However, among the various
17
phosphates available, dipotassium hydrogen phosphate was found to be predominantly favoured and
subsequently consumed [82]. Microorganisms in autotrophic cultivation depend on the energy from
light for energy production. The only carbon sources that are utilized in autotrophic culture are
sodium bicarbonate and CO2. The use of atmospheric CO2 helps in lowering manufacturing costs and
aiding in CO2 reduction, particularly in large-scale cultures [62].
Carbon sources for heterotrophic culture include fructose, acetate, glucose, sucrose, and glycerol.
A mixture of these two carbon sources (inorganic and organic) is used in mixotrophic culture [66].
The pH of the system will usually increase to 11 due to the accumulation of OH - during
photosynthesis, which is unfavourable for algae production. As a result, CO2 must be introduced into
the system to minimise and sustain the initial pH system in the microalgae community [83]. The
micronutrients Mo, Co, K, Fe, Mn, Mg, Zn, and B are only required in minute quantities but have a
profound effect on microalgae growth as they can create an impact on numerous enzyme-related
activities in algal cells [84]. Nutrient deficiency influences the growth rate of microalgae greatly and
results in reduced biomass [85, 86]. Lipid and carbohydrate synthesis and accumulation in microalgae
are strongly influenced by nutrient supply [86]. The culture must grow quickly to produce microalgal
biomass at a large scale. Therefore, providing the right nutrients is critical for accelerating algal
growth. Certain bacteria can help microalgae grow faster as they can provide essential nutrients.
Microalgae absorb nutrients that are in a particular form and bacteria can convert nutrients into those
specific forms, such as ammonia and nitrate [87].

2.5. Harvesting and drying techniques of microalgae

Harvesting of microalgae is the process of biomass recovery from the culture medium [88] and
contributes 20–30% of the total expenditure in biomass manufacture [89]. Suitable harvesting
methods include a variety of physical, biological, or chemical ways of performing the intended solid-
liquid separation, extracting large amounts of water, and processing large volumes of algal biomass.
Filtration [90], centrifugation, flotation, flocculation [91], electro flocculation [92, 93], gravity
sedimentation, electrophoresis, and magnetic separation [94, 95] are the most popular harvesting
methods. The benefits and challenges of different harvesting techniques have been presented in Table
3. To maximize efficiency and reduce operating costs, harvesting methods can be combined by
including an initial phase of biological or chemical coagulation/flocculation thickening [96].
Since the microalgae technique is fast, effective, and universal, continuous centrifugation is
extensively used for biomass separation [97, 98]. Microalgal biomass is detached from the growth
medium using centrifugal forces and the excess water is drained off once isolated from the biomass
[99, 100]. However, this method cannot be used for large-scale harvesting purposes as it is so energy-
intensive and lacks economic feasibility [101]. Gravity sedimentation, another common method of
18
biomass harvesting, is simple, works for a variety of algae, and is very energy efficient [102].
Sedimentation and filtration may be used in addition to flocculation [103]. Filtration is a popular
process for separating solids from liquids [104]. The key costs of this process include the cost of
regularly replacing filtration membranes, as they get clogged from time to time, and the energy used
by the pumping system [105]. Nevertheless, in contrast to other harvesting methods, filtration is
considered one of the economical and effective harvesting techniques. Membrane micro-filtration or
ultrafiltration is needed to effectively harvest smaller algae [90, 106].
The mechanism of conventional flocculation is charges dispersion. In general, microorganisms
have a negative charge. Iron(III) sulphate (Fe2(SO4)3), aluminium sulphate salts (Al2(SO4)3), and
iron(III) chloride (FeCl3) are examples of flocculants, among many others, that are used to neutralize
or reduce the repulsion charge between microalgae and allowing the species to clump together,
resulting in more effective sedimentation or filtration [102, 107]. Alum, which is commonly used for
traditional methods of treating wastewater, can effectively flocculate algal biomass [103, 107]. It can
obstruct oil extraction depending on the cultivation of the dominant strain of algae [103, 107].
Flocculation can also be accomplished using cationic polymers or substances that are alkali to
increase the pH [106].
Some species of microalgae will naturally cluster and undergo flocculation (self-flocculate) in
response to stimulations caused by the environment, like alterations in the pH of the culture medium
[108]. Other species of algae cause colloidal suspension, which is difficult to settle, by synthesizing
chemicals that are excreted in the outer cells. Furthermore, some organisms are motile and do not
undergo natural settling [109]. In a recent report, Yin et al. [110] compiled a collection of flocculants
used on various microalgae organisms for the harvesting process. The combination of flocculation
and mechanical harvesting technique can enhance the recovery of biomass at a low cost [91].
Laboratory scale experiments have shown that flotation of algal biomass using the formation of
charged bubbles and ozonation-dispersed flotation can be implemented for harvesting algae that are
smaller and unicellular [111-113].
The efficacy of dissolved air flotation can be enhanced by lowering the negative charge borne by
air bubbles. A net positive charge can be obtained by applying a cationic surfactant, or other
chemicals, into the mixture [111]. Chlorella saccharophila has been harvested using dispersed air
flotation [114]. To harvest concentrated flocs, flotation is commonly combined with flocculation
[115] and bioflocculation [116]. Following the harvesting process, the biomass slurry is dried to keep
it in a stable state which can also be stored. The harvesting process extracts moisture and concentrates
the biomass slurry to a range of 15–25 percent of the total solid matter [117]. This is high enough to
enable spoilage of the slurry, which is kept at room temperature, in a few hours [117].

19
 The drying process is critical because it aids in the concentration of the biomass, which can reach
90–95 percent of total solid matter [118]. However, the drying phase requires a lot of energy and
therefore might not be cost-effective for the whole (cultivation to recover energy) process [113]. The
most frequently used drying methods are spray drying, freeze-drying, drum drying, and sun-drying
[96]. The most basic drying technology is sun drying, which has a low initial and ongoing cost [119].
However, to remove water effectively from the harvested biomass, a long process time of up to 72
hours is required [120]. Many microalga species are commonly dried using spray drying [121]. Spray
drying has many benefits, including high flexibility, the ability to pack directly after drying, the ability
to produce powder without the need for grinding, and the ease of taking control of size [113]. This
enables the uniformity of the product to be maintained during the process. However, its key
drawbacks are energy costs and high capital [113].

Table 3: The benefits and challenges of different microalgae harvesting techniques [122-124]
Harvesting Sample image Benefits Challenges
techniques
Coagulation/  The fast and simple technique  Expensive chemicals
flocculation  Better suited for large scale requirement
production  Dependence on pH
 Nominal cell damages  Difficulties in isolating the
 Widely applicable to different coagulant from harvested
species biomass
 Low energy demand  Efficiency varies with types
 Auto and bio-flocculation of coagulant
may be inexpensive methods  Limited recycling of culture
medium
Flotation  Appropriate for large scale  Additional requirement of
 Low cost and low land surfactants
footprint  Ozoflotation is costly to
 Brief operation time implement

Electrical  Widely applicable  The extra cost from metal

based  No requirement of chemical electrodes requirement
processes use  Possibility of metal
contamination
 High CapEx and OpEx

20
Filtration  Superior recovery efficiency  Protracted process; entails
 Cost-effective pressure or vacuum
 No requirement of chemical  Not suited for small-sized
use algae
 Nominal energy use  Membrane
 Low shear stress fouling/clogging and
 Optimum use of water replacement surges
through recycling operational and
maintenance costs
 High energy demand
(vacuum filter)
Gravity  Nominal energy requirement  Large land area
sedimentation  Efficient use of water requirement
 No need for chemicals  Time-consuming process
 Low operation expense  Not applicable to all species
 Output needs further drying
Centrifugation  A rapid and efficient  Not economical due to
technique excessive energy
 High rate of recovery requirement
(>90%)  High OpEx
 Ideal for pilot scale and lab-  Suitable for recovery of
based study. high-value products
 Applicable to the entire  Too slow and costly to
microalgae species implement on a large scale
 High probability of cell
damage

2.6. Cell disruption and lipid extraction techniques

There are many collateral benefits of using microalgae for manufacturing biofuel other than reducing
CO2 levels in the atmosphere. Microalgae are convenient as a feedstock as they can grow in any
environment, irrespective of the type of land needed for cultivation [125]. Additionally, they produce
a higher biomass per acre and grow without the use of pesticides. However, there is a drawback to
this production process. Producing biofuel on a large scale requires high capital costs and provides a
slow return on investment [126]. Biofuel production continues to face challenges due to the

21
competition in the market and ongoing demands for fossil fuels. Further innovative and advanced
technologies in microalgae cultivation will pave the way for biofuel production to reach a price scale
and manufacturing capacity such that it can compete with fossil fuels.
Since passive diffusion through the cell wall is sluggish, disrupting the cell walls is an effective
way for augmenting the efficacy of lipid extraction. Disrupting cell walls and membranes enables
solvents complete access to intracellular lipids, increasing the lipid recovery efficiency. There are a
variety of available techniques for disrupting cells, which are categorised as mechanical and non-
mechanical. The different methods of cell disruption are outlined in Figure 6.

Figure 6: Different cell disruptions and lipid extraction methods of microalgae [127]

Microalgae lipid can be extracted from broken cells using organic solvent extraction, which is the
most common and simplest method. However, organic solvents such as chloroform, methane,
benzene and others are toxic. Several environmentally friendly solvents are now being used in place
22
of organic solvents due to their toxicity, volatile nature, and difficulty in the recycling of organic
solvents [128]. Examples of these include bio-based solvents, ionic liquids and convertible solvents,
supercritical fluids, subcritical water, and pressurised solvents [129, 130]. The most common method
is supercritical fluid extraction. A supercritical fluid has a higher temperature and pressure than its
critical point. CO2, the most frequently used supercritical fluid, exists in the gas phase at atmospheric
pressure, allowing the solvent to be largely removed to form a solvent-free extract [131].Though
many studies have been conducted to reduce solvent usage, reduce extraction time, increase yield
from extraction, and enhance final product characteristics, there are as yet no well-defined methods
for extracting lipids from microalgae [132]. Extraction efficiency, reactivity with lipids, process
duration, and the costs associated with capital and operations, safety, and the generation of waste
have all been described as critical factors for large-scale lipid extraction [133].
The hydroenzymatic method is a novel sort of gentle, low-energy-consumption, highly specific
approach that does not produce hazardous or volatile organic compounds but produces a large number
of beneficial chemicals, and has garnered interest among scholars in recent years [134, 135]. Although
direct enzymatic cell disintegration is not only costly but also inefficient, pretreatment procedures
such as ultrasound-assisted hydroenzymatic oil extraction are frequently required before
hydroenzymatic oil extraction [136].The advantage and disadvantages of different cell disruption and
lipid extraction methods are shown in Table 4. A summary of the findings on the utilisation of
different lipid extraction techniques to extract lipid from microalgae has been presented in Table 5.

Table 4: Advantages and disadvantages of different cell disruption and lipid extraction processes from
microalgae [129, 137, 138]
Category Types Advantages Disadvantages

Autoclaving  Efficient method of  High OpEx

cellular disruption  Time-consuming cycles
 The limited volume inside the
Mechanical method

chamber
Bead beating  An effective and  The limited volume inside the
economical method of bead beater
cellular disruption when  Removal of beads from the
cell concentrations are culture may be time-
high consuming
 High biomass loading  High energy demand
capability

23
  Satisfactory temperature
control
 Wide commercial
application
Microwave  Reduced reaction time  Extraction at high temperature
radiation  Low OpEx may necessitate additional
 Uniform heating cooling
 Potential for direct  Selective to polar solvents
transesterification
 Higher oil yield
Osmotic shock  A simple method to carry  Large scale implementation
out not feasible due to high
 Not time-consuming energy demand
 More applicable for the  Not applicable to all
strains cultivated in microalgal cell walls
aquatic conditions
Ultrasound  Low power input required  It does not suit microalgae
for small samples with thick cell walls
 Can be performed at low  Prolong use of ultrasound
temperatures may emulsify oils released
 Possible adaptation to from microalgal cells
become part of microalgal  Not appropriate for large scale
photobioreactors
Hydrothermal  Need less time to extract  Energy-intensive
cavitation  Better of wet biomass  Set-up is expensive
 Efficient
 Scale-up is easy
Electric pulse  Simple and easy method  Sensitive to the conductivity
 Environmental-friendly of the medium
 Need lower energy
consumption

24
 Organic solvent  Simple and inexpensive  Pollution arising from
(Hexane,  Convenient for noxious and volatile solvents
chloroform, uninterrupted operation  Residuals difficult to separate
methanol,  Suitable for diverse
acetone) production scale
Surfactants  Biodegradable and non-  The selection of surfactants is
toxic problematic
 Energy-intensive
Bio-base solvent  Biodegradable and non-  Large scale application not
(Terpenes) toxic developed yet
 Renewable
Ionic liquids  Low toxicity  Lack of knowledge regarding
Non- Mechanical method

([Bmim(C),  Non-volatile commercial viability to date.

[Bmim][BF4),  Thermally stable: non-
[Bmim[MeSO,], flammable, and liquid at a
[Bmim[HSO4), wide temperature range (0-
[Bmim)CF3SO3), 140°C)
[Emim|[Ac},  Can be adapted for specific
[Emim][MeSO4,], solubility, polarity,
(Propy-mim] conductivity and relative
(Br, [Cyno-mim]) hydrophobicity.
Supercritical  Higher yield with reduced  Complex operation
fluids extraction time  High CapEx and OpEx
(CO2, ethanol,  Low toxicity
glycerin, H2O)  The solvent-free extract
produced in the process,
 Can preserve the natural
quality of bioactive
compounds
 Energy saving
Switchable  Higher yield with reduced  Complex operation
solvent extraction time  High CapEx and OpEx

25
(NN dimethyl  Low toxicity, the solvent-
cyclohexylamine, free extract produced in
ethyl butylamine, the process,
secondary  Can preserve the natural
amines, tertiary quality of bioactive
amines) compounds
 Energy saving
Subcritical water  Non-toxic/low toxic,  Large scale application not
Extraction  Low energy requirement, developed yet
(Water)  Eco friendly,
 High selectivity of
required compounds
(lipids), and
 Reduced extraction time.
Pressurized liquid  As a clean solvent, water is  Harsh conditions
Extraction environmentally friendly  Water needs to be disinfected
(CO2, ethanol) and easy to recycle to remove dissolved oxygen
 Abundant and cheap  The high temperature on the
source equipment may cause
corrosion.
Aqueous  Equipment automation  High voltage
enzymatic  Reduced extraction time  High CapEx and OpEx
method  Less consumption of
(Cellulase, solvent
pectinase,  Airtight extraction
amylase) minimizes pollution

Three phases  Low energy consumption  Time-consuming,

Partitioning  Environmentally friendly,  Enzyme emulsification
(Tert-butanol and safe and hygienic problem
ammonium,
sulphate)

26
Wet extraction  Mild, economical and can be  Influenced by different factors
directly used in crude plant which need to be further
materials. studied.

27
Table 5: Summary of the findings on the utilisation of different lipid extraction techniques to extract lipid from microalgae
Microalgae Treatment Solvent Temperature Duration Frequency Acid Enzyme Lipid Refs.
(ratio) (℃) (min) (kHz) (%)
Chlorella Vulgaris Ultrasound C:M (2:1) × 20 40 × × 19 [139]
Acid hydrolysis H × 120 × HCL × 54.20 [140]
Acid hydrolysis H 60 120 × H2SO4 × 33.74 [141]
Oxidation E × 3 × *FeSO4 × 17.40 [142]
Microwave C:M (2:1) 140 ± 9.0 1 × × × 41.31 [143]
Ultrasound C:M (2:1) 44.0 ± 1.7 1 24 × × 35.28 [143]
Supercritical H:E (1:1) 40 60 × × × 75.54 ± [144]
CO2 1.12
Nannochloropsis sp. Microwave C:M (2:1) 65 5 × × × 8.47 [145]
Hydrodynamic H:M 34 10 × × × 8.90 [146]
cavitation (2.3:1)
Surfactant H 50 120 × × × 78.20 [147]
Enzymatic H:P (3:2) × × × × Cellulase, 73 [148]
Mannanase
Enzymatic S:B (1:2) × × × × Cellulase 90.40 [149]
Scenedesmus sp. Microwave C:E (1:1) 65 5 × × × 8.47 [150]
Microwave H 45 ± 1 2 × × × 50.90 [151]

28
 Enzymatic C:M (1:1) × × × × Cellulase, 13.80 [152]
Xylanase,
Pectinase
Enzymatic H × × × × Lysozyme, [153]
12.50
Cellulase
Enzymatic H × × × × Lysozyme 16.60
Enzymatic H × × × × Cellulase 15.40
Botryococcus Microwave C:M (2:1) 140 ± 9.0 1 × × × 56.42 [143]
braunii Ultrasound C:M (2:1) 44.0 ± 1.7 1 24 × × 39.61 [143]
C=Chloroform; M: Methanol; H=Hexane; P= Propanol; E= Ethanol; * Iron (II) sulphate; S=slurry of hydrolized algae, distilled water and ammonium
sulphate; B: tert-butanol; ×Either not applicable or Not found.

29
3. Production of biofuel from microalgae
Biofuel, like any other hydrocarbon fuel, is produced from organic matter which undergoes
photosynthesis in a similar way to photosynthetic bacteria, vascular land plants, and microalgae [8].
Biofuel research and development has been underway for quite a while since it can be considered a
practical alternative to traditional fossil fuels like diesel and gasoline. Biofuel is categorized as a
renewable energy source due to its consumption of biomass as feedstock [154]. In daylight hours, a
continuous and controlled supply of CO2 with the correct pH measurements needs to be provided to
microalgae [155]. Power plant emissions can be utilized in microalgae cultivation to supply CO 2
which would help reduce environmental degradation caused by burning fossil fuels.
Different technologies including thermochemical, biochemical or biological can be used to
produce various forms of biofuel from microalgae biomass (Figure 7) [156]. Thermochemical
conversion is the process by which the organic content in algal biomass is decomposed in order to
produce biofuels. Based on temperature, pressure, and heating time, thermochemical conversion is
classified as pyrolysis, liquefaction, gasification [48]. It is generally agreed that thermochemical
conversion is the most straightforward method of turning microalgae into biofuel when compared to
chemical and biochemical conversion processes [157]. Biochemical conversion technologies refer to
the conversion of biomass into corresponding products through certain physical, chemical, and
biological pretreatments. Pretreatments of biomass are intended to aid in the achievement of optimal
conversion effects rather than the production of final products, which is the primary distinction
between the aforementioned physical and chemical conversion of biomass technologies. Table 6
shows the distinctive features of thermochemical and biochemical technologies of converting
microalgae into biofuel.

30
 Figure 7: Thermochemical, biochemical/chemical conversion of microalgae biomass into biofuel
[158]

Table 6: Distinctive features of thermochemical and biochemical conversion technologies of

microalgae biomass [159]
Thermochemical conversion Biochemical conversion
 This conversion route is unaffected by  Environmental factors significantly control
environmental factors. the conversion process.
 Operating temperature and pressure are  Low processing temperatures and higher
much higher compared to biochemical selectivity of products created are two
routes. notable advantages of biochemical
processes.
 Can be used on practically any biomass  Particularly suited to convert wet algal
feedstock. biomass with a moisture content above 50%.
 Feedstock pretreatment is avoided in many  Feedstock pretreatment is an integral part of
cases. this conversion route.
 Reactions proceed at a faster rate, leading  The reaction rate is slower, requiring more
to a much shorter residence time. residence time for product generation.
 Due to the reaction's fully chemical nature,  Productivity is limited due to biological
it has higher productivity. conversion. The increase would require
higher capital investment such as bigger
reactor

31
  Using fractional separation, numerous  The use of bacteria, enzymes, and/or
high-value products are possible. chemicals is a prerequisite to get reasonable
output.
 Unwanted wastes in the form of sludge can
 Waste generation is marginal. be generated, which requires further
processing.
 Product output is comparatively low, owing
 Energy-intensive.
to the biological nature of conversion.
 Normally, confined to one or a few products,
 Commercially well-established. requiring more microbes and enzymes for
further products.

3.1. Microalgae as a biodiesel source

Biodiesel is one of the most significant biofuels produced because there is favourable demand for it,
and it can replace non-renewable fuels used in industry and transportation. Biodiesel is a liquid fuel
that is known to be environmentally safe because it is non-toxic, biodegradable, and reusable and can
be used in diesel or standard engines requiring no alteration [160]. Biodiesel is generally produced
through the transesterification process [161]. The chemical reaction of oil/fat with alcohol to produce
glycerol and methyl esters, which is usually aided by a catalyst, is known as transesterification [162]
as shown in Figure 8. The process of conventional transesterification involves separating and refining
microalgae biomass prior to transesterification, with refining steps comprising drying, oil extraction,
and cell disruption [163]. Such refining phases constrain the commercialization of third-generation
biodiesel tremendously because: (i) based on certain microalgae’s width and cell wall strength, oil
extraction and cellular disruption methods require high energy; and (ii), wet biomass adversely
impacts extraction of oil due to the presence of water, requiring the need for time-consuming drying
[163-165]. The various mechanisms of transesterification in the conventional transesterification
process are determined by the form of catalyst used. Several studies determined the appropriate
operating conditions for producing biodiesel from algae using enzymatic catalyst [166, 167], acid
catalyst [168, 169], homogenous alkaline [170-172], or heterogeneous catalyst [173, 174]. The
experiments for the algae-to-biodiesel transesterification method using various catalysts are
summarized in Table 7.

32
 Figure 8: The transesterification reaction of biodiesel production [175, 176]

Because of the minimal pressure and temperature requirements, transesterification was most
commonly performed using a homogeneous alkaline catalyst. Furthermore, high lipid production is
obtained comparatively short time. Nevertheless, alkaline catalyst partially saponified by the free
fatty acids contained in microalgae lipids and is unfavourable for producing biodiesel as it leads to
issues in downstream purification and separation [177]. Therefore, a homogeneous acidic catalyst is
best utilized for microalgal lipids with a free fatty acid content of more than 1%. The reaction process
of this method is more complex, and the temperature is usually higher. Both acid and alkaline catalysts
have been used in research, with free fatty acids decreased by esterification utilizing the acidic
catalyst, followed by transesterification with the alkaline catalyst until the free fatty acid content was
lower than 1% [177]. Removing and separating homogeneous catalysts after a reaction is complex
and sometimes results in catalyst reduction. As a result, heterogeneous catalysts are applied because
they are easy to recycle and reutilize.
Sometimes enzymes are used as a catalyst because of their natural viability in the environment.
High energy input is required for the purification and production process of biodiesel from
microalgae, and for esterification and transesterification, hence research has focussed on optimizing
the use of energy in these processes [178]. Song et al. [179] studied the optimization and simulation
of the development process of biodiesel from microalgae in his research using process integration
and heat recovery of waste. The optimized process was simulated using PRO/II software. It involved
restoring latent gas vapour recompression which is later replaced with cooling water. According to
their research, the cost of producing biodiesel was minimized by 0.172$/L to 0.592$/L.

33
Table 7: Summary of the research done on the microalgae-to-biodiesel transesterification process
Types of Microalgal Biodiesel
Algae Species Conditions Ref.
transesterification biomass moisture yield
Aurantiochytrium sp. Enzyme catalysed, In situ 0% to 2% (v/v) At 50° C for 12 hours, the DMC to 89.5% [180]
esterification in dimethyl biomass ratio is 5:1 (v/w) and the
carbonate enzyme to biomass ratio is 30% (w/w).
Botryococcusbraunii Continuous methanol 7.8% methanol: mol lipid = 151:1, 5 hours, 95% [181]
reflux with an acid catalyst 47 % v/v hexane, 75% catalyst/total
and a co-solvent lipid
Chlamydomonas sp. Alkaline catalyst 68.7% sample = 12ml/8ml/2g, 15 mins, 45° C, 101% [182]
600rpm 0.5wt% NaOH in MeOH,
hexane/MeOH/
Chlorella sp. Acid catalyst 0% to 72.5% Varied - [183]

Chlorella pyrenoidosa Acid catalyst with co- 80% 60° C for ±10mins, 4ml methanol, 4ml 10.5% [184]
solvent and microwave chloroform, 0.2ml H₂SO₄,
irradiation
Co-solvent and acid 90% 4mL methanol, 0.5 M H2SO4 and 8 mL 92.5% [185]
catalyst n-hexane at 120° C for 180 minutes
Chlorella vulgaris Supercritical method 80% Temperature= 325° C 100% [186]
Time=120mins
Ionic liquid catalyst 40% Temperature= 85° C 51.54% to [187]
Time= 60 mins 93.89%

34
 Stirring speed: 1000 rpm
Methanol to biomass ratio: 0.15:1 to
18:1
Acid catalysed 75% Temperature= 60° C 96.0 wt.% [188]
Time= 120 mins
Stirring speed: 200 rpm
Ettlia sp., Acid catalyst 80% Pre-treatment at 80° C for 30 minutes - [189]
Nannochloropsis oceanica,
Nannochloropsis salina,
and Golenkinia sp.
Nannochloropsis gaditana Acid catalyst with co- 65% 0.3ml H₂SO₄, 2ml chloroform, 1ml 96.4% [190]
solvent ethanol, ethanol to biomass ratio =
3.3:1, 125° C, 2 hours
Nannochloropsis oculata Supercritical CO2 80% Dry microalgae to ethanol ratio: 1 to 15 25% [191]
(wt.%/v.%)
Temperature = 150° C, different
reaction times (80 to 160 min) and
pressures (100 to 200 bar)
Nannochloropsis sp. Co-solvent and microwave 80% Temperature= 65° C 36.79% [192]
irradiation Time=15 mins

35
Combined acid catalyst - Methanol plus H₂SO₄ (20 min, 90° C), - [193]
and alkaline in microwave methanol plus
method NaOH (20 min, 90° C)
Supercritical method 90% wet algae to methanol = 1:9 (wt./vol), 85.8% [194]
255° C, 25mins
Microwave and ultrasound - SrO catalyst (0.3 g), 20.9% [195]
radiation with alkaline 60° C for microwave, 5 mins at 50° C 37.1%
catalyst for sonication methanol–chloroform
(1:2 v/v)

36
3.2. Microalgae as a source of bioethanol and biobutanol
Bioethanol, one of the major biofuels, comes in three groups: (1) sugar and starch bioethanol, (2)
lignocellulosic bioethanol, and (3) microalgal bioethanol [42]. Because of its abundance and
simplicity, sugarcane is commonly used as a raw material for the processing of bioethanol [196].
Biomass and lignocellulosic starch are cheaper; however, they must be hydrolysed to produce
fermentable sugars. Due to their high carbon content, macroalgal bioethanol might be fermented using
various microalgae through the formation of essential or fundamental sugars from their
polysaccharides [42]. Various carbohydrates of microalgae, such as cellulose, agar, starch, laminarin,
and mannitol are used in bioalcohol including ethanol, butanol, propanol processing, and the
performance of fermentation is determined by microalgae preference [197].
Microalgae with a lot of starch, like Chlorella, Dunaliella, Chlamydomonas, and Scenedesmus,
are great for making bioethanol [198]. In the fermentation of ethanol, algae such as Chlorococcum
and Chlorella Vulgaris is easy to convert and offer higher conversion efficiency. Brown algae are
often used as the main feedstock in modern agriculture due to their high carbohydrate content [199,
200]. Scenedesmus, Dunaliella, Chlorella, Chlamydomonas, and Spirulina are microalgae that are
considered to generate significant starch and glycogen (more than 50 percent of their dry weight),
making them viable to use as raw material in the manufacture of ethanol [201]. Bioalchol is produced
through a series of steps that include drying, cell disruption, saccharification, and fermentation as
shown in Figure 9.
In the case of bioalcohol production, it is possible to use directly harvested wet biomass;
nevertheless, it is believed that dried biomass will perform significantly better [202]. Seon et al. [203]
investigated post-treatment processing and various acid hydrolysis of Chlorella sp. They found that
the neutralization using Ca (OH)2 and hydrolysis using H2SO4 have led to higher outturn (o.43 g
ethanol/g sugars) than NaOH (0.27 g ethanol/g sugars). They also found that reducing the number of
reactive species can lead to an increase of yielding to 0.25 g ethanol/g sugars. Yu et al. [204] studied
the bioethanol yield of two indigenous microalgal under microwave-supported heating wet
torrefaction. They found that microalgae C. Vulgaris offer excellent performance in producing
bioethanol. Kim et al. [205] studied the bioethanol and biodiesel co-production using the microalga
Chlamydomonas sp. KNM0029C. The maximum bioethanol (0.22 g/g residual biomass) was found
by pretreatment with amyl glucosidase enzyme after sonication. A summary of the recent research on
bioethanol production from microalgae has been presented in Table 8.

37
 Figure 9: Series of steps involved in the production of bio-alcohols from microalgae through
fermentation [202]

Table 8: Summary of the recent research on bioethanol production from microalgae

Bioethanol
Microalgae species Process Parametric study Ref.
yield
Chlorella sp. Hydrolysis using Effect of reactive 0.43 g [203]
H2SO4 and oxygen, nitrogen ethanol/g
neutralization using Ca species, time and sugars
(OH)2. depletion of cations
C. Vulgaris Hydrolysate using wet Effect of acid 0.07 g [204]
torrefaction concentration, ethanol/ g
temperature and time microalgae
Chlorella Fermentation Effect of acid 0.19 g/g [206]
concentration, glucose
temperature and time
Chlorella Vulgaris Wastewater treatment Effect of water quality 33.21 g/L [207]
effluent from tilapia and time
culture pond and
fermentation
Arthrospira platensis, Hydrolysis and Effect of acid 45.49% [208]
chlorella marina, fermentation concentration and
and ulva linza time

38
 Saccharomyces Hydrolysis and Effect of acid 0.46 g/g [209]
cerevisiae fermentation concentration, time, glucose
microalga biomass
concentration and
different catalysts
Nannochloropsis Municipal wastewater Effect of culture 7.26% [210]
oculata and tetraselmis treatment and duration and
suecica fermentation fermentation time

Porphyridium Hydrolysis and Effect of incubation 2.98 mg/mL [211]

cruentum fermentation and time and reaction time
simultaneous
saccharification and
fermentation
Saccharomyces Hydrolysis Effect of algal 18.57 g/L [212]
cerevisiae fermentation process biomass amount, yeast
volume, and time of
fermentation
Saccharomyces Enzymatic hydrolysis Kinetics study and 0.46 g [213]
cerevisiae and fermentation effect of time ethanol/g
glucose
Chlamydomonas sp. Fermentation Effect of Incubation 0.22-g/g [205]
time and yield of residual
biodiesel along with biomass
bioethanol
Chlorella Acid pre-treatment on Effect of time and 0.28 g [214]
microalgae and acid concentration ethanol/g
fermentation microalgae

3.3. Microalgae as a source of biohydrogen

Hydrogen is an abundant, long-lasting, and cost-effective source of clean energy. Innovative studies
on hydrogen production, processing, and technical advancement, could lead to hydrogen becoming a
sustainable bioenergy source in the near future [215]. Biohydrogen is produced by a photobiological
reaction that involves water and sunlight. Under various conditions of the microalgae community, the

39
synthesis of hydrogen and ethanol, as well as the proportion of carbon dioxide produced, is dependent
on the starch produced by the oxidative reaction [216].
Hydrogen may be produced in several ways, including water electrolysis, methane (CH4)
reforming by steams, coal gasification, oil and natural gas oxidations, and thermal process, indirect
and direct bio photolysis [217, 218]. Photo-fermentation and dark fermentation produce hydrogen
from organic compounds [43, 217]. Photo fermentation uses the most abundant materials, light and
water, to produce hydrogen gas. Unfortunately, this method of hydrogen production is ineffective
because the oxygen released at the same time halts the hydrogen production mechanism by inhibiting
the hydrogenase enzyme [156]. During the process of dark fermentation, fructose, xylose, strict
anaerobes, and oxygen releases hydrogen gas. Chlamydomonas has been widely researched for its
ability to produce hydrogen, however, it produces very little hydrogen and oxygen [219].
Different researchers have used different techniques to produce biohydrogen from microalgae. For
example, Nurdiawati et al. [220] studied the biohydrogen production from Acutodesmus
obliquus microalgae using supercritical water gasification and they proposed an integrated system
that can produce up to 45.3% hydrogen. However, they highlighted that selection of feedstock is one
of the critical cost drivers for generating hydrogen. Saka et al. [221] reported that biohydrogen can
be produced from Spirulina platensis microalgae through NaBH4 hydrolysis. Ibrahim et al. [222]
recently studied the production of hydrogen from Galdieria sulphuraria microalgae under pyrolysis
state in a membrane reactor. They observed that the addition of the Pd77Ag23 membrane in the
reactor resulted in the recovery of hydrogen in the permeate stream and which also improved the
conversion of biochar to gaseous fuels. Arun et al. [223] explored the Scenedesmus obliquus
microalgae as a source of biohydrogen through the catalytic conversion and reported a 37% yield of
biohydrogen. A summary of the recent research on biohydrogen production from microalgae has
been presented in Table 9.

Table 9: Summary of the recent research on biohydrogen production from microalgae

Parametric Biohydrogen
Species Process Ref.
study yield
Acutodesmus Supercritical Effect of moisture 13,944 kg/h [220]
obliquus water contents of wet
gasification and algae, temperature,
syngas chemical pressure and
looping hydrogenation on
process integration

40
Cyanobacterium Photobiological Effect of the 118 kg/m3/h [224]
anabaena in microalgae
variablilis photobioreactors concentration, solar
irradiance, bubbles
scattering and
concentration
Wastewater born Ammonia Effect of NH3 18.2 L H2/kg VS [225]
microalgal biomass pretreatment and pretreatment and microalgae
anaerobic dark digestion time
fermentation

Chlamydomonas Photoproduction Effect of time, 56% [226]

reinhardtii oxygen level and
presence of bacteria
Spirulina platensis Catalytic Kinetics study and 3940 mL min1g−1catalyst [221]
hydrolysis effect of
temperature, the
concentration of
H3PO4 and Co
amount
Chlorella Vulgaris Catalytic Catalytic activity and 17833 mLmin1g−1catalyst [227]
methanolysis effect of ZnCL2,
CuB loading,
NaBH4 concentration
and temperature
Parachlorella Photosynthesis Organic carbon 2.2 mmol/L [228]
kessleri and dark sources and lighting
incubation regime
Chlorella Vulgaris Catalytic Catalytical activity, 13232 mLmin1g−1catalyst [229]
methanolysis the effect of HCl
concentration,
times and
temperature

41
Galdieria Liquefaction Effect of time and 45.70 mol% H2 [222]
sulphuraria and pyrolysis in pressure
a membrane
reactor
Enterobacter Fermentation Kinetics study and 40.9 mL H2/g alga [230]
aerogenes effect of sterilization
in an autoclave, gas-
liquid ratio, bacterial
growth and
inoculation
conditions
Chlamydomonas sp. Anaerobic Effect of time and 17.02 ± 3.83 μmol [231]
phototrophic concentration L−1 h−1
Scenedesmus Catalytic Effect of time, 37 wt.% [223]
obliquus hydrothermal temperature and
liquefaction catalyst

Anabaena sp. Anaerobic Effect of 57.6% [232]

photobioreactors temperature, pH,
under nitrogen light and glucose
deprivation supplementation
Chlorella PTCC Catalytic Effect of 41.46% [233]
6010 supercritical temperature, biomass
water loading and reaction
gasification time

3.4. Microalgae as a source of biomethane/biogas

Biomethane/biogas is one of the biofuels that can be produced from different feedstocks e.g. biomass,
organic waste through an anaerobic digestion process [234]. Pre-treated and nontreated microalgae
biomass, as well as residues following lipid extraction, can be used as a methane-producing substrate,
[39]. Carbon dioxide, methane, and digestate (fertilizer) are produced via anaerobic digestion of
defatted microalgae [234]. Microalgae must be pre-treated before they can begin producing methane
[235]. Pre-treatment is divided into four categories: biological, mechanical, chemical, and thermal
[236]. Microalgae biomass is biologically pre-treated through enzymatic actions on the algal cell wall
and either fungal or bacteria is used as an enzyme to pretreat.
42
 Various approaches are used for the enzymatic pretreatment of microalgae biomass to produce
biomethane (Figure 10). Despite the fact that the use of enzymes considerably boosts methane
productivity, the cost of utilising enzymes prevents it from being used in a real-world situation [237].
To get around this problem, crude enzymes can be used to pretreat microalgae biomass. Prajapati et
al. [238] have found that fungal crude enzymes are extremely effective at digesting the algal cell wall
and liberating soluble sugars. Hom-Diaz et al. [239] found that Laccase-containing fungal broth from
Taramites versicolor is effective in increasing 74% of microalgal biogas production. Even while the
use of crude enzymes helps to alleviate the problem of high enzyme costs, it requires an additional
step of producing and extracting the enzymes. To address this issue, it may be necessary to incubate
algal biomass with enzyme-producing organisms [240].

Figure 10: Detailed enzymatic pre-treatment of microalgae biomass, Reprinted with the permission
of Elsevier from [240]

The hydraulic retention, organic loading parameters, pH, and temperature are all optimized to produce
the best possible product [42]. Currently, most biogas research focuses on wastewater treatment and
combining the production of biogas with several other algal-based production processes (e.g., high-
value products, bioethanol, biodiesel) [241, 242]. Microalgae-produced methane can be used to
produce energy in manufacturing facilities, and surplus energy could be marketed to offset biodiesel
production costs. However, because of high harvesting, culturing, cultivation and refining costs,
biomethane generated by anaerobic digestion of microalgae has not yet been compared to commercial

43
fuels [39]. A summary of the recent research on biogas production from microalgae has been
presented in Table 10.

Table 10: Summary of the recent research on biogas production from microalgae using anaerobic
digestion technology
Microalgae Operating condition Reactor type; Methane yield Refs.
Temperature HRT Volume
(°C) (Days)
Porphyridium 55 20 Anaerobic reactor; 179 mL CH4/g VS [243]
cruentum 500 m3
Chlorella 35 20 Continuous stirred 168.9 mL CH4 /g [244]
vulgaris tank reactor; 1.5 L COD
Haematococcus 37 30 Mesophilic 91.9 mL CH4/g VS [245]
pluvialis anaerobic digester;
0.118 L
Chlorella 37 18 Batch reactors; 147 mL CH4/g VS [246]
pyrenoidosa 300 mL
Chlorella sp. 37 12 Anaerobic 194.63 mL CH4/g [247]
stainless-steel VS
reactor; 500 mL
Chlorella sp. 35 60 Batch, 0.16 L 0.14 m3/kg [248]

Chlorella vulgaris 37 35 Batch, 0.0715 L 0.062 m3 CH4/kg VS [249]

Scenedesmus 37 35 Batch, 0.0715 L 0.16 m3 CH4/kg VS [249]

obliquus
Scenedesmus sp. 39 70-100 Continuous stirred 0.185 m3 CH4/kg VS [250]
tank reactor, 14 L
Nannochloropsis 53 15 Continuous stirred 0.41 m3 CH4/kg VS [251]
limnetica tank reactor, 1 L
Chlorella sp. 37 20 Lab-scale reactor, 0.2 m3 CH4/kg VS [252]
2L
Nannochloropsis 53 24 Batch, 0.337 L 0.41 m3 CH4/kg VS [251]
limnetica
Chlorella sp. 37 100 Batch, 0.16 L 0.26 m3 CH4/kg VS [253]

44
3.5 Microalgae as a source of bio-oil
One popular way to use algae energy is to produce crude bio-oil which is dark, viscous and energy-
dense fuel. Bio-oil can be produced from microalgae through the utilisation of pyrolysis and
hydrothermal liquefaction process. It has been reported that the bio-oil yields from several microalgae
were 5–25 weight percent higher than their lipid content [254]. Aquatic processing of microalgae
biomass is attractive because it reduces the energy requirements associated with drying.
Pyrolysis involves heating organic compounds at a high temperature (less than 700 °C) in the absence
of oxygen. Slow, intermediate, and fast pyrolysis are the three basic subcategories of pyrolysis [255].
Chemically, the bio-oils contain aldehydes, cresols, and acids. Bio-oils obtained through the
utilisation of pyrolysis from lignocellulose biomass are rich in oxygen but complex and viscous. It is,
therefore, necessary to improve bio-oils by hydrogenation and cracking. Bio-oils derived from
pyrolysis of microalgae biomass, on the other hand, contain linear hydrocarbons and nitrogenous
species, which are generated as a result of the pyrolysis of lipids and proteins [256]. Hydrothermal
liquefaction is the thermochemical process in which hot compressed or sub-critical water are used to
transforms wet microalgal biomass into liquid fuel. Liquification is typically carried out at
temperatures between 300 and 350 ℃ and pressures ranging from 5 to 20 MPa, with microalgal mass
fractions ranging from 5 to 50% in the slurry feed [257].
Pressures are maintained at high levels to keep water in the liquid phase, and the reaction
temperature is typically maintained for 5–60 minutes. Catalysts are being used to speed up the
liquefaction process. The main products are bio-oils, but there are also gaseous, aqueous, and solid
bi-products that are produced during the conversion process [258]. Following completion of the
liquefaction process in an autoclave, the process is allowed to cool down to standard room
temperature before collecting the gas for further analysis. Then the mixture is treated with a
chloroform-based solvent. In order to treat the reaction mixture, chloroform is used as a solvent.
Evaporation of the solvent is used to extract the bio-oil from the mixture while filtration is used to
distinguish the aqueous phase from the insoluble solids [259]. A summary of the research findings
on bio-oil production from microalgae using different thermochemical technologies has been
presented in Table 11.

Table 11: Summary of the research findings on bio-oil production from microalgae using pyrolysis
and hydrothermal liquefaction technologies.
Technologies Microalgae Operating condition Bio-oil Refs.
yield
T RT HR CG, (%)
(℃) (min) (°C/min)

45
 GFL
(L/min)
Hydrothermal Chlorella 300 60 × × 46.60 [260]
liquefaction vulgaris
Desmodesmus sp. 375 5 × × 49.40 [261]
Spirulina 290 30 × × 43.60 [262]
Chlorella sp. 300 6 × × 32.10 [263]
Chlorella 280 30 × × 32.10 [264]
pyrenoidosa
Chlorella sp. 300 6 × × 32.10 [265]
Chlorella 299.7 65 × × 21.70 [266]
sorokiniana &
Chlorella sp.
Echinacea spp 272 35 × × 57.80 [267]
Coelastrum sp. 320 120 × × 36 [268]
N. chlorella 360 60 × × 82.80 [269]
Pyrolysis Chlorella 500 0.03– 36000 N2, 6.67 57.90 [270]
protothecoides 0.05
M. aeruginosa 500 120 20 N2, 0.6 32.50 [271]
ml/min
Chlorella sp. 550 180 15.46 --- 50.20 [272]
Chlorella 375 30 25 N2, 83 26-38 [273]
vulgaris ml/min
Scenedesmus 375 30 25 N2, 83 28-50
obliquus ml/min
Desmodesmus sp. 600 --- 20 N2, 10 28.20 [274]
ml/min
Nannochloropsis 600 --- 20 N2, 10 74.80
oculata ml/min
Halamphora 600 --- 20 N2, 10 58.70
coffeaeformis ml/min
Nannochloropsis 380 60 10 N2, 180 L/h 24.60 [275]
gaditana
Nannochloropsis 480 60 10 N2, 180 L/h 31.20
gaditana
Scenedesmus 380 60 10 N2, 180 L/h 20.30
almeriensis
Scenedesmus 480 60 10 N2, 180 L/h 20.30
almeriensis
Chlorella 550 --- ---- Helium 62 [276]
sorokiniana
46
 Scenedesmus 550 20 10 N2, 200 55.59 [277]
obliquus ml/min
×Not applicable; T: Temperature; RT: Reaction time; CG: Carrier gas; GFL: Gas flow rate

4. Fine chemicals and bioactive compounds production from microalgae

Microalgae has considerable industrial importance when it comes to non-fuel materials [278]. Lipids,
proteins, carbohydrates, polyunsaturated fatty acids (PUFAs), cellulose, starch, vitamins, food,
cosmetics, pesticides, organic fertilizer, livestock feed, animal feed, natural dyes, nutraceuticals,
pigments, and even combustion-based electricity generation [60, 279] are just a few of the industrially
significant co-products or value-added products that algal biomass can be used for. Figure 11 shows
the application of algae strains to generate value-added products.

Figure 11: Application of microalgae strains to generate value-added products

47
4.1. Microalgae in agriculture
In modern agriculture, microalgae are an eco-friendly option to supplant chemical fertilizers since
they can be used as biofertilizers, soil modifiers, and feed additives[280, 281]. Cultivation of rice
inoculated with marine or aquatic microalgae like Spirulina Platensis and Chlorella Vulgaris as
biofertilizers increased rice yield by 7–20.9 percent [282]. Microalgae as a soil additive increase root
capacity, chlorophyll development, sprout dry weight, and plant height significantly [196, 283]. As a
soil supplement, microalgae enrich soil health by fixing CO2 and N2 and through the secretion of
extracellular polysaccharides, thus lowering the cost of the macro and micronutrients needed to
achieve high yields. Auxin, cytokinin, and jasmonic acid are plant growth hormones produced by
microalgae that act as biostimulants [281]. Carrots cultivated using 0.5 percent Algaminoplant®,
algae-derived biostimulants, showed improved sprouting, nitrate reduction capacity, and carotenoid
build-up [284]. Microalgal species are found to improve the plant immune system by generating
antimicrobial compounds which constrain or kill pathogenic bacteria, fungi, or nematodes and hence
can be used as an alternative to harmful chemical fungicides [281].

4.2. Microalgae as a source of animal feed

Microalgae provide a wide range of high-quality compounds, including carbohydrates, lipids, and
proteins as well as vitamins, antioxidants, and minerals which illustrates their value as food sources
for aquaculture or animal feed [285-287].In general, the microalgae must be of a size suitable for
ingestion with a digestible cell wall, free of any toxicity, and contain ample biochemical ingredients.
Proteins, a major source of human and animal nourishment, constitute 50–70% of microalgae
structure [60]. Microalgae are used as commercial feed for many aquatic animals, both marine and
freshwater, such as larvae, molluscs, finfish, and adolescent shellfish [288]. Microalgae are also used
to produce the zooplankton necessary as live food for juvenile crustacean animals and finfish [288].
Aquaculture’s most commonly used microalgae organisms are Spirulina, Isochrysis, Chaetoceros,
Chlorella, Nannochloropsis, Tetraselmis, Thalassiosira, and Skeletonema. Spirulina has a high
pigment content and is widely used in aquaculture feed, notably for tropical fish.
Fish cultured using Spirulina as feed have been found to grow faster, have improved palatability,
contain more flesh and less fat, and have brighter skin [288]. The addition of small quantities of
microalgae biomass in the feed enhances physiological characteristics such as gut function, disease
tolerance, and antiviral and antibacterial action, and as a result, the animal's development, feed
conversion ratio, weight, and overall reproductive performance will improve [286]. Microalgal
biomass which is abundant in bioactive compounds and proteins including polyunsaturated fatty acids
(PUFAs), especially EPA and DHA, has been shown to boost meat quality in pigs, broilers, and lambs
[289]. When poultry is fed PUFA-rich microalgae as a feed supplement, PUFAs settle in the eggs.
48
 Arthrospira sp., Chlorella sp., Schizochytrium sp. have been found to develop potential functional
elements in broiler nutrition [286, 290]. The addition of microalgal supplements such as
Schizochytrium sp. to the diet of ruminants and pigs results in higher PUFA concentrations in meat
and milk, thereby improving nutritional value [290, 291]. Wastewater with high nutrient content
could be used to grow microalgae for aquaculture or livestock feed while lowering greenhouse gas
pollution and wastewater remediation, resulting in a self-sustaining system [196]. Table 12 shows
the summary of the research findings on microalgae as animal feed.

Table 12: Summary of the recent research on microalgae as animal feed. Microalgae can be used as
feed for fish, poultry, cow, lamb, sheep etc.
Microalgal species Types of Effect of microalgae as a feed Refs.
animal
Aurantiochytrium Pig DHA levels were higher in pork loin and back fat, and [292]
limacinum EPA levels were higher in pork loin fat.
Pig There was no discernible difference in growth; [293]
however, there was a decrease in cortisol response and
an increase in cytokinin and chromogranin.
Arthrospira Pigs Meat's physical-chemical properties were maintained [294]
platensis along with an increase in PUFAs. Pigs' growth
performances and meat technological quality were
unaffected.
Chlorella Vulgaris Fish Improved colour factors were observed. [295]
Ducks More feed intake and body mass gain; improvement of [296]
meat colour
Cows Enhanced milk production; higher daily protein build- [297]
up; lower milk fat content
Goats More milk production; greater energy and protein [298]
content in milk; slightly higher omega-3 fatty acid
content
Chlorella sp. Fish Higher dry mass and raw protein content in the carcass; [299]
positive effect on growth, feed conversion ratio, and
protein productivity.
Chlorella Cows More feed intake and daily body mass gain [300]
pyrenoidosa

49
Chlorella kessleri Goats Milk was found to contain more protein and lower fat. [301]
Desmodesmus sp. Pig Growth followed the control measures applied, with no [302]
notable differences
Pig Hb levels improved; ADG increased with higher ADFI; [303]
feed efficiency was unchanged with control.
Dunaliella salina Shrimp Beta-carotene in microalgae offered protection against [304]
the white spot syndrome virus.
Cows More feed intake and daily body mass gain [300]
Dunaliella Sea-urchin There were no alterations in the larvae's growth [305]
tertiolecta timeline; the larvae were larger and had a better chance
of surviving.
Sea-urchin There was no change in survival and development rates; [306]
however, the growth rate was faster.
Nannochloropsis Pig Hb levels improved; ADG increased with higher ADFI; [303]
oceanica feed efficiency was unchanged with control.
Nannochloropsis sp Fish Weight increase, specific growth rate, protein efficiency [307]
. ratio, and hepatosomatic index were all unaffected.
Increases in the essential amino acids, activities of
pepsin, trypsin, and lipase, as well as total superoxide
dismutase and glutathione peroxidase and total
antioxidant capacity in serum and liver.
Porphyridium sp. Chickens No discernible difference in body mass; improved yolk [308]
colour, lower cholesterol in eggs.
Scenedesmus sp. Fish Higher dry mass and raw protein content in the carcass; [299]
positive effect on growth, feed conversion ratio, and
protein productivity.
Fish Several factors such as weight gain, specific growth [309]
rate, thermal growth coefficient, feed conversion rate,
condition factor, and protein efficiency ratio were all
decreased at higher doses.
Spirulina platensis Fish Around 20% raw alga or enzyme-treated alga can be [310]
used, having no adverse effects on the animals' growth
or health.

50
 Fish Weight increase, specific growth rate, protein [311]
efficiency, and lipid efficiency were all significantly
greater in the diets containing 6 and 8% algae. The feed
conversion ratio was lower.
Chickens Omega-3 fatty acids content increased; improved [312]
colour of the yolk, lower cholesterol levels.
Cows Milk with low fat [301]
Lambs Body mass enhancement [313]
Spirulina sp. Chickens Unaltered growth rate; visual appearance of fillets [314]
improved.
There was no noteworthy variation in body weight; [315]
antioxidative enzyme activity was raised (SOD and
Glutathione peroxidase)
Cows Enhanced milk production; higher daily protein build- [297]
up; lower milk fat content
Sheep Supplementing with 3% algae lowered body fat, blood [316]
cholesterol, triacylglycerol, and oxidative stress while
also increasing antioxidant capacity. Increased IgG
content in serum, as well as red and white blood cell
counts, are among the immune-related metrics that were
improved.
Cows More feed intake and daily body mass gain [300]
Schizochytrium Chickens There was no significant change in body weight; the egg [317]
limacinum yolk had a higher DHA content, and the colour was also
darker.
Schizochytrium sp. Cows Body weight and milk yield unaltered; fat yield [318]
lowered; saturated fatty acids in milk reduced; DHA
levels in milk enhanced.
Shrimp Growth and feeding efficiency unaffected. [319]
Sheep DHA and EPA levels in animal muscle and liver tissues [320]
enhanced.
Sheep The fatty acid composition of different parts such as [321]
intramuscular, subcutaneous, and kidney knob and

51
 channel fat is altered. The most affected deposit was
intramuscular fat.
Lambs Reduced daily weight gain; increased DHA levels; [322]
higher lipid oxidation in meat; lesser meat flavour and
smell ratings.
Lambs Superior digestibility values of PDF and ADF; [323]
Pigs Better DHA amounts; [324]
Pigs Pigs' growth performances and meat quality were [325]
unaffected.
Pigs Growth performance, total cholesterol, white blood cell, [326]
and triglyceride levels were unaffected by feed
variations. The number of lymphocytes was found to be
higher.
Tisochrysis Fish Growth or feed conversion efficiency is unaffected by [327]
lutea and Tisochrys feed variation.
is Suecica

4.3. Microalgae as a source of medicine

The therapeutic effects of microalgae are incorporated in a variety of human medicinal products,
including anticancer, antiviral, antibacterial, antifungal, cardiovascular health, anti-inflammatory,
and others [328-330]. Medicines that inhibit disease and promote wellbeings, such as pills, tablets,
powders, beverages, and gels, can contain microalgal bioactive components [330]. Carotenoids are a
type of organic pigment found in the chromoplasts and chloroplasts of plants and photosynthetic
organisms. Photosynthetic bacteria, plants, and algae produce over 600 carotenoids that occur
naturally, a few of which have found use in commercial applications such as astaxanthin, lutein, β-
carotene, zeaxanthin, lycopene, and bixin [330]. Various microalgae were used to produce
commercially viable pigments, such as β-carotene originating from astaxanthin and Dunaliella salina
from Haematococcus pluvialis [279].
The algae Dunaliella salina is considered to be the best source of β-carotene [331]. Astaxanthin
is one type of carotenoid which is found in both fresh and saltwater [332]. Astaxanthin is a powerful
antioxidant with anti-inflammatory, sun protection, anti-aging, anticancer, and immune system
enhancing properties, making it suitable for use in nutraceutical products, food sources, and feed
[333]. Astaxanthin prevents UV-induced photo-oxidation occurring in the skin and oxidative
degradation in the retina and is used to treat nerve damage correlated with age-related macular
degeneration. It also lowers the risk of neurodegenerative disorders like Alzheimer’s and Parkinson’s
52
disease [334, 335]. It is also known as a natural superfood that can increase stamina and speed up
muscle recovery and ultimately boost athletic performance [336]. Chlorella zofingiensis and
Haematococcus Pluvialis are commonly used for astaxanthin processing [337, 338]. Another form of
the carotenoid present in the pigmentation of animal tissues is lutein. It is utilized as a colourant in
pharmaceuticals, cosmetics, and foods, and is considered a significant nutraceutical. Chlorella
protothecoides, Muriellopsis sp., Chlorella zofingiensis Scenedesmus almeriensis are identified as
natural producers of high lutein content, potentially making them a suitable substitute for marigold.
Microalgal components have been used to strengthen the immune response and lower cholesterol
levels in the blood, making them effective in the treatment of hypercholesterolemia. Taurine, a non-
proteinaceous amino acid found in marine red algae, lowers blood cholesterol levels [339]. Several
species of microalgae contain 70% protein that can fulfil all essential amino acids for human health
[339]. Microalgae consist of long-chain polyunsaturated fatty acids essential for neurological and
cardiac function, as well as helping the body combat heart disease, hypertension, cancer, and
cholesterol issues [279, 340, 341]. Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), α-
linolenic acid (ALA), linolenic acid and arachidonic acid (AA) are examples of long-chain PUFAs
[196]. Some essential fatty acids including DHA and EPA cannot be synthesized by higher animals
or plants due to a lack of enzymes.
Various essential fatty acids occur in microalgae and can be processed to produce effective
nutritional supplements [342]. Odontella, Porphyridiumcruentum, Cryptothecodiniumcohni,
Isochrysisgalbana, and A. platensis are examples of PUFA-producing microalgae [196]. According
to studies, consuming DHA and EPA promotes health by controlling inflammation and lowering the
occurrence of cardiovascular diseases, as well as aiding in the growth of the nervous system in
children and improving brain function [343, 344]. Recent research [345] has revealed the significance
of polyunsaturated fatty acids (PUFAs) in the immune system, stating the positive effects of PUFA
supplementation on reactions that occur due to allergies and as new therapeutic methods for
eosinophilic disorders. Regulated EPA/AA absorption might be beneficial in the treatment of a
number of diseases and metabolic disorders [196]. Gamma-linolenic acid (GLA) decreases LDL
cholesterol in people with hypercholesterolemia, reduces premenstrual syndrome, and cures atopic
dermatitis and rheumatoid arthritis. Studies of humans and animals have shown that GLA dietary
supplementation suppresses inflammation [343]. Figure 12 shows the nutrients contain in microalgae
and the therapeutic effects of microalgae.

53
Figure 12: Nutrients contains and therapeutic effects of microalgae. Microalgae offer several benefits
as medicine including immune level improvement, reduce anemia, heart failure, hypertension etc
[346].

4. 4. Microalgae as a source of pigment

Since microalgae contain a diverse range of naturally coloured pigments, they can be used to produce
food colourants sustainably. Microalgal cultivation is not only environmentally friendly and safe, but
it also represents a cost-effective alternative to artificial colourants as a source of natural food dyes.
Carotenoids, chlorophylls, and phycobiliproteins are natural algal pigments that are precursors to
vitamins found in food and animal products, as well as to colouring agents and biomaterials,
cosmetics, and pharmaceuticals [347]. Most microalgae species contain 0.5–1 percent chlorophyll
and 0.1–0.2 percent carotenoid. All microalgae contain chlorophyll, a pigment that is important for
photosynthesis [348].

54
 Carotenoids are important components in oxygenic photosynthesis, either as light-harvesting
pigments or as protein folding structural molecules in the photosynthetic apparatus, keeping it stable
and playing a major role in commercial microalgal pigment production[349]. Carotenoids obtained
from commercially viable algae are used in livestock and fish feed, food product colourants, and
cosmetics [278]. Phycobiliproteins are a type of hydrophilic protein similar to photosynthetic
pigments called phycobilins which are present mostly in red algae and cyanobacteria [350]. In the
health sector, phycobiliproteins are used as anti-inflammatory, anti-allergenic, antioxidant, antiviral,
anticancer, and neuroprotective substances, and in other pharmaceutical applications [351].
Phycocyanin, one type of phycobiliprotein produced by Arthrospira platensis, is used to improve
the visible appearance of food items such as colouring fermented milk products, carbonated
beverages, alcoholic beverages, candy, ice cream, chewing gum, milkshakes, and fish feed. [352].
The US Food and Drug Administration (FDA) regulates phycocyanin pigment extracted from
Spirulina platensis used as a colouring pigment in gum, toffee, and other foods [353]. Salmon
pigment is made from Chlorella zofingiensis [354]. Despite the diverse benefits of microalgae,
industrial production of microalgal pigments has been limited to only a few species. At present, there
are two carotenoids (Dunaliellasalina producing β-carotene and astaxanthin from Haematococcus
Pluvialis), one phycobiliprotein (Arthrospira platensis producing phycocyanin), and chlorophyll
(from Chlorella Vulgaris) which are commercially cultivated [348]. Table 13 presents the summary
of the research finding on pigment extraction from microalgae.

Table 13: Summary of the research finding on the pigment extraction from microalgae
Algal species Extracted product Extraction method Yield (mg.gdw) Ref.
Chlorella vulgaris β-carotene Pressurized liquid extraction 0.67 [355]
Chlorophyll Pressurized liquid extraction 10.83 [355]
Carotenoids Pulsed electric fields 1.00 [356]
Dunaliella salina β-carotene Pressurized liquid extraction 34.60 [357]
β-carotene Supercritical CO2 extraction ̶ [358]
Carotenoids Supercritical CO2 extraction 115.43 [359]
β-carotene Cell milking 0.25% of TC* [360]
β-carotene Cell milking 5.30% of TC* [361]
Haematococcus Astaxanthin Pulsed electric fields 18.30 [362]
pluvialis Astaxanthin Supercritical CO2 extraction 19.72 [363]
Astaxanthin Supercritical CO2 extraction 1.80 [364]
Astaxanthin Cell milking 85 mg Lculture−1 [365]

55
 Astaxanthin Cell milking 76 mg Lculture−1 [366]
Nannochloropsis sp. Carotenoids Supercritical CO2 extraction 7.61 [367]
Carotenoids Supercritical CO2 extraction 0.34 [368]
Chlorophyll Supercritical CO2 extraction 2.23 [368]
β-carotene High-pressure 10.07 [369]
homogenization
Carotenoids Pulsed electric fields ̶ [370]
Porphyridium cruentum Phycoerythrin Pulsed electric fields 32 [371]
* TC= total carotenoids

4. 5. Microalgae as source of human food

Spirulina, Chlorella, and Dunaliella are some of the most common microalgal species used as
additives to popular foods such as pasta, noodles, cookies, ice cream, bean curd candies, and bread to
increase the nutritive values and health qualities of these foods [372]. Chlorella strain is widely used
as a food substitute in many countries around the world [288, 373, 374]. Alginates are one type of
polysaccharide found in brown algae that have been used as a human food source due to their
numerous health benefits, including the ability to absorb contaminants, produce short-chain fatty
acids, alter characteristics of bacterial colonies and reduce the absorption of cholesterol [96, 278].
Microalgae such as Thalassiosira, Isochrysis, Tetraselmis, Chaetoceros, and Nannochloropsis,
contain beneficial health additives such as DHA and EPA [60]. Since it has been shown to have
cardioprotective effects in several trials, DHA is an additive in several food types, including infant
meals, baby formula, eggs, dairy, bakery, and non-alcoholic drinks. DHA isolated from
Schizochytrium sp. and Crypthecodinium cohnii has been shown to have a cardioprotective effect
[375]. Microalgae have certain properties such as their green colour, which negatively affects
consumers’ perceptions of taste and consistency and limits their use in everyday goods [376]. For
instance, despite the various health benefits of Arthrospira and Chlorella, they change the taste and
texture of foods, which are often regarded as undesirable by customers [346, 376]. Table 14
highlights the potential application of different microalgae extracts in human food preparation.

Table 14: Application of different microalgae extracts in human food preparation

Microalgae Products Improvements Refs.
Diacronema vlkianum  ω3-PUFA* [377]

56
 Dunaliella salina  Fat [378]
 Protein
 Minerals
Haematococcus  Bioactive [379]
pluvialis composition

Isochrysis galbana  ω3-PUFA* [377]

Spirulina platensis  Nutritional [380]

properties

 Protein [381]

 Protein [382]

 Protein [383]
 Antioxidant

*ω3-PUFA=omega-3 polyunsaturated fatty acids

4.6. Microalgae as a source of cosmetics

Microalgae-derived components may have the potential to be of interest to the cosmetics industry to
produce personal care products. Cosmetic products are designed to improve the structure and
appearance of the skin with the help of active ingredients that are specifically formulated for types of
skin. Antioxidative enzymes found in algae products can help mitigate the adverse effects of reactive
oxygen species, which are one of the leading causes of aging [384]. Extracts from microalgae
organisms are widely used in the cosmetics industry in various sectors: anti-aging agents
(Colpomenia, Halymenia, Padina, Polysiphonia, Spirulina), moisturizing and skin softening agents
(Ulva Lactuca, Codiumtomentosum, Postelsiapalmaeformis), wound healing agents
(Kappaphycusalvarezii), and hair growth promoters (Ecklonia cava) [96, 279, 384]. In addition to
anti-aging skin care products, microalgae extracts have potential as antimicrobials, expanding the
already broad spectrum of applications in the cosmetics field [385].
It has been reported that eye shadow can be produced from thermophilic blue-green
microorganism's pigments as well as pink and purple colours from colourants of red microalgae
natural pigments [386, 387]. Extracts from Chlorella vulgaris have been shown to enhance collagen
repair pathways in anti-aging supplements and cosmeceuticals [5]. Also, the utilisation of extracts
from these microalgae in products promotes tissue restoration and wrinkle reduction.

57
The extracts from Arthrospira, Nannochloropsis oculata and D. salina microalgae can repair the
indications of skin ageing, tighten the skin, and prevent the development of striae [388, 389].
Additionally, Dunaliella tertiolecta and Tetraselmis suecica microalgae may be used to
produce vitamin E which is a powerful antioxidant that is commonly utilised in cosmetics [390, 391].
Because of the potential application of extracts from microalgae as cosmetics, recently, different
industries (Codif, France; Greentech, USA; Pentapharm, Switzerland) have begun to invest in the use
of microalgae extract in producing a variety of cosmetic products [392].
Recently, companies have registered new patents for new topical administration containing mic
roorganism’s exopolysaccharide particles [392]. For example, Solazyme Inc. (San Francisco, CA,
USA) has registered new patents for producing exopolysaccharides by green microalgae
(Parachlorella) for improving the health and appearance of skin. Another company, Terravia
Holdings, Inc. (San Francisco, CA, USA), has recently introduced GoldenChlorella TM and AlgaPürTM
Algae Oils, which it claims will provide significant cosmetic benefits to skin and hair. The list of
registered patents on the utilisation of microalgae extracts as a cosmetic and available cosmetic
product already in the market has been presented in Table 15.

Table 15: List of registered patents on the utilisation of microalgae extracts as a cosmetic and list of
available cosmetic products already in the market
Microalgae Activity as cosmetics Patent Refs
.
Haematococcus sp.  Inhibits UV WO2018/062427) [393
 Anti-aging ]
 Skin hydration
Phaeodactylum  Anti-aging US2018/0078521 [394
tricornutum ]
Haematococcus  Skin hydration US2018/0042978 [395
pluvialis  Inhibits UV ]
Haematococcus spp  Colorant US2018/002711 [396
.; ]
Chlamydomonas sp
p.;
Chloromonas spp.;
Dunaliella spp.;

58
 Chlamydocapsa spp
.

Chaetoceros  Preventing and treating hair loss KR20140062249 (A) [397

gracilis ]
Prototheca  Improves hair shine US20150352034 (A1) [398
moriformis,  Inhibits UV and pollution damage ]
Chlorella  Prevent moisture loss
protothecoides
Tetraselmis  Preventing and treating hair loss KR20150100302 (A) [399
tetrathele ]
Microalgae Activity as cosmetics Products Refs
.
Dunaliella salina  Prevent from oxidative damage Pepha®-Ctive [400
 Stimulates cell proliferation ]
Chlorella vulgaris  Inhibits UV Dermochlorella D® [401
 Boost the production of collagen ]
Haematococcus  Prevent from oxidative damage AstaPure® [402
pluvialis ]
Phaeodactylum  Prevent from oxidative damage FucoVital™ [402
tricornutum ]
Phaeodactylum  Inhibits UV, photo-aging and age- Megassane® [403
tricornutum spots ]

5. Future directions
The potential of microalgae to minimize carbon dioxide pollution while also treating wastewater and
producing biofuel paves the way to a more sustainable future. The applications of microalgae in the
industrial sector nevertheless face a challenge in terms of energy input and production cost, which
must be solved to allow commercial viability. For microalgal biofuel production to be economically
and technologically feasible, other essential bioproducts must also be produced. A significant amount
of biomass is required for the successful extraction of valuable products from microalgae, which
should be considered in future research. The development of an optimized biorefinery system must
be prioritized because this would increase the cost-effectiveness and efficiency of microalgae
production, cultivation, and refining as compared to the existing biorefinery system.

59
 All stages of microalgae exploitation should be developed with the aim of long-term
sustainability and environmental benefit. Future implementation should focus on the processes being
simple to apply in the pharmaceutical, cosmetic, or biofuel industries, especially in developing
countries. This is because growing microalgae for biofuels, bioproducts, and other chemical
production is also about ensuring food and energy stability for large populations, and not just about
generating revenue and protecting the environment. Further analysis is necessary to determine the
appropriate microalgae organisms to include in the biofuel production process if the commercially
viable system is to be implemented in the future.

6. Conclusion
Much focus has been placed on producing biofuel from microalgae over the last decade, thus limiting
the discovery of microalgae applications in other areas like cosmetic and pharmaceutical industries
and agriculture. Utilising effluents from biogas processing units during cultivation results in the
optimization of bioprocesses and the production of a variety of bioproducts such as proteins, biofuels,
biofertilizers, and other useful chemicals. Choosing the appropriate microalgae species for the
production of high-quality biofuel and value-added products depends on their carbohydrate, lipid,
and protein composition. The microalgae species comprise 8–69.7% carbohydrates, 5–74% proteins,
and 7–65% lipids, proving their capacity to be utilized as a source of value-added commodities in
several industries such as agriculture, animal husbandry, medical, gastronomic, and cosmetics. The
algal consortium may be used to treat wastewater effectively and quickly. Another way to develop
this technique is to stimulate specific genes of attention in algae. Specific approaches should be based
on appropriate genome sequencing and editing techniques which will aid in interpreting gene
networks and address the limited understanding related to the culture and growth of microalgae.
Therefore, the combination of algal genetic modification and strain selection is crucial to the
development of new technology keeping in mind several factors, including biomass productivity and
efficiency, downstream technologies, and cost-effectiveness

Funding: The authors extend their appreciation to the Deanship of Scientific Research at King Khalid
University, Saudi Arabia for funding this work through a research groups program under grant
number RGP.1/101/42 and Strategic Research Fund at the University of Technology Sydney,
Australia.

60
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