Journal of Biotechnology 345 (2022) 1–16

Contents lists available at ScienceDirect

Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Algae biorefinery: A promising approach to promote microalgae industry

and waste utilization
K. Chandrasekhar a, Tirath Raj a, S.V. Ramanaiah b, Gopalakrishnan Kumar c, J. Rajesh Banu d,
Sunita Varjani e, Pooja Sharma f, Ashok Pandey g, Sunil Kumar f, Sang-Hyoun Kim a, *
a
School of Civil and Environmental Engineering, Yonsei University, Seoul 03722, Republic of Korea
b
Food and Biotechnology Research Lab, South Ural State University (National Research University), Chelyabinsk 454080, Russian Federation
c
Institute of Chemistry, Bioscience and Environmental Engineering, Faculty of Science and Technology, University of Stavanger, Box 8600 Forus, 4036 Stavanger,
Norway
d
Department of Life Sciences, Central University of Tamil Nadu, Thiruvarur, 610005
e
Gujarat Pollution Control Board, Gandhinagar, Gujarat 382 010, India
f
CSIR-National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440020, India
g
Centre for Innovation and Translational Research, CSIR-Indian Institute of Toxicology Research, Lucknow 226 001, India

A R T I C L E I N F O A B S T R A C T

Keywords: Microalgae have a number of intriguing characteristics that make them a viable raw material aimed at usage in a
Biorefinery variety of applications when refined using a bio-refining process. They offer unique capabilities that allow them
High-value products to be used in biotechnology-related applications. As a result, this review explores how to increase the extent to
Bioprocessing
which microalgae may be integrated with various additional biorefinery uses in order to improve their main­
Techno-economic analysis
Biocomposites
tainability. In this study, the use of microalgae as potential animal feed, manure, medicinal, cosmeceutical,
Bioplastic ecological, and other biotechnological uses is examined in its entirety. It also includes information on the
boundaries, openings, and improvements of microalgae and the possibilities of increasing the range of micro­
algae through techno-economic analysis. According to the findings of this review, financing supported research
and shifting the focus of microalgal investigations from biofuels production to biorefinery co-products can help
guarantee that they remain a viable resource. Furthermore, innovation collaboration is unavoidable if one wishes
to avoid the high cost of microalgae biomass handling. This review is expected to be useful in identifying the
possible role of microalgae in biorefinery applications in the future.

1. Introduction danger to the environment. Additionally, because effluent handling is an

Increased population demands food and energy, while depleted re­
sources have a detrimental effect on productivity and supply chains,
identified as the key global issues to address in the twenty-first century
(Abomohra et al., 2016; Mehariya et al., 2021). By 2030, the world’s
population will reach 9 billion, with 13% of the population experiencing
food shortages (de Souza et al., 2019). At the moment, food crops cannot
meet the population’s nutritional and functional dietary demands. As a
result, sustainable alternative food sources are necessary to avoid food
scarcity. Aside from these issues, the world faces another critical prob­
lem in the form of wastewater production and carbon dioxide (CO2)
emissions (Siddiki et al., 2022; Talebi et al., 2013). Globally, over 380
trillion liters of wastewater are produced each year, posing a significant
expensive and energy-intensive procedure, approximately 80% of waste
is released short of appropriate treatment (Mehariya et al., 2021). As a
result, it is critical to develop affordable and energy-effective methods
for waste remediation (Chandrasekhar et al., 2021). As a consequence, a
microalgae-based technology is developed, which is considered as a
green solution that can be readily incorporated into wastewater treat­
ment operations to remove pollutants, recover nutrients, and generate
high-value commodities (Hemalatha et al., 2019; Wuang et al., 2016).
Microalgae are unicellular eukaryotic phototrophs/heterotrophs that
may thrive in various climatical and marine environments (Chaturvedi
et al., 2020). In the presence of light energy (sunshine), it fixes atmo­
spheric CO2 and transforms it to biomass via photosynthesis. It stores
several valuable components such as lipids, carbohydrates, carotenoids,

* Corresponding author.
E-mail address: sanghkim@yonsei.ac.kr (S.-H. Kim).

https://doi.org/10.1016/j.jbiotec.2021.12.008
Received 1 July 2021; Received in revised form 9 December 2021; Accepted 14 December 2021
Available online 24 December 2021
0168-1656/© 2021 Elsevier B.V. All rights reserved.
K. Chandrasekhar et al.
and a few other naturally occurring pigments to be used in nutrition,
animal feedstuff, nutrient supplements, and also in the manufacture of
health products (Yadavalli et al., 2021, 2020). Even though it is believed
that around 0.2–0.8 million species of microalgae live in nature, only a
minimal number of species are explored for commercial purposes and
scientific studies (Barati et al., 2021; Wobbe and Remacle, 2015). The
pace of biomass synthesis and the capacity to accumulate biomolecules
enables microalgae to completely replace petroleum-based energy re­
sources or goods and build a sustainable circular bioeconomy (Goswami
et al., 2021; Oey et al., 2016). Recently, the microalgal biotechnology
industry has attracted considerable attention due to the development
and application of biorefinery, which allows for the production of a wide
range of products from the same harvested microalgal biomass in as
little as a few days, including chlorophylls, carotenoids, carbohydrates,
lipids, proteins, nucleic acids, and nutraceuticals, among other things
(Mutanda et al., 2020). In addition, due to the nutraceutical and cos­
meceutical uses, microalgae-based carotenoids and other natural pig­
ments become more critical (Wang et al., 2015a,2015b). A wide variety
of microalgae produce these high-value chemicals (such as β-carotene,
astaxanthin, phycocyanine, and polyunsaturated fatty acids (PUFA)),
which may be used as storage components or as functional compounds
in the bioenergy and pharmaceutical sectors, among others (Mehariya
et al., 2021; Yadavalli et al., 2021, 2020). Due to its medicinal benefits,
such as possible cancer therapy agents, the synthesis of omega-3 fatty
acids, carotenoids, and phycobiliproteins (PBPs) from microalgal
biomass is gaining a great deal of interest in recent years. Because of the
high costs and low biomass productivity associated with microalgal
bioprocessing, the economic competitiveness of the industry and the
industrialization of microalgal biotechnology are severely hampered. As
a result, high-value products for microalgal biorefinery are being sug­
gested (Mehariya et al., 2021; Wobbe and Remacle, 2015). The cost of
capital of the microalgal process can be lowered by the integration of
multiple methods and fractionation, separating distinct high-value
products (Singh et al., 2022). While a specific molecule (e.g., flava­
noids or astaxanthin) is being isolated at this time, other potentially
significant substances (e.g., proteins and carbs) are being wasted or
otherwise underrated.
It has been stated by Espinoza Pérez et al. (2017) that the concept of
a biorefinery is comparable to that of an oil refinery, with the end aim
being the conversion of biomass into lucrative fuels, chemicals, or
compounds. Oil refinery and biorefinery basically differ in the crude
feedstock, which can be either unprocessed petroleum or biomass and
the technology used (Chen et al., 2021; Espinoza Pérez et al., 2017).
Achieving the greatest possible overall monetary gain out of algae re­
quires producing large quantities of bulk chemicals and high-value
chemicals out of the algae produced. It is proposed that microalgal
biomass be treated similarly to that of an oil/petrochemical refinery to
accomplish a full fractional process and valorization, with the notion of
employing a microalgae-related multi-product biorefinery being used as
an example (Moncada et al., 2015). The ability of this type typically
consists of multiple phases, including cell culture, collecting and inter­
ruption of algae cells, and desired product separation (Bhattacharya and
Goswami, 2020).
The present review article aims to provide an overview of the most
recent information on standards and learning in the field of algae bio­
refinery in the direction of the formation of high-value products.
Microalgae biorefinery’s fundamental criteria and essentials are exam­
ined, and both the benefits and drawbacks of these methods are
considered and weighed against one another. Further, microalgae pro­
ducing a variety of value-added compounds such as carbohydrates,
lipids, pigments, amino acids, antioxidants, PUFA, vitamins, etc., are all
included in the product profile studied in this study (Lum et al., 2013;
Siddiki et al., 2022). This review article also discussed the likelihood of
establishing a microalgae biorefinery regarding economic valuation and
maintainability, along with possibilities and restrictions.
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Journal of Biotechnology 345 (2022) 1–16
2. Microalgae as a biorefinery capable of producing a variety of
products
There is no singular, agreed-upon definition for the biorefinery idea.
The biorefinery facility does for renewable resources what the typical oil
refinery does for fossil fuels (Alishah Aratboni et al., 2019; Nagappan
and Kumar, 2021). Nevertheless, a biorefinery may utilize raw materials
from renewable sources for industrial applications (Andersen et al.,
2018). As a result, there are many methods for implementing it,
increasing the number of ways it may be used. The primary raw material
for biorefineries is biomass, organic material derived from living or
recently living organisms that may be utilized for industrial purposes
(Meixner et al., 2018). Several conversion processes such as physical,
chemical, and biological methods are required to change the physical
and chemical characteristics of biomass resources into a diverse variety
of feedstock materials, owing to the diversity of biomass resources
(Damtie et al., 2021; Raj et al., 2021; Siddiki et al., 2022). Biorefineries
may produce a variety of products, including energy, fuels, chemicals, or
materials (Espinoza Pérez et al., 2017; Meixner et al., 2018).
The possible candidates for biorefineries include microalgae, which
may produce several valuable products. As a result of its capacity to
grow more quickly and provide a far lower risk to the food supply chain
than other resources, biomass has long been regarded a sustainable
resource. (Catone et al., 2021). In the process of making value-added
goods such as chemical components, fuels, feed, biogas, and other sig­
nificant algae-derived components, mineral, oil, carbohydrates, and
protein may be employed (Singh et al., 2022). Similarly, as an example,
glycerine may be converted into value-added goods during the
manufacturing process. Low-value components of proteins and carbo­
hydrates may be able to be converted into energy in the biorefinery
through consolidated heat and power generation (Assacute et al., 2018;
Sirajunnisa et al., 2021). As illustrated in Fig. 1, the whole biorefinery
process may be described as follows.
The four categories of microalgae biomass transformation include
thermochemical reactions, biochemical processes, transesterification
process, and light energy mediated photosynthesis process (by har­
nessing solar energy, which initiates a series of reactions to generate
protons, electrons, and oxygen (O2) through the splitting of water) in
algal fuel cells/photosynthetic microbial fuel cells (Fig. 1). Several
research reports state that the thermochemical conversion process uses
specific heat energy to transform algae biomasses into biofuel. These
instances employ gasification (syngas production), thermal liquefaction
(bio-oil production), pyrolysis (bio-oil production, syngas formation,
charcoal production), and burning for the production of energy in the
form of heat and electricity (Alishah Aratboni et al., 2019). Under
suitable microenvironment conditions, the biochemical process, which
involves biomass conversion via microorganisms or biocatalysts to
manufacture biofuels, may be successful (Sirohi et al., 2021; Trivedi
et al., 2015). Examples of biochemical transformation include alkaline
hydrolysis, alcoholic fermentation, and photobiological hydrogen gen­
eration (biohydrogen production). Algal triglycerides will be trans­
esterified with alcohol in the presence of powerful catalysts, resulting in
the production of biodiesel. In this approach, fatty acid methyl esters
(FAME) and glycerol may be provided by the use of methanol and
ethanol, as well as through an acid or base-catalyzed procedure (Sharma
et al., 2018). Usually, a similar or varied synergist method, or even
super-critical CO2 extraction, is employed to obtain biodiesel. Microbial
fuel cells (MFCs), which generate electricity by the biological oxidation
of carbon-rich organic matter in anaerobic environments, are one of the
most important components of microalgae biomass transformation.
Microalgae inclusion in MFC has an enormous possibility in O2 gener­
ation since photosynthesis expels O2 through green algal development
(Enamala et al., 2020; Kannan and Donnellan, 2021). A few things have
an impact on the process of transformation: raw feedstock biomass,
monetary considerations, kind of activities, and intended product
prototype.
K. Chandrasekhar et al.
Fig. 1. A simplified flow diagram of a microalgae biorefinery.
Biorefineries based on microalgae have made significant strides in
the conversion of algal biomass into biofuels, cosmetics, biochemicals,
food, and feed, as well as high-value end goods such as pharmaceuticals
(Wang et al., 2015a,2015b; Xu et al., 2021). The carbohydrates derived
from microalgae are mainly composed of cellulose and starch, with very
little or no lignin present. These carbohydrates serve as a readily
available carbon source for the brewing sector and the production of
biobutanol and bioethanol preparations (Chew et al., 2017). Several
types of microalgae, such as Nannochloropsis sp, as well as Chaetoceros sp,
may also supply long-chain unsaturated fatty acids, which are valuable
nutritional supplements, for instance, docosahexaenoic acid (DHA) and
eicosapentaenoic acid (EPA) (Sirohi et al., 2021). Furthermore, proteins
and pigments produced from microalgae have tremendous future po­
tential for use in various medical applications (Sathasivam et al., 2019).
Despite the considerable potential of microalgae for energy and chem­
ical synthesis from renewable resources in biochemical processes, there
is no threat to food security or agricultural issues due to this potential
(Sharma et al., 2018).
In comparison, the desire for revenue generation in a given amount
of land is compared to diverse harvests, decreased water consumption,
extreme oil content, and the capacity to grow in dry environments,
among other factors. Microalgae-based biofuel generation has sparked
significant interest, much like the synthetic chemicals, food, and phar­
maceutical sectors have done in the past (Bjornsson et al., 2012; Xu
et al., 2021). On top of that, continuous research has revealed that the
current advances in economic conditions and advancements in
biochemical expertise have made the production of algae-derived bio­
fuels and biochemicals economically feasible.
3. Microalgae-derived high-value products and their industrial
uses
The microalgae are unicellular autotrophic organisms, and it is
common to find microalgae in both fresh water and marine water. The
production of composite molecules (such as lipids, carbohydrates, and
proteins) is made possible by the use of CO2 or other carbon-rich organic
materials found in the environment (Sirajunnisa et al., 2021). They
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Journal of Biotechnology 345 (2022) 1–16
typically make use of CO2 from the atmosphere to fuel their growth and
energy generation; on the other hand, certain microalgae utilize
carbon-rich organic matter to generate biomass as well as other
algae-derived biofuels and biochemicals (Ren et al., 2021). Conferring to
taxonomy information, above 3,00,000 species of microalgae have been
identified so far. However, only around 30,000 species of microalgae are
recorded in the scientific literature, which is concerned to live in normal
environments that are complicated, and they can adapt fast to extreme
circumstances, for example, changes in salinity, temperature, nutrition,
light strength, and so on (Low et al., 2014). As a result, they may
generate an incredible variety of interesting secondary metabolites, such
as bioactive compounds with complex conformations and biological
effects that are not often observed in other species (Singh et al., 2022).
Japan began exploiting the microalgae species Chlorella for commercial
uses for the first time in the 1960 s, more than five decades after the
country’s first commercial development of microalgae was initiated. The
worldwide market for a limited volume of algal growth is estimated to
be worth around 6.5 billion US dollars, with approximately 2.5 billion
US dollars created by the nutrition and food segment, 1.5 billion US
dollars generated by the production of docosahexaenoic acid, and 700
million US dollars generated from aquaculture sector, according to the
estimates. The annual production of microalgae is around 7.5 million
metric tons (Sathasivam et al., 2019). Spirulina is the most well-known
microalgae, and it is also the most often used. Chlorella vulgaris is the
second most widely used microalgae, followed by Dunaliella salina and
Haematococcus pluvialis, the third and fourth most commonly used
microalgae (Pagels et al., 2021; Ren et al., 2021; Reshma et al., 2021).
3.1. Algae derived lipids and their applications
While growing under optimal growth circumstances, microalgae
accumulate a variety of lipids, which can account for up to 3–20% of
their total dry biomass. During the photosynthesis process tri­
acylglycerol (TAGs) are accumulated in the microalgal cells. Production
of these lipids is believed to provide energy storage for microalgae cells
(Fig. 2) (Alishah Aratboni et al., 2019). As a result of their energy-storing
abilities, TAGs are essential for photosynthesis in microalgae cells,
K. Chandrasekhar et al.
Fig. 2. Simplified photosynthesis process and the three main possible biochemical pathways for nonpolar lipids like triacylglycerols formation: in the chloroplasts, in
the ER, or in the cytosol.
where they convert sunlight into molecules that are helpful to the cell to
generate essential energy that can be used for other metabolic processes
(Alishah Aratboni et al., 2019). By considering the length of the carbon
chain, algae-derived lipids can be divided into two different categories:
the first type is fatty acids with the total number of carbon atoms in the
chain lengths ranging from 14 to 19, which can be converted into bio­
diesel; and the second type is polyunsaturated fatty acids (PUFAs) with
the total number of carbon atoms in the chain lengths greater than 19,
such as EPA and DHA (Lai, 2015). Because they are considered to be
essential in human nutrition, these omega-3 lipids are of specific
attention for use as health sustenance complements. Generally, these
omega-3 fatty acid capsules derived from fish oil are very expensive and
are available in drugstores for the public (Kim et al., 2021; Kraic et al.,
2018). Fish oil prices are increasing day to day, and the Organization for
Economic Cooperation and Development predicts that they will
continue to rise, but there is a growing international interest in the
product. This way, algae-derived PUFAs are considered a viable and
vegetarian alternative to fish oil while also potentially filling a market
demand in the medium-cost product categories (Nagappan et al., 2021;
Sathasivam et al., 2019).
Furthermore, it cannot be denied that bio-oils derived from algae, on
the whole, have the potential to significantly increase the vegetable oil
industry (Lai, 2015). Even yet, these chemicals have a lot of commercial
value. The TAGs may be readily transformed into fatty acid methyl es­
ters, which are an essential and flexible type of biodiesel and the
cornerstone for its manufacturing (Alishah Aratboni et al., 2019).
However, in addition to PUFAs, additional microalgae-derived lipids can
be used for commercial purposes in the form of chemical compounds
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Journal of Biotechnology 345 (2022) 1–16
such as waxes used in the cosmetics sector, ointments, or even lubri­
cating components for polymers (Michalak et al., 2015).
3.2. Algae derived proteins and their applications
The growth of the world’s population, along with a rise in consumer
interest in poultry and dairy products, will raise the need for animal
feed, which will put further strain on the supply of protein in the food
system (Sathasivam et al., 2019). Worldwide meat consumption is
anticipated to upsurge from 233 million tons in the year
2000–300 million tons by the year 2020, as per the Food and Agriculture
Organization of the United Nations. As a result, to meet the forthcoming
demands, researchers are taking on the task of identifying alternative
protein sources to those now accessible (Caporgno and Mathys, 2018).
As a possible renewable protein alternative, microalgae have arisen. As a
result of their ability to grow without arable land, microalgae are
regarded as a potential source of protein. For human consumption, the
amino acid profile of microalgae ranges from 20% to 70% according to
species, growing conditions, and other factors (Bertsch et al., 2021). By
the middle of the century, 18% of protein sources may be provided by
algae. However, several elements of algal food safety, such as the
presence of pollutants, allergies, or dangerous chemicals, aren’t well
recognized. Microalgae and other protein sources have differing
time-to-market estimates (Caporgno and Mathys, 2018).
Consequently, microalgal proteins give excellent nourishing value by
transporting each single amino acid, which includes all of the basic
amino acids, to the human body (Bertsch et al., 2021; Lum et al., 2013).
Commercialization of microalgae-based products does, however, bring
K. Chandrasekhar et al.
certain difficulties. Large-scale operations allocate their bulk products to
aquaculture or create high-value chemicals. Other hurdles include
gaining regulatory approval for new goods (van der Spiegel et al., 2013).
In the future, algae might be used for humanoid nourishment in the
same way that animal feed is now being used.
3.3. Algae derived carbohydrates and their applications
Carbohydrates are the principal component of biomass that may
account for around half of the entire dry weight of the biomass and serve
primarily as storage and structural molecules for the majority of the
biomass (Arad and Levy-Ontman, 2013; Xu et al., 2021). These carbo­
hydrates are stored as reserve substances (e.g., starch) or are mostly part
of the cell wall (e.g., cellulose, pectin, and sulfated polysaccharides).
Nevertheless, microalgae’s carbohydrate metabolism (mostly starch and
cellulose) may vary substantially between species. Microalgae with high
carbohydrate production as well as an appropriate sugar composition
should be chosen for use in biofuels or chemical synthesis. Algae that
contain glucose-based carbohydrates are a good candidate for bio­
ethanol synthesis (Chen et al., 2013; Magalhães et al., 2021). For the
most part, algal-derived carbohydrates consist of starch and cellulose,
which may be utilized as a source of potential feedstock for the pro­
duction of bioethanol. Moreover, starch generated from microalgae may
be utilized as a renewable raw substrate for the fermentation process as
an alternative to sugarcane molasses, therefore contributing to the
conservation of farming land (Maia et al., 2020).
Fig. 3. Schematic representation of carbohydrate metabolism in algae.
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Journal of Biotechnology 345 (2022) 1–16
Microalgal extracellular coatings (e.g., cell walls) are another
carbohydrate-rich component, which may be converted into biofuel.
Algal extracellular coatings are unique to each species, although, in
green algae, cellulose is one of the primary fermentable carbohydrates.
Cellulose production requires a complex series of enzymatic processes.
UDP-glucose (Uridine diphosphate glucose) is the beginning substrate
for cellulose synthesis. Sucrose synthase is required for the process
(Fig. 3) (Chen et al., 2013). Although algae lack complex structures
compared to plants, such as hemicellulose or cellulose bound with
lignin, they do contain a diverse and progressively composite intracel­
lular assembly and intriguing extracellular sulfated polysaccharides,
which are responsible for a number of biological processes. Sulfated
polysaccharides have high bioactivity, allowing them to produce
bioactive substances, cosmetics, nutritional and medicinal formulations
(Arad and Levy-Ontman, 2013). Microalgal polysaccharides may also be
used to make commercially valuable components such as hydrophilic
greases and thickening agents, such as agar, which can be used in
various applications (Sathasivam et al., 2019).
3.4. Algae derived pigments and their applications
Carotenoids, chlorophylls, and phycobiliproteins are three essential
kinds of standard microalgal pigments that should be highlighted in this
context. Despite the fact that the majority of these pigments are known
as precursor molecules for vitamins in the diet, these pigments are also
used in the manufacturing of food additives as well as make-ups,
K. Chandrasekhar et al.
medicines, edible coloring compounds, and biochemicals (Lum et al.,
2013; Nagappan et al., 2021; Sharma and Sharma, 2017). Carotenoids
are microalgae pigments, which are highly soluble in lipids and provide
coloration to various plant parts. These carotenoids are also identified as
decoration pigments. Generally, Soxhlet extractions with organic sol­
vents are used in microalgae-derived carotenoid extraction strategies
(Chen et al., 2016). In recent years, however, super critical fluid
extraction (SCFE) has been the most preferred method because it pro­
vides more control over the solvent, allowing greater control over the
amount of affinity and faster carotenoid extraction. Astaxanthins are
pigments that can be employed in the food and nutraceuticals sectors
with possible antioxidant activity, such as anti-aging, anti-in­
flammatory, and immunological enhancements (Hu et al., 2018; Yada­
valli et al., 2021). Astaxanthin is one of those pigments that may be
employed in the food and nutraceutical industry and have potential
antioxidants such as anti-aging and immune-increasing properties
(Yadavalli et al., 2021, 2020).
It should be noted that chlorophylls are similarly lipid-soluble with
low extremity; these are necessary pigments of photosynthesis, which
are more often found in plants to harvest solar energy (Sirajunnisa et al.,
2021). The typical approach for extracting these photosynthesis pig­
ments needs a few critical stages using appropriate solvents, similar to
the procedure for removing carotenoids. Multiple factors can impact the
pigment yield during solvent extraction (Assemany et al., 2015). These
factors include the kind of solvent used, the technique for cell wall
rupture, the extraction duration, as well as the use of numerous
empirical correlations to determine the pigment yield. Phycobilipro­
teins, which are prevalent in microalgae and aid in photosynthesis, are
fitment pigments that aid in photosynthesis (Ahmed et al., 2021). The
most critical applications of phycobiliproteins are incorporating
distinctive hues into food products and medicinal products. The phy­
cobiliproteins possess anti-cancer, anti-viral, antioxidant,
anti-inflammatory, and neuro protective characteristics, indicating its
possible use in health industry-associated products (Chang et al., 2015;
Sathasivam et al., 2019). Centrifugation, drying, homogenization, and a
recurring freeze-thaw technique are used in a continuous cycle to extract
phycobiliproteins from microalgae.
3.5. Algae derived vitamins and their applications
The existence of high concentrations of essential trace metals and
vitamins in algae, in comparison to commercial foods, is supported by
algae’s high bioavailability. Biological availability experiments on vi­
tamins and trace metals are often necessary in combination with feed
quality studies in order to establish the effect of these components after
they have been ingested (Lum et al., 2013). When the cyanobacteria
were exposed to low quantities of nitrogen (N), they generated less
vitamin B12 per cell than when exposed to high N levels. Moreover, both
the amount of N supplied and the amount of N existing in the culture
medium had an influence on the total quantity and process of vitamin
formation (Sathasivam et al., 2019). Other vitamins such as riboflavin,
which may be produced from microalgae, are essential for the survival of
marine-cultured organisms since it is a nutrient that they lack.
3.6. Algae derived polyunsaturated fatty acids and their applications
In general, PUFA is regarded in the algae and is considered an
essential health complement. This PUFA serves to prevent the effects of a
variety of heart-related conditions. Previously, sea fish has been
considered as a major source for the recovery of PUFA, but this practice
has been discouraged in recent years due to the rapid depletion of ma­
rine resources, where raising demand for PUFAs has prompted the
finding of alternate sources of EPA and DHA for human use (J. J. Wang
et al., 2015; H.-M.D. Wang et al., 2015). This is because microalgae are
genetically programmed to produce PUFAs (Sathasivam et al., 2019).
Developments in algae cultivation and bioprocessing can be put to use
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Journal of Biotechnology 345 (2022) 1–16
for the production of PUFAs because algae is a maintainable source that
can be cultivated all over the year and produces a high amount of oil as
well as a large amount of biomass. For the qualitative analysis of fatty
acids in marine microalgae, a few methods were used (including Bligh
and Dryer extraction process, solvent extraction method), all of which
produced comprehensive findings (Wang et al., 2013a,2013b).
4. Microalgae-derived biofuels and biochemicals
Climate change due to the widespread usage of fossil fuels and their
derivatives have prompted consumers to turn to algae as an environ­
mentally friendly renewable energy source in the contemporary world.
The work’s overall goal is to boost energy output while increasing the
availability of energy resources to all of humanity (Sharma and Sharma,
2017). In energy production, the phrase "biofuel" refers to any solid,
liquid, or gaseous substance that may be converted into energy produced
from live or dead biomass (Jagadevan et al., 2018; Li et al., 2018). On
the other hand, an overall produced energy depends on the kind of
biomass, the quantity of biomass utilized as a substrate, and the tech­
nique employed for the energy production (Norsker et al., 2011). Sugar,
starch, and vegetable oil are used to produce first-generation biofuels.
The fermentation of sugar, starch, or cellulose by various microorgan­
isms produces ethanol, propanol, and butanol as biofuel. Although
first-generation biofuel methods are beneficial, there is a limit beyond
which they cannot generate enough biofuel without jeopardizing food
supply and biodiversity (Vigani et al., 2015). In contrast to biofuels
generated from residual agricultural feedstock, algal biomass produc­
tion does not require agricultural land use. Photosynthesis is the primary
engine behind all biofuel synthetic processes, turning solar energy into
biomass, carbon storage products (e.g., carbohydrates and lipids),
and/or biohydrogen (Fig. 4 & 5) (Beer et al., 2009; Gurav et al., 2021).
The efficiency of algal biomass production may be increased by the se­
lection of less expensive substrates such as carbon-rich waste­
water/industrial effluents (Bhatia et al., 2021; Prudkin-Silva et al.,
2021; Vigani et al., 2015).
4.1. Algae derived bio-oils and their uses
However, even though the use of bio-oils has environmental ad­
vantages by lowering CO2 emissions, the poor quality of bio-oil, such as
thermal instability, high viscosity and acidity, and low heating value,
makes it impossible for directed applications such as drop-in fuels to be
successful (Zhang et al., 2021). Examples include pyrolysis bio-oil,
which has a water content of 15–30% by weight, which is much
higher than the water content in petroleum crude oil (which is less than
a percent by weight). The presence of high water content in the oil may
create issues with the engine’s ignition, not to mention the fact that the
oil has a substantially reduced energy content (Zhang et al., 2021).
Bio-oils can be generated by heating dried biomass thermally in
oxygen-restricted conditions and then submitting it to a cooling process,
also known as bio-crude oil or pyrolyze oil (Zhang et al., 2015). They are
equal to petroleum-derived oils regarding energy production and may
be used as alternates (Sathasivam et al., 2019). Thermal-chemical
liquefaction and pyrolysis are the two phases of bio-oil production
that may be divided into two categories. Pyrolysis is a thermochemical
conversion process that converts dry biomass into valuable liquid
(bio-oils), solid (charcoal), and gas. It is typically conducted at
350–530 ℃. By using the pyrolysis or thermal liquefaction methods,
several microalgae have been explored for their potential to produce
bio-oil for human use (Pan et al., 2018). Spirulina (up to 41%), Scene­
desmus (24–45%), Dunaliella (around 37%), and Desmodesmus (up to
49%) are only a few examples.
4.2. Algae derived biodiesel and its uses
According to the existing literature, oil seed crops such as soybean,
K. Chandrasekhar et al.
Fig. 4. Metabolic pathways in green algae related to biofuel and biohydrogen production.
palm, and rapeseed can be utilized as a source for biodiesel
manufacturing in recent years, with palm oil being the most common
(Godwin et al., 2017). The kind of crude material used in the manu­
facture of biodiesel has a significant impact on the fuel cost, accounting
for 50–85% of the overall fuel cost. Nevertheless, for a profitable bio­
diesel production process, the use of feedstock is a critical component
that may be used to optimize production costs (Nagappan et al., 2021;
Selvaratnam et al., 2015). It is usually created by a transesterification
process, which includes the transformation of triacylglycerols into
non-toxic molecules that are composed of fatty acids and other com­
ponents. Refining algal oil is critical because it allows the transformation
of higher molecules into relatively very low molecular weight molecules
(for example, fatty acid alkyl esters), which are essential for human
health. Many researchers have noted the presence of significant oil
content in the cells of the two algae species, which have been identified
as Chlorella vulgaris and Chlorella protothecoides. The process factors that
impacted the yield of methyl ester were the proportions of methyl
alcohol and oil, the proportions of catalyst and oil, and the reaction time
(Godwin et al., 2017). According to the results of the studies, the optimal
conditions for biodiesel generation were a 9:1 methanol: oil molar
proportion, a 1:5 potassium hydroxide catalyst: oil proportion, and a
10 min reaction time. The biodiesel produced from microalgae, on the
other hand, is often derived from unsaturated fatty acids (Sharma and
7
Journal of Biotechnology 345 (2022) 1–16
Sharma, 2017). It is possible to get distinct fatty acid profiles from algal
biomass generated from wastewater because the biomass comprises a
combination of different alga species.
4.3. Biohydrogen production from algae and their uses
Increasing interest in biohydrogen generation has been sparked by
the demand for alternative energy sources (Jafari and Zilouei, 2016). To
be successful, producing hydrogen from microalgae must be both highly
efficient and economically feasible. As a result, bioreactors are in charge
of regulating large-scale biohydrogen production. The design of the
bioreactor, as well as the manner of operation, have a major impact on
the yield of biohydrogen produced (Ahmed et al., 2021). Despite several
efforts, the generation of biohydrogen on a large scale has not been
successful because of the high cost of production (Kadier et al., 2021),
the requirement of specialized machinery, and the low amount of
biomass fixation (Ahmed et al., 2021). According to several studies,
biodiesel synthesis can be achieved by subjecting specific green algae
species to various stress conditions, such as lack of light, which can
result in an apparent output of hydrogen gas. Many papers were
reviewed to know better the process and factors intricate in the gener­
ation of hydrogen gas from algal biomass (Bhatia et al., 2021). It is
possible to produce hydrogen via three different paths. The first is direct
K. Chandrasekhar et al.
photolysis, the second is indirect photolysis, and the third is an
ATP-driven process (Fig. 5). In order to turn water into O2 and hydrogen,
algae must either exploit their photosynthetic capabilities or engage in
direct photolysis. The simultaneous synthesis of hydrogen and O2, on the
other hand, may result in increased security risks, as well as an increase
in the total cost of the manufacturing process (Milledge and Heaven,
2017). Hydrogenase enzymes are also involved in the process, however
owing to their O2 sensitivity, they are vulnerable to damage when
exposed to O2 (Jones and Mayfield, 2012). Because of these drawbacks,
indirect photolysis is preferred for hydrogen generation over direct
photolysis. Several studies describe the production of biohydrogen
under strict oxygen-restricted (anaerobic) and sulfur-restricted condi­
tions, in which the algae cell wall containing carbohydrates is converted
into biohydrogen (Singh et al., 2020). Most investigations have found
that cyanobacteria are the primary producers of hydrogen over the
ATP-driven route, with the enzymes hydrogenase and nitrogenase
serving as catalytic agents in the process.
4.4. Biomethane production from algae and their uses
The generation of methane from biomass is gaining in reputation
these days, both as a result of customer demand and increased public
awareness of the benefits of using environmentally friendly sources of
energy (Klassen et al., 2016). The generation of biogas may be accom­
plished by the use of an anaerobic digester that contains a synergistic
microbial community that converts algae biomass components (lipids,
proteins, and sugars) into methane and CO2 for use in a variety of ap­
plications (Klassen et al., 2015; Mussgnug et al., 2010). When this oc­
curs, methane may be used effectively as a fuel and as a chemical
feedstock (Chandrasekhar et al., 2020). According to the literature,
several researchers investigated the capabilities of several substrates for
biomethane production (Chandrasekhar et al., 2021), such as algae,
forest wood, and leaf biomass, agriculture waste biomass, and industrial
Fig. 5. Photosynthetic and glycolytic pathways in green algae related to biofuel and biohydrogen production.
8
Journal of Biotechnology 345 (2022) 1–16
effluents/wastewater, among other things (Andersen et al., 2018; Kruse,
2015; Ma et al., 2021; Uggetti et al., 2016). According to their findings,
algal biomass might serve as a viable candidate for the production of
biomethane. Even though algae have a greater biomass output than
terrestrial plants on average, the growth of algae is hindered by a variety
of reasons (Klassen et al., 2015). According to a recent study,
pre-treatment with the heat of microalgae (Chlorella sp.) can increase the
production of biomethane from them, therefore increasing their pro­
ductivity. When the temperature is raised from 70 ℃ to 90 ℃ for 0.5 h,
the biomethane production increases from 37% to 48% when compared
to the control algal biomass.
4.5. Bioethanol production from algae and their uses
Numerous factors, counting high efficacy, a greater diversity of
algae, the existence of diverse quantities and combination of starch and
carbohydrate, slim cell wall assemblies, and the photosynthesis rate,
have prompted increased interest in the use of algal biomass for the
biosynthesis of ethanol (Klassen et al., 2016; Tsarpali et al., 2021).
Ethanol production from algae is typically accomplished through two
approaches: the fermentation process and the gasification process
(Uggetti et al., 2016). In many nations, wide-scale fermentation of
agricultural products comprising starch is used to produce the
value-added product (Klassen et al., 2016). In most cases, instead of
using directly, the agricultural/forest biomass is exposed to various
pretreatment methods such as wrinkled/ground with the assistance of
various enzymes, yeast, acids, and alkali, wrinkled/ground to transform
the starch found in it into sugars. Sharma and Sharma (2017) investi­
gated the feedstock competence of microalgae biomass in ethanol pro­
duction and reassured its usage as a sustainable fuel to provide
consumers with a genuine alternative. Many different algae are utilized
for ethanol generation, including Chlorella, Chlamydomonas, Spirulina,
and Scenedesmus. The higher quantity of carbohydrates in the algal cell
K. Chandrasekhar et al.
and the effortlessness with which the algal cell can be cultured distin­
guish brown algae from other kinds of algae, for instance, green algae
and red algae. Further, brown algae are regarded to be a viable source to
produce bioethanol, among other forms of algae, including brown,
green, and red algae (Jung et al., 2013).
Microalgal biomass can be utilized as a supplement to augment main
feedstocks using current methods and infrastructure. A conceptual
procedure is shown in Fig. 6. Starch accumulation in algal cells can be
used to make ethanol via the dark fermentation process, even though the
ethanol production rate and yield are much lower, making this tactic
scientifically important in determining the basic metabolism of photo­
synthetic algal cells in the absence of a light source (Chen et al., 2013).
Further, reports are available regarding the usage of red algae for bio­
ethanol production by acid hydrolysis, although the yield was extremely
poor in this case (approximately 45%). Additionally, seaweeds have
been conveyed to generate ethanol around 0.08–0.12 Kg/Kg of dried
seaweed, based on the species of algae and the procedures used for
biomass pretreatment/hydrolysis (Shimakawa et al., 2016). Compared
to land-based harvests for biofuel production, another set of employees
generated a possible bioethanol output of around 90 L per tonne
biomass.
Nevertheless, the overall yield was very low regarding land-based
harvests for biofuel generation. Microfiltration is a less energy utiliz­
ing technology that has been developed. Biologically produced ethanol
is released into the market in Brazil as pure ethanol or as a mix of bio­
ethanol and oil, and it is found in around 86% of all marketed cars.
However, because of their little vapor pressure, less energy density, and
little fire retardancy, they have several drawbacks in the manufacturing
process as well (Puschner, 2018).
4.6. Biobutanol production from algae and their uses
Butanol, four-carbon alcohol, is made from ethanol’s same
Fig. 6. A conceptual process of ethanol production coupled with the cultivation of starch-based microalgae.
9
Journal of Biotechnology 345 (2022) 1–16
feedstocks, including maize and other biomass. The word biobutanol
refers to butanol produced by feedstocks of biomass. Biobutanol has the
advantage of having a larger energy content and lower Reid vapor
pressure compared to ethanol because it is immiscible in water. Several
countries in Asia, Europe, and South America grow algae culture on a
large scale to produce bioethanol and biogas, while in the United States,
they are being employed to manufacture biobutanol (Sharma and
Sharma, 2017). Because of its great energy density, butanol has been
used as a transportation fuel for the past 100 years and proposed biofuel
as a possible basis (Cho et al., 2017). Because the bacteria utilized for
butanol synthesis employ algal biomass as potential raw material, bio­
butanol production can be as financially lucrative as bioethanol pro­
duction. Several Clostridium sp. are proficient for the formation of
biobutanol and bioethanol by anaerobic fermentation process utilizing
the carbohydrates present in algae biomass, which is referred to as acetic
acid, butanol, and ethanol (ABE) fermentation in the scientific com­
munity (Orona-Navar et al., 2021; van der Wal et al., 2013).
On the other hand, Butanol hinders the fermentation process,
resulting in lower yields and production. It has been shown that Ulva
lactuca can be utilized by Clostridium sp. to produce butanol, which may
then be used as a substratum for the fermentation of biobutanol. The
substrate has been exposed to enzymatic hydrolysis and used to produce
ABE among other products. As a result of these findings, Ulva lactuca has
the potential to be used as a feedstock for the ABE fermentation process
(Cho et al., 2017). Nevertheless, throughout the course of the ABE
fermentation progression, propylene glycol was produced as a
by-product, which reduced the overall butanol production efficiency of
the process.
4.7. Algae derived bioplastics production and their uses
Algal biomass has the potential to be utilized in the manufacturing of
a variety of plastics, including mixed-plastics, bio-polyethylene,
K. Chandrasekhar et al.
polylactic acid, and cellulose plastics (Meixner et al., 2018). The mixed
plastics are created by combining algae biomass as a filler ingredient by
petroleum-based polymers to form a composite material. These mate­
rials offer biodegradability qualities, which helps to reduce the number
of petroleum derivatives used in the plastics manufacturing process even
more. The cellulose-based polymers are mostly produced from the cel­
lulose residues left over after extracting algal oil from algal biomass
residue (Meixner et al., 2018). The method is less expensive; addition­
ally, the algae biomass waste assists as an excellent raw material for the
manufacture of cellulose plastics (Haniewicz et al., 2018). An alternative
type of bioplastic is polylactic acid, which is synthesized by the poly­
merization of lactic acid produced during the fermentation of natural
biomass. Hence, the algal biomass can be a more efficient feedstock for
lactic acid generation by fermentation process (Rhim, 2013). The
polymerization of ethylene results in the formation of bio-polyethylene.
Because most ethylene synthesis is performed through ethyl alcohol
cracking and dehydration (Orona-Navar et al., 2021), algal biomass may
serve as a potential feedstock/carbon source for bioethanol vial
fermentation process, which could be used as an alternate feedstock for
bio-polyethylene production.
4.8. Algae derived nanocellulose production and its uses
If you compare algae to terrestrial plants, they are becoming
increasingly popular by way of a beneficial source aimed at nano­
cellulose (NC) synthesis in the near forthcoming. There are just a few
studies available on the effective taking out of NC fibers from algal
biomass that have been published so far (Liao et al., 2015). Before oil
extraction, the cellulose content existing in algae remnants could be
converted into NC, a biopolymer with unique properties such as
renewability, environmental friendliness, and mechanical properties.
NC is a high-value biopolymer with industrial significance with inimi­
table properties such as renewability, environmental friendliness, and
mechanical properties. Using algae for biopolymer production has
several advantages over using woody biomass, including a lower con­
centration of lignin in the cell wall, which requires less chemical usage
for pretreatment for delignification process to avoid the lignin hindrance
and less time for physical pretreatment events that result in a higher
gradation of crystallinity in the formed NC (Prakash Menon et al., 2017).
As a result of its wide variety of potential uses in different industrial
sectors such as food, pharmaceuticals, agricultural, water purification,
and others, the market demand for NC has risen dramatically in recent
years.
4.9. Algae derived biocomposites production and their uses
The manufacture of microbial biodiesel has garnered considerable
attention in recent years, and the industry is thriving due to the capacity
of certain microalgae to supply large volumes of lipid under carefully
regulated growth conditions (Chandra et al., 2019). Even though there is
limited research on the effective exploitation of microalgal residues,
which are left behind after lipid extraction yet contain large amounts of
proteins, carbohydrates, and cellulose, there is a growing body of
knowledge (Chandra et al., 2019). Because of its cheap and environ­
mentally benign nature may be utilized as a filler option in bioplastics
manufacturing. Researchers such as Toro et al. (2013) have recently
described the manufacture of biopolymers with an optimal configura­
tion of polybutylene succinate (PBS), and algae biomass remains
following oil extraction in the range of 20–30% by using a process
known as polymerization (Toro et al., 2013). Similar findings were made
by Torres et al. (2015), who employed poly(butylene
adipate-co-terephthalate) and algae biomass remains in a 4:1 ratio for
the production of biocomposites in a laboratory setting (Torres et al.,
2015).
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Journal of Biotechnology 345 (2022) 1–16
5. The bioprocessing of algae for successful biorefinery
A large number of research articles have demonstrated that micro­
algae may be utilized efficiently for bioproduct production. The primary
phase of a successful biorefinery includes both upstream processing (UP)
as well as downstream processing (DP), with the efficiency of UP being
determined by different factors such as the type of algae used, CO2
source, nutritive additions (e.g. sources of phosphorus (P) and N), and
light source (Parsons et al., 2020). The sources of supplements are
critical for the growth of microalgae culture development because they
provide the necessary conditions for cultivating microalgae (Van­
thoor-Koopmans et al., 2013). Furthermore, it was taken into consid­
eration that both light intensity as well as source were very relevant
since they had a substantial impact on the photosynthetic rate of algae
and, consequently, their growth rate (Aravantinou and Manariotis,
2016).
Microalgae extraction and filtering techniques are consistent DP unit
procedures that take place in an innermost portion of the bioreactor, and
they are used to obtain essential mixtures from the algae (Parsons et al.,
2020). Ordinary procedures, for example, homogenizers, bead pound­
ing, heating, and chemicals usage, can result in startling costs of pro­
duction, and the lack of a conservative approach has prompted the need
for a combination of many ways to achieve the desired results (Jaco­
b-Lopes et al., 2009; Orona-Navar et al., 2021; Vanthoor-Koopmans
et al., 2013). It has been found that the collection of biomass pursued
by means of biorefinery reactors has made a significant contribution to
the mixture of algae biomass-changing methods (Parsons et al., 2020).
Further, gentle separation methods, such as those that do not need sol­
vents or high pressure, are critical for producing perfect compounds that
do not cause damage to different sections of the body (Aravantinou and
Manariotis, 2016). Algae biorefineries may benefit from a variety of
technological developments, depending on the quantity of energy they
demand and the availability of new ideas (Parsons et al., 2020). Inte­
gration of upgrading skills into the DP is also expected to improve
cost-effectiveness and ease of use operations.
Nevertheless, to uphold a tactical distance from microbe contami­
nation or the close vicinity of contaminating impacts while fulfilling
administrative criteria, the development of micro-algae should be car­
ried out under fully standard circumstances for restorative biotech­
nology usage. Open development frameworks, e.g., open lake or
raceway lake, may not be suitable for algae production for restorative or
pharmaceutical use, even if they may be economically feasible for large-
scale growth. This would increase the feasibility of a closing cultural
framework (for example, photobioreactors), enabling comprehensive
development control situations even though the initial expenditures for
investments and running would be greater.
5.1. Cultivation of algae
When it comes to the development of renewable resource-based
enterprises, the large-scale culture of microalgae may make a signifi­
cant contribution to the production of cost-effective value goods in
addition to biomass generation. There are numerous species of micro­
algae that have the potential to be grown on a large scale, but there is a
lack of data from commercial trials of these organisms. Microalgal
biomass is necessary for bioethanol production in order to compete with
other feedstocks such as agricultural biomass. A large volume of
microalgal biomass is required in order to compete with agricultural
biomass (Lum et al., 2013). As a result, microalgae culturing technolo­
gies are forced to provide a high biomass yield, making the use of
agricultural/forest biomass for bioethanol synthesis a less attractive
option. In order for algae culture to be economically viable, the algae
biomass production rate must be more than 30 g/m2 per day (Bhatta­
charya and Goswami, 2020).
Various techniques for cultivating microalgae may be used in a va­
riety of environments and under a variety of environmental conditions.
K. Chandrasekhar et al.
They transform water and CO2 from the environment into biomass
through a biochemical process known as photosynthesis, with the
assistance of light as an associated energy source (Bhattacharya and
Goswami, 2020). Synthesized bioproducts (starch and lipids etc., in the
algal cells) are stored in various forms, such as cell components or store
food supplies, and account for anywhere from 20% to 50% of total
biomass in various forms. Algae prefer nitrogen and phosphorus as main
nutrients, and nitrogen and phosphorus together account for 10–20% of
alga biomass. Other requirements for the photosynthesis process and
growth of algae include macronutrients in the form of magnesium,
calcium, and potassium salts, micronutrients such as molybdenum,
manganese, boron, cobalt, iron, and zinc. The preference of various
species for different growth medium components may be determined by
looking at their DNA. Essential nutrients (N, P, K, etc.), as well as an
associated carbon source, temperature (20–30 ℃), pH (6− 8) and O2,
light supply (16 h of light/8 h of darkness), and CO2, are all required by
virtually all life (Nagao et al., 2019). Microalgae culturing technologies
may benefit from wastewater from industrial and agricultural sectors,
which may provide the specific nutrients required for their growth and
development (Bhatia et al., 2021).
Using different types of bioreactors for algal cultivation is another
important element to consider while growing algae. The design of bio­
reactors may be done by the species and in accordance with the type and
nature of the end product. On an enormous scale, microalgae are grown
in open ponds to provide food for livestock. Morales-Sánchez et al.
(2017) found that this method was particularly more suitable aimed at
the types of microalgae that grow in heterotrophic culture. Open culture
systems are generally inexpensive; however, they are susceptible to
contamination. As an alternative, either continuous or batch photo­
bioreactor amenities can be used. Wastewater treatment plants using
microalgae can be cultivated for industrial culture in order to achieve
the dual advantages of waste water remediation and biomass production
while also reducing costs. Apart from that, marine water is an appro­
priate medium for the industrial cultivation of algae, and the marine
cultivation system is conquering fame between scientists and capitalists
due to its numerous compensations, including natural mixing of the
culture by ocean waves, availability of organic nutrients, and so on. The
use of sea water instead of pure water for microalgae production has the
potential to lower the end product’s value. Marine water might be a
good option of media for algae culture since it can reduce production
media costs while improving product output, which could contain lipids
and other value-added compounds in the biomass. Sea water has the
ability to lower the cost of production medium while boosting product
yield.
5.2. Harvesting of algae
An open culture system is often linked with the energy-intensive and
expensive centrifugation process, which accounts for 20–25% of total
culture costs (Chandra et al., 2019). This is another major reason why
many diverse and affordable harvest technologies exist. Open pond
culture methods, such as open ponds, employ flocculation as a low-cost
harvesting approach that prior to harvesting, concentrates the culture
(Chandra et al., 2019). Flocculants such as inorganic flocculants,
cationic polymer flocculants, and nanoparticles have been utilized.
Microfiltration is the second most major harvesting technique, and
current research shows that it consumes about 1 kWh/m3, making it the
second most energy-efficient technology (Ruiz et al., 2016). Micro­
filtration is used to concentrate algal biomass in open culture systems by
Bilad et al. (2012). Pre-concentration is done by submerging membranes
with a lower energy input of 0.27–0.41 kWh/m3. Although the process
of pre-concentration is essential before algae produced in open ponds
may be harvested to lower costs, it is not required if microalgae have
been cultured in a targeted cell suspension system bioreactor, such as the
photobioreactors. At this size, centrifugation is the preferred technique
of harvesting microalgae due to it eliminating the requirement for a
11
Journal of Biotechnology 345 (2022) 1–16
pre-concentration phase and being expected to contribute for less than
5–7% of overall production expenses (Ruiz et al., 2016). Thus, collecting
microalgae from closed systems is more preferred than over-harvesting
algae from open systems. Collecting microalgae from open culture sys­
tems may soon become a newly emerging market segment. This opinion
predicts that harvesting algal biomass from closed photobioreactors will
become more lucrative in the near future. Further study is required, as
current information on alternative harvesting techniques is limited.
Biological variables specific to strain must be considered in order to
perform harvest methods consistently across different microalgal spe­
cies, regardless of the anticipated requirements (such as the kind of
photobioreactor, species of microalgae, etc.) (Bilad et al., 2012).
5.3. Microalgal cell disruption methods
Due to the fact that lipids are stored inside cells or attached to
intracellular membranes, cell disruption is a critical stage in the process
of lipid extraction from microalgae (Wang et al., 2021). Polysaccharides,
uronic acid, proteins, algaenan, glycolipids, and minerals are common
cell wall constituents in most organisms (Sandani et al., 2022). Because
the plasma membrane and membrane provide a hindrance to the release
of intra-cellular substances, microalgal cell rupture may be a need for
the whole biorefinery. Three techniques for microalgae cell disruption
are available, including biochemical, physical, and mechanical tech­
niques (Enamala et al., 2018). Cell disruption should be carried out in a
controlled environment rather than in a high-risk environment. In order
to maintain the original conformation of individual cell components, it
is necessary to maintain these circumstances (Günerken et al., 2015).
Because of their technological-functional characteristics, soluble pro­
teins, in particular, are sensitive to high temperatures and pH, and they
must maintain their integrity in order to maintain their high value
(Wang et al., 2021). Unlike chemical procedures, mechanical methods
do not require high temperatures or acidic conditions to operate; as a
result, they are an excellent choice for sensitive cell disruption
(Günerken et al., 2015). Recent research has revealed that native algal
proteins are released during the process of bead beating; nevertheless,
the energy consumption of these activities is substantial (Coons et al.,
2014). In accordance with certain published research, the systematic
improvement of operating conditions may result in a significant
decrease in the amount of energy used by mechanical interruption
operations.
5.4. Extraction of diverse value-added components from algae
Because the hydrophobic lipids and pigments are extracted using an
organic solvent, only the hydrophilic components are left as residual
solids. It is frequently used through an initial drying procedure to boost
the lipid extraction process (Sharma and Sharma, 2017). In order to
valorize all of the biomass fractions, it is important to discover alter­
natives to organic solvents because proteins have a propensity to
denature when extracted by polar or amphiphilic organic solvents.
Using a two-stage extraction process, where the removal of soluble
proteins happens first, followed by the extraction of lipids from the
biomass, is more effective (Desai et al., 2014). Newly, a novel approach
for the extraction of soluble proteins was developed, which makes use of
aqueous and biphasic systems to accomplish the extraction. Future
research should concentrate on improving the characterization of these
systems, which will allow for a more logical design of sensitive
high-value molecular extraction methods in the future (Desai et al.,
2014). Despite the fact that there are several compelling ways to extract
each fraction from microalgae biomass, this strategy of
single-component extraction may be required for a biorefinery that
produces many different products.
K. Chandrasekhar et al.
6. Product portfolio based on the present approach
Here several publications are describing current understandings of
microalgae-derived goods. Whole dry powder (extracted from algae) has
several health claims, such as improved immunity, reducing the risk of
cardiovascular disease, and cancer prevention. The use of algal biomass
by humans is still quite limited (Rodrigues et al., 2015). Commercially
available food items are the sole way to include microalgal biomass into
meals because most alga species will have a robust green hue, fishy
scent, and powdery consistency. Currently, most algae components that
are valuable for pigments and PUFAs are found in markets for nour­
ishment, therapeutic, and aqua-feed production (Wang et al., 2015a,
2015b). On the market, there are currently only two pigments, astax­
anthin, and β-carotene widely available. Carotenoids derived from the
plant are utilized in many ways, such as natural sources of pigmentation
for horticulture or food coloring (Yadavalli et al., 2021). Japan,
Thailand, and China are the primary consumers of pigments. Lipids may
be used in a variety of ways across many sectors (Trivedi et al., 2015).
The additional carbohydrates found in algae comprise just a small
quantity of compounds that are still being investigated. These poly­
saccharides are very beneficial since they may possess medical charac­
teristics (e.g., utilized in the manufacture of pharmaceuticals, NC, and
bioethanol), yet their elimination (through excretion) could impact
one’s overall health. To date, complete dried biomass is needed to make
powders, pills, and tablets (Chew et al., 2017). Mostly, only spray drying
processes are employed in the majority of algae processes. For pilot
production, bead edge or expeller pressing cell breakup was done
automatically. Carotenoids and lipids can be extracted using organic and
sub- or supercritical solvents. It is possible to obtain the best yield of
antioxidant recovery by using the supercritical CO2 gas extraction
technology, however it demands high pressures and energy (Yadavalli
et al., 2020). This approach seems to be the most cost-effective choice
for manufacturing nutraceutical product formulations. Outside of the
nutraceutical industry, there is a lack of quantifiable data.
7. Industries engaged in the development of algae-based
technologies
There are several investigators, investigating groups, institutions,
and corporations at work across the world to develop algal-based fuel
and additional high-value biochemicals in a more cost-effective and
environmentally friendly manner than is now available (Chandra et al.,
2019). The fact is that there are just a few firms at work on algal bio­
refineries that are engrained and have a significant position in the
worldwide market. Industries such as Algatechmologies, BioReal Inc.,
etc., to mention a few, are engaged in the production and marketing of
pigments, which are utilized as a biotherapeutic and are derived from
algal sources (Chandra et al., 2019). Mera Pharmaceuticals Inc., a ma­
rine biotechnology business, has also successfully produced astaxanthin
from the Haematococcus pluvialis. Cyanotech Corporation is well recog­
nized as the world’s largest manufacturer of Spirulina as a food
component that the FDA has authorized and it is sold in over 30 nations
across the world, together with the United States. Algeon Inc., based in
the United States, was formed in 2011 and has since become a
well-known manufacturer of Spirulina and Chlorella. Many of their goods
are made from the photosynthetic protist Euglena gracillis, specifically
b-glucan and whole-cell extracts. TerraViaHoldings, Inc., amongst other
things, is a publicly-traded American corporation specializing in pro­
ducing food components derived from eukaryotic algae (Chandra et al.,
2019). Another prominent microalgal biotechnology business is based in
Germany, BlueBioTech International GmbH, which has been in opera­
tion for more than ten years and produces huge amounts of Spirulina and
Chlorella.
12
Journal of Biotechnology 345 (2022) 1–16
8. Microalgae biorefinery has both advantages and limitations
Although the algal biorefinery has shown great potential, the current
disadvantages must be addressed if the facility is to continue to operate
successfully. Although microalgal biomass is produced in industrial
processes, the total volume of biomass produced is very small (about
15,000 tons per year), and the techniques used to produce it are not
always economically feasible. High-value goods derived from algae are
frequently pointedly pricier than their chemical counterparts, and they
cater to specific biomarkets (Sharma and Sharma, 2017). Bulk products,
for example, biofuels, are positioned at the lower end of the price
spectrum, despite the fact that biofuels should be equivalent in price to
low-priced fossil fuels, they must nonetheless be forced to require far less
supplementary energy, which is ultimately included within the biomass
itself. This need has not been met satisfactorily (Koyande et al., 2019).
Additionally, a breakthrough might be on the horizon in the sector of
medium-value goods for feed, such as those used in agriculture, food
production, or chemical building blocks. How would the biorefinery
idea help to overcome current economic constraints? The main principle
of the biorefinery approach is to employ leftover biomass to produce
high-or medium-value bioproducts (Zeng et al., 2014). In comparison to
plants, the microalgae do not have roots or stems, and they do not have
highly resistant plasma membrane components such as lignocelluloses,
which are exceedingly difficult to break down and exploit with chemical
agents. However, the grouping of the usage of goods derived from algae
and leftover biomass is made significantly easier as a result (Zeng et al.,
2014). Using algae as a dynamic feedstock for biogas production is the
most apparent use of this technology. Another option is to extract and
sell secondary biochemicals or just dry the leftover biomass in order to
use it for animal feed purposes (Wang et al., 2013a,2013b).
Another alternative is the reprocessing of the cellular components at
regular intermissions throughout the process of regeneration. The
nucleic acids derived from leftover biomass might be used to provide a
source of N for the production of current, product-forming algal
biomass. To that degree, the disadvantages of algal manufacturing
procedures that must be overcome depend on various fundamental
factors. The composition of an algal cell is not a static feature; rather, it
depends on the type of strain and the cultural circumstances, which
include nutrients, temperature, and light exposure. As a result, the ratio
is customized according to process alternatives such as energy con­
sumption and the yield on the one hand, and promotional demands on
the other hand (Juneja et al., 2013). According to the literature, this
appears to be a hindrance in numerous stoichiometrically connected
co-products in different divisions of chemical engineering. Glycerine
production from the oilseed-based biodiesel synthesis process might also
be used as an example. As a general rule, moving the metabolism of
microalgae toward the production of a high-value product, such as by
increasing light intensity, leads to the next productivity, but at the price
of the overall method’s energy efficiency (Sharma and Sharma, 2017).
This is often due to the fact that light inside the saturation range does not
drive the next energy turnover at the cellular level but rather just en­
courages product synthesis. Nevertheless, it is not only necessary to
consider the distribution of a wide range of high-value products to a
diverse range of markets. The biorefinery’s outputs, such as nutrients,
water, CO2 gas, and light, should be used in innovative ways to ensure its
success. As many energy and material flow pathways as feasible inside
the plant should be sealed off and protected. Biorefineries, in their
widest meaning, have a third feature that is driven by the need for closed
material and energy cycles in their production.
9. Techno-economic analysis and life cycle assessment (LCA)
Microalgae have demonstrated a number of characteristics,
including the ability to produce high lipid content, the ability to thrive in
dry regions, and the usage of CO2 sources. These traits have attracted
researchers’ attention in light of the potential for improving the process
K. Chandrasekhar et al.
steps used to harvest microalgae that might be used to produce high-
value biochemicals and biofuels (Koyande et al., 2019). Currently,
research is focused on developing and upscaling the method and
determining the industrial feasibility of fuel generation using micro­
algae as a feedstock source. In order to establish the feasibility of
algae-based fuels and other useful biochemicals, the basic analytical
methodologies of techno-economic assessment (TEA) and life cycle
assessment (LCA) are often employed in combination to determine the
viability of such products. These tools make it possible to assess the
selection of process knowledge and build production processes focused
on the goal and investigation efforts necessary to achieve industrial
usableness in a cost-effective manner (Quinn and Davis, 2015). Bio­
refinery systems are being tested and assessed for their industrial
viability using technologies such as the open pool system (OP), and
photobioreactors for algae culture (Rajesh Banu et al., 2020). By means
of excellent commercial and ecological performance, a fruitful bio­
refinery system should employ remaining biomass as an animal feed
alternative and lipids for the production of vegetable oils, in combina­
tion with each other. Compared to photobioreactors, OPs are less chal­
lenging to construct and operate, resulting in reduced capital and
operating costs. Thomassen et al. (2016) conducted a techno-economic
study that concluded that the use of a specialized filtration membrane
for medium reprocessing and OP culture of algae were the most tech­
nologically viable and lucrative options available. It was discovered that
integrating photobioreactors into the system increased capital invest­
ment costs while decreasing the need for medium-term financing
(Thomassen et al., 2016). Further, the course of land occupancy con­
tinues to be a source of contention since environmental consequences
must be considered when determining land usage. Excessive water use
will also result in water scarcity, which will have an impact on water
pricing (Koyande et al., 2019). In order to get ambient water quality
suitable for use in food applications, it may be necessary to add water
treatment methods to the mix. The further problem is that, due to un­
certainties surrounding the overall process scale-up of the new algal fuel
sector, there is a lack of public consensus on whether algae fuels are
economically viable in the short term. The process of cultivating and
harvesting the algal biomass is the most significant element contributing
to the high production costs of microalgae. Whether in an OP or in an
artificial culture environment, algae growth conditions are equally
important factors in determining the overall amount of product pro­
duced (Thomassen et al., 2016). Additionally, as the price of oil pro­
duction and its utilization continues to rise, the use of
microalgae-derived products for energy generation is expected to rise
as a result.
It is also critical to conduct life cycle assessments (LCAs) to deter­
mine the environmental effect of algal biomass production. LCA studies
examine the influence of substrate, techniques, by-products produced
while biomass formation, kinds of algal strains for certain products, and
a variety of additional factors on the overall process of producing a given
product (Adesanya et al., 2014). These ideas encourage the appropriate
usage of algae in a variety of applications. Numerous investigations have
demonstrated that the retrieval of lipid in conjunction with anaerobic
digestion results in the production of gas. The combustion of these gases
is employed to generate electricity and heat. Furthermore, the algal
strains used in the research of the environmental impact of microalgae
and the lipid content of the algal cells are important considerations. It
was discovered in the life cycle assessment research for anaerobic
digestion heat generation that the amount of heat generated is relatively
low when compared to photobioreactors. A number of additional ad­
justments are required in order to increase the use of algae as a potential
substrate for power generation (Koyande et al., 2019). Based on an LCA
research conducted by Soh et al. (2014), they discovered three major
variables impacting the environment: the demand for energy; green­
house gas emissions; the existence of dissolved O2 and mineral content
in water; and the need for energy. Numerous algae were screened from
fresh and salt water, and they were then grown under various growth
13
Journal of Biotechnology 345 (2022) 1–16
circumstances for the purpose of the investigation. The availability of
nutrients was the only one of the three environmental variables
described above that increased biomass content without negatively
influencing the environment or the ecosystem’s health. Whatever the
case, the consequences on the life cycle of microalgae production are
sensitive to factors such as the availability of renewable resources and
the public demand for goods derived from microalgae. This technique
may modify the LCA of the algal process for fuel production and
high-value biochemicals.
10. Conclusions
When summarizing marketed microalgae products, factors such as
algal strains and cell lipid composition, product synthesis techniques,
overall process simplicity, substrate utilization to carry out reactions,
product, and by-product generation, and product and by-product gen­
eration are all taken into consideration. While process integration may
reduce the number of purification operations required, the end product
may sometimes be of low purity because the range of the steps needed to
attain the specified purity threshold varies from industry to industry.
Integration of Processes: Product degradation losses and the impacts of
algal culture growth circumstances, in addition to the tiny market for a
wide variety of goods, must be addressed. The degradation of micro­
algae pigments occurring due to exposure to light, high temperatures,
and microbial contamination is inevitable. It is essential to conduct these
studies to make the whole process economically feasible. To conclude, a
life cycle analysis of high-value microalgae compounds may be carried
out to determine the process’s long-term viability. In order to reduce
reliance on petroleum-derived commodities, efforts should be made to
eliminate further waste and energy costs, particularly in the case of
important downstream processes for high-value chemical extraction
from microalgae, which are now being investigated.
CRediT authorship contribution statement
K. Chandrasekhar: Conceptualization, Methodology. Tirath Raj:
Writing- a part of the paper. S. V. Ramanaiah: Writing- a part of the
paper. Gopalakrishnan Kumar: Writing- a part of the paper. J. Rajesh
Banu: Writing- a part of the paper. Sunita Varjani: Writing- a part of
the paper. Pooja Sharma: Writing- a part of the paper. Ashok Pandey:
Writing- a part of the paper. Sunil Kumar: Writing- a part of the paper.
Sang-Hyoun Kim: Supervision, Data curation, Writing – reviewing &
editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The research was supported by the National Research Foundation of
Korea (NRF) grant funded by the Korean government (Ministry of Sci­
ence & ICT) (No. NRF-2020R1A2B5B02001757).
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