Science of the Total Environment 814 (2022) 152795

Contents lists available at ScienceDirect

Science of the Total Environment

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

Cyanobacterial biorefinery: Towards economic feasibility through the

maximum valorization of biomass
Syama Prabha a, Aravind K. Vijay a, Rony Rajan Paul b, Basil George a,
⁎
a
Department of Botany, CMS College (Autonomous), Kottayam 686001. Kerala, India
b
Department of Chemistry, CMS College (Autonomous), Kottayam 686001. Kerala, India

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Multiple product cyanobacterial biore- A sustainable, multiple product recovering, minimum waste cyanobacterial biorefinery model.
finery with minimum waste output for
the effective valorization of biomass
has been validated.
• Cost-effective strategies for enhancing
cyanobacterial cultivation, metabolite
production has been presented.
• Evaluated the integrated approaches in
cultivation and downstream processing
for improving the economic feasibility of
cyanobacterial biorefinery
• Effective management of cyanobacterial
blooms by nanotechnological approach
• Possibility of uplifting new cyanobacterial
strains to biorefinery concept has been
evaluated.

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

Article history: Cyanobacteria are well known for their plethora of applications in the fields of food industry, pharmaceuticals and
Received 15 September 2021 bioenergy. Their simple growth requirements, remarkable growth rate and the ability to produce a wide range of
Received in revised form 24 December 2021 bio-active compounds enable them to act as an efficient biorefinery for the production of valuable metabolites. Most
Accepted 27 December 2021
of the cyanobacteria based biorefineries are targeting single products and thus fails to meet the efficient valorization
Available online 1 January 2022
of biomass. On the other hand, multiple products recovering cyanobacterial biorefineries can efficiently valorize the
Editor: Huu Hao Ngo biomass with minimum to zero waste generation. But there are plenty of bottlenecks and challenges allied with
cyanobacterial biorefineries. Most of them are being associated with the production processes and downstream strat-
Keywords: egies, which are difficult to manage economically. There is a need to propose new solutions to eliminate these tailbacks
Cyanobacteria so on to elevate the cyanobacterial biorefinery to be an economically feasible, minimum waste generating multiprod-
Multi-product recovery uct biorefinery. Cost-effective approaches implemented from production to downstream processing without affecting
Integrated approach the quality of products will be beneficial for attaining economic viability. The integrated approaches in cultivation sys-
Minimum-waste biorefinery tems as well as downstream processing, by simplifying individual processes to unit operation systems can obviously
Economic feasibility
increase the economic feasibility to a certain extent. Low cost approaches for biomass production, multiparameter op-
timization and successive sequential retrieval of multiple value-added products according to their high to low market
value from a biorefinery is possible. The nanotechnological approaches in cyanobacterial biorefineries make it one
step closer to the goal. The current review gives an overview of strategies used for constructing self-sustainable- eco-
nomically feasible- minimum waste generating; multiple products based cyanobacterial biorefineries by the efficient
valorization of biomass. Also the possibility of uplifting new cyanobacterial strains for biorefineries is discussed.

⁎ Corresponding author at: Department of Botany, CMS College, Kottayam, India.

E-mail address: basil.george@cmscollege.ac.in (B. George).

http://dx.doi.org/10.1016/j.scitotenv.2021.152795
0048-9697/© 2022 Elsevier B.V. All rights reserved.
S. Prabha et al. Science of the Total Environment 814 (2022) 152795

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Value-added products from cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1. Phycobiliproteins and other pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2. Cyanotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.3. UV-protectants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Cyanobacterial biorefinery: From chances of failure to overcome the shortcomings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4. Cultivation of cyanobacteria: low cost approaches for feasibility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.1. Major cultivation strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.2. Cultivation systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.2.1. Photobioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.3. Stress mediated accumulation of bioactive compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5. Downstream processing: feasibility through cost-effective strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5.1. Biomass harvesting: approaches for overcoming the challenges through economic feasibility . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.2. Cell disruption and extraction of value-added compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.2.1. Cell disruption methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.2.2. Extraction of bioactive compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.2.3. Multiple product recovery with sequential extraction of products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6. Advancements of nanotechnology in cyanobacterial biorefinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6.1. Role of nanoparticles in cyanobacterial biorefinery- upstream to downstream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6.2. Cyanobacterial factories for the production of nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6.2.1. Cyanobacteria mediated green synthesis of nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
6.2.2. Cyanobacterial bloom to boon – a nanotechnological approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
7. Integrated approaches to increase the feasibility of multiple product recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
7.1. Integrated cultivation systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
7.2. Integrated harvesting and downstream strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
8. Possibility of uplifting cyanobacterial strains for minimum waste biorefineries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
8.1. Minimum waste cyanobacterial biorefinery models for a circular bio-economy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
9. Promising solutions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
CRediT authorship contribution statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

1. Introduction
Cyanobacteria marked a milestone in the evolutionary history of life on
planet earth. They succeeded in transforming the earlier anaerobic “terra”
to a planet that supports life, by their formerly evolved distinct capability
for oxygenic photosynthesis (Demoulin et al., 2019). The carbon dioxide
fixation rate of cyanobacteria is several times higher than that of terrestrial
plants and thus they possess an immense role in the mitigation of carbon in
the atmosphere (Singh et al., 2016). The cyanobacteria stores captured car-
bon dioxide in the form of organic molecules and convert it into fuels, bio-
chemicals and other value-added compounds using sunlight as the energy
source (Chittora et al., 2020;Singh et al., 2016). Thus cyanobacterial cells
can act as an efficient system for the production of unique metabolites
which are having a wide range of applications (Sonani, 2016).
Cyanobacteria are mainly exploited for the production of phycobiliproteins,
which are brilliantly coloured pigment proteins with applications in phar-
maceutical, food, and nutraceutical fields (Pagels et al., 2019; Paswan
et al., 2016). Besides phycobiliproteins, they are identified to be a good
source of several other products, like lipids, carbohydrates, carotenoids,
cyanotoxins, polysaccharides, UV protectant compounds like scytonemin,
mycosporine-like amino acids (MAA) etc., (Demay et al., 2019; Noreña-
Caro and Benton, 2018). Apart from this, the past few years witnessed
the discovery of plenty of biomolecules such as antibiotics, anti-
inflammatory, anti-cancer agents from cyanobacteria. Table 1 shows sev-
eral bioactive compounds obtained from different strains of cyanobacteria.
The majorities of these potent metabolites are reported to have extensive
bioactivities and can have therapeutic and pharmaceutical applications.
Hence, the demand for distinct metabolites from cyanobacteria leads to
their industrial production and commercialization, which further explicit
the scope of cyanobacterial biorefinery (Cuellar-Bermudez et al., 2015b;
Tiwari, 2018; Trivedi et al., 2015). A cyanobacterial biorefinery is targeting
the conversion of biomass into high-value products, along with the genera-
tion of several other co-products. Fig. 1 shows the diagrammatic represen-
tation of a typical cyanobacterial biorefinery. The major advantage of the
biorefinery concept is the maximum valorization of available raw material
(biomass) while preventing the loss or damage of retrieving products
(Chandra et al., 2019a). The current bio-economy challenges demand for
the production of multiple value-added products with less time. The appro-
priate solution to this challenge is the multiple product recovery and max-
imum valorization of biomass. Most of the value-added products from
cyanobacteria can be concurrently retrieved from the same biomass by se-
quential extraction techniques based on their order of high to low market
value and can of course step towards zero-waste biorefinery through a
green circular bio-economy (Chandra et al., 2019b; Mitra and Mishra,
2019).
However, there is a lack of exclusive reports on the cyanobacterial
biorefineries, maximum valorization of cyanobacterial biomass and the
possibility of uplifting novel strains for the biorefinery. The available re-
ports cover mainly the Arthrospira platensis/Spirulina platensis. S. platensis
is considered as an efficient multiple product proffering strain for decades
(Chia et al., 2019; Costa et al., 2019; Mitra and Mishra, 2019). Exploring
the cyanobacterial world for uncovering more possible strains for the recov-
ery of value-added products would be necessary, since many of them are ac-
tive producers of several valuable bio-active compounds, which are having
immense scope in the pharmaceutical, nutraceutical, cosmetic and food in-
dustries. Inter alia most of these cyanobacteria remain unnoticed and are
not studied properly. Hence, there is an increasing need to discuss different
aspects of a cyanobacteria based biorefinery, by considering the whole
cyanobacterial genera in general.
Most of the cyanobacterial biorefineries are aiming for single product
recovery, which usually fails to meet the maximum valorization of biomass.
It is uncertain about the industrial success of those multiproduct based

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S. Prabha et al. Science of the Total Environment 814 (2022) 152795

Table 1
Different metabolites produced by some selected strains of cyanobacteria.
Cyanobacteria Metabolites Application

Microcystis Aeruginosin Antiviral

Microcystin Cyanotoxin
Microviridin Antiviral
Oscillatoria Aeruginosin Antiviral
Microcystin Cyanotoxin
Venturamide A,B Antimalarial
Acutiphycin Anticancer
Agardhipeptin Enzyme inhibitor
Anabaena Anatoxin Larvicidal
Cylindrospermopsin Cyanotoxin
Shinorine UV-protectant
Minutissaminde A-D Anticancer
Microcystin Cyanotoxin Fig. 1. – Diagrammatic representation of a cyanobacterial biorefinery.
Bastadin Antibiotic
Bis-(χ-butyrolactones) Antibiotic
Circinamide Enzyme inhibitor
of their unique physiological and metabolic adaptations. (Noreña-Caro
Aphanizomenon Anatoxin Cyanotoxin
Cylindrospermum Anatoxin Cyanotoxin
and Benton, 2018). Cost-effective cultivation of cyanobacteria can be easily
Planktothrix Anatoxin Cyanotoxin achieved by integrating the cultivation with wastewater treatment plants.
Lyngbya Apratoxin Anticancer The distinct capability of these organisms to survive in wastewater can be
Curasin A Anticancer utilized for biomass production in a win-win approach, as it reduces the
Desmethoxymajusculamide C Anticancer
production cost and can be efficiently used for the treatment of wastewater
Dolastatin Anticancer
Homodolastatin Anticancer and flue gas (Singh et al., 2016)
Hectochlorin Cyanotoxin The process of eutrophication by the enrichment of phosphorous and ni-
Jamaicamide Anticancer trogen in the water bodies often leads to cyanobacterial blooms which not
Lyngbyatoxin Cyanotoxin only harm the water bodies but also have detrimental effects on human be-
Majusculamide Anticancer
Barbamide Antiparasite
ings (Martens, 2017). The transformation of the cyanobacterial bloom to a
Carmabin A,B Antiparasite boon can be achieved by the efficient valorization of biomass from blooms.
Scytonemin UV-protectant The harvested biomass from the cyanobacterial bloom can be used for the
Dragomabin Antimalarial retrieval of high-value compounds. The part of the income generated
Dragonamide A,B Antimalarial
from the commercialization of extracted high-value products can be uti-
Aplysiatoxin Anticancer
Grassypeptolide Antiproliferative lized for adopting strategies for the prevention of blooms (MacÁrio et al.,
Sulfolipids Antiviral 2021). The successful stories of the development of efficient nanoparticles
Pitipeptolides A, B Anticancer with pharmaceutical applications from cyanobacterial blooms opens up a
Pahayokolides Larvicidal new path for the handling of blooms as well as the valorization of biomass
Calothrix Calothrixin A,B Antiparasite
Nostoc Cryptophycin Anticancer
(Lee et al., 2015). Recent advances unveiling the efficient participation of
Cyanoviridin Antiviral nanoparticles in cyanobacterial and micro-algal research, especially in
Microcystin Cyanotoxin downstream processing, have made implications in reducing the cost in-
Borophycin Anticancer volved in the multiproduct recovery (Nguyen et al., 2019b; Pathak et al.,
Muscoride Antibiotic
2019). Thus, a closed-loop approach to cyanobacterial biorefinery and val-
Nostocine A Antibiotic
Cylindrospermopsis Cylindrospermopsin Larvicidal orization of biomass will be self-subsistent which encourage multi-product
Nodularia Spumigin Antiviral recovery and environmental sustainability.
Nodularin Cyanotoxin The success of a multi-product proffering cyanobacterial biorefinery is
Scytonemin UV-protectant determined by several sequential factors, which includes different modes
Prochloron Patellamide A Anticancer
Scytonema Scytonemin UV-protectant
of cultivation strategies for the accumulation of desired bio-products,
Didehydromirabazole Antibiotic methods of downstream processing and most importantly, the manner
Aphanothece Mycosporine 2 glycine UV-protectant and order of extraction of different value-added products (Mitra and
Symploca Symplocamide A Antiparasite, Anticancer Mishra, 2019). There is a need to notice every single aspect of a biorefinery
Symplostatin 3 Anticancer
from production to downstream, to check wherever the cost-effective mea-
Tolypothrix Tjipanazoles Anticancer
Toyocamycin Antifungal sures have to be implemented. The biomass production can be improved in
Dysidea arenaria Arenastatin A Anticancer different ways by increasing the efficiency of photobioreactors (PBR)
Plectonema Radiosumin Enzyme inhibitor (Johnson et al., 2018). It has been shown that the economic viability of
Phormidium Fischerllin Antifungal, Herbicidal cyanobacterial biorefineries can be increased by raising the product titers
Hapalosiphon fontinalis Anhydrohapaloxindole Antifungal
above a significant concentration in open ponds (Fasahati et al., 2019).
Constructed based on (Arif et al., 2012; Noreña-Caro and Benton, 2018; Rajneesh Stress mediated accumulation of bioactive compounds by using low-cost
et al., 2017; Rastogi and Sinha, 2009). measures can be also used for economic feasibility (Paliwal et al., 2017). In-
tegrated approaches in cultivation, harvesting and downstream processing
biorefineries due to expensive methodologies adopted for production and can increase the economic viability of cyanobacterial biorefineries. Integra-
downstream processing. Cost-effective strategies for the recovery of the tion of those several complex processes, which needs more time and capital
maximum possible number of intact products from a single cyanobacterium investments can be turned into single unit operation and can bring eco-
and thereby valorizing the biomass can show way towards the industriali- nomic viability to a greater extent (Lam et al., 2018). This review aims to
zation of a multiproduct biorefinery. Examining novel techniques and pro- analyze the scope of maximum valorization of cyanobacterial biomass, in
posing new solutions for expunging the glitches encountered while dealing the light of cyanobacteria based multiple product biorefineries. The strate-
with cyanobacterial biorefineries are crucial for developing it to the indus- gies for increasing the economic feasibility of biorefineries, valorization of
trial level. The optimization of culture conditions and strategies for the ac- biomass from cyanobacterial bloom, along the possibility of uplifting the
cumulation of desired products in cyanobacteria is relatively easy because cyanobacterial strains are also discussed in detail.

3
S. Prabha et al.
2. Value-added products from cyanobacteria
2.1. Phycobiliproteins and other pigments
Phycobiliproteins are the major accessory protein pigments found in
cyanobacteria. The phycobiliproteins are the key factors responsible for
the regulation of machinery behind the inimitable complementary chro-
matic adaptation in cyanobacteria, where they manage to adapt with differ-
ent intensities of light by the synthesis of these pigment proteins in response
to light (Lee et al., 2017a; Singh and Montgomery, 2013). These naturally
occurring brilliant blue coloured, water-soluble, fluorescent protein
pigments are having high market value owing to their potential applica-
tions in food, pharmaceuticals, and nutraceutical biomedical industrial
fields (Li et al., 2019). The classification of phycobiliproteins are strictly
based on spectral characteristics, attributable to the tetrapyrrole prosthetic
groups known as the bilin chromophores, attached with cysteine
residues in the biliproteins by thioether linkages. Based on the spectral
properties they are majorly divided into four types; Phycocyanin (C-PC;
λA max − 610–620 nm), Phycoerythrin (C-PE; λA max − 540–570 nm),
Allophycocyanin (C-APC; λA max − 650–655 nm), Phycoerythrocyanin
(C-PEC; λA max − 560–600 nm) (Paswan et al., 2016; Safaei et al., 2019;
Wang et al., 2014). The structure of bilin chromophore in phycocyanin
shares structural similarity with the naturally occurring antioxidant biliru-
bin and accordingly, it has substantiated antioxidant, anticancer, anti-
inflammatory, hepatoprotective, neuroprotective, immunomodulation
roles and it can also act as a substitute for artificial colouring agents in
food, which imparts health benefits (Jiang et al., 2017; Kannaujiya et al.,
2017a; Li et al., 2019; Pagels et al., 2019; Prabakaran et al., 2020). Spirulina
platensis has been regarded as the most investigated and industrially
exploited cyanobacterial species for the production of phycobiliproteins
and the explorations for new biotechnological applications are still
proceeding with this strain (Prabakaran et al., 2020).
Phycobiliproteins have been isolated from different strains of
cyanobacteria for examining their pharmaceutical activity. The role of
these pigment proteins in anti-ageing research is also an interesting area
of study. The anti-ageing property of phycobiliproteins from Lyngbya sp
was tested in model organism Caenorhabditis elegans and found that phyco-
erythrin was successful in increasing its life expansion rate (Sonani et al.,
2014). By emphasizing ‘oxidative stress theory of ageing’, which details
the interconnection between oxidative stress and ageing, the anti-oxidant
activity of phycobiliproteins can be useful for the development of anti-
ageing drugs (Sonani et al., 2015).
The cyanobacteria also possess other pigments such as carotenoids and
chlorophylls, with several industrial applications. The most studied carot-
enoid is ß-carotene, which is having applications in food, cosmetic and
nutraceutical industries, owing to its yellow–deep orange colour and anti-
oxidant properties (Chandra et al., 2019b).
2.2. Cyanotoxins
Apart from nutraceutical and pharmaceutical agents, cyanobacterium is
a good source of cyanotoxins. The cyanotoxins can be divided into five clas-
ses according to their biological activities. They are hepatotoxins, neuro-
toxins, cytotoxins, dermatotoxins and lipopolysaccharides (Rastogi and
Sinha, 2009). According to their chemical structures, cyanotoxins com-
monly fall into three groups: cyclic peptides (Microcystins and Nodularin),
heterocyclic compounds (alkaloids) (Cylindrospermopsin, Anatoxins,
Saxitoxins, Lyngbyatoxins, Aplysiatoxin) and lipid compounds (Lipopoly-
saccharide), etc. Most of them contain an unusual beta-amino acid which
is responsible for the acute toxic effects of cyanobacteria (Sanseverino
et al., 2017). Microcystins are cyclic heptapeptide cyanotoxins with vari-
able L-amino acid residues at two positions (2 and 4). Most common is
the MC-LR with leucine and arginine at positions 2 and 4 respectively.
Others are MC-LA, MC-RR and MC-YR. Nodularins are cyclic pentapeptides
with many similarities to microcystins. Cylindrospermopsins are guanidine
alkaloid cyanotoxins. Anatoxins are similar in structure with synthetically
4
Science of the Total Environment 814 (2022) 152795
produced organo phosphate-based insecticides. Saxitoxins (STXs), also
called paralytic shellfish poisoning (PSP) toxins are classified into several
groups and a total of about 57 saxitoxins have been reported so far
(Salmaso et al., 2017).
These toxic secondary metabolites from cyanobacteria can be consid-
ered as an excellent source for the production of novel biopesticides. Exu-
dates and aqueous extracts of different species of cyanobacteria, Alusira
fertilisima and Nostoc muscorum, Microcystis aeruginosa, etc. can cause inhibi-
tion in hatching activity and mortality of juveniles of root-knot nematode,
Meloidogyne triticoryzae, Daphnia pulicaria etc., (Chandel, 2009).
2.3. UV-protectants
Cyanobacteria were the first photosynthetic inhabitants on the surface
of the earth to establish their prokaryotic kingdom at the ever-worst time
in the evolutionary history of the earth, which was devoid of the ozone
layer and hence, constantly exposed to the harmful ultraviolet radiation
from the sun. Consequently, these organisms develop several preventive
strategies and defensive mechanisms to cope with this complex situation
(Singh et al., 2010). One of the methods adopted by the cyanobacteria to
withstand the harmful UV (Ultraviolet) radiation was to synthesize some
chemical compounds that can absorb UV light and can act as UV protec-
tants. Mycosporine like amino acids (MAA) and scytonemins are well-
known UV protectants from cyanobacteria, which usually absorbs both
UV-A and UV-B radiations. They are lipophilic compounds that are usually
present in the sheath of cyanobacteria. Due to their strong UV-absorbing
and photoprotective nature, these compounds can be used in cosmetic
products having high market value (Rastogi et al., 2015).
Scytonemins are yellowish-brown lipid-soluble, compounds present in
the extracellular polysaccharide sheath of different cyanobacteria, and are
exhibiting a maximum absorption peak at 386 nm (Rastogi and Sinha,
2009). An increase in temperature along with oxidative stress in presence
of UV-A radiation can increase the production of scytonemins in
cyanobacteria. It was reported that Nostoc punctiforme PCC 73102 produce
maximum scytonemin when it is grown diazotrophically rather than non-
diazotropically (Singh et al., 2010). Rather than photoprotective roles,
scytonemin has also been found to be an excellent anti-proliferative and
anti-inflammatory agent. Apart from this, scytonemin exhibit extreme
stability at different temperatures and in the presence of different com-
pounds (Singh et al., 2017). Three derivatives of scytonemin are,
dimethoxyscytonemin, tetramethoxyscytonemin, and scytonin, which
have been isolated from the extracts of Scytonema sp. (Rastogi et al., 2014).
The strongest UV-absorbing compound in nature is the mycosporine-
like amino acids (MAA). They are water-soluble compounds, which usually
possess an absorption maximum at a range of 310–360 nm (Carreto and
Carignan, 2011). MAA producing cyanobacteria are likely to be found in
hypersaline environments (Rastogi and Sinha, 2009). They are water-
soluble compounds, with a structure composed of a chromophore conju-
gated with nitrogen substituent of amino acid. The chromophores can be
cyclohexanone or cyclohexenimine and usually possess an absorption max-
imum at 310 nm (Carreto and Carignan, 2011; Rajneesh et al., 2017). MAA
has been commercialized under the brand name Helioguard 365, which
contain shinorine and mycospora 365, which are the most common
MAAs (Borowitzka, 2013). Apart from their UV- protectant nature, like
scytonemins, MAA also exhibits certain pharmaceutical activities. MAA ex-
tracted from the cyanobacterium Aphanothece halophytica, exhibited
considerable antioxidant, collagenase inhibition and osmoprotectant activ-
ities (Hibino et al., 2018).
3. Cyanobacterial biorefinery: From chances of failure to overcome
the shortcomings
The concept of cyanobacterial biorefinery does make sense, even
though it sounds a little unmanageable while dealing with the drawbacks
related to the economic aspects of its successful establishment (Koyande
et al., 2019). Multiple bottlenecks are present in many of the unit
S. Prabha et al.
operations from cultivation to downstream processing. The currently em-
ployed methods for recovery and further processing of the biomass are ex-
pensive and energy-consuming, which can seriously affect the economic
feasibility of a biorefinery (Deprá et al., 2018). Most of the downstream pro-
cessing techniques are highly expensive; however, they are targeting single
products. The remaining biomass left after the extraction process gets
wasted and fails to get valorized. Fig. 2 represents an overview of the max-
imum valorization of cyanobacterial biomass. The primary target from a
cyanobacterial biorefinery can be high value-low volume products,
followed by low value-high volume products. The spend biomass, after
the extraction of value-added products can be further converted to several
bio-energy based products. One of the main challenges of a biorefinery is
the successful recovery of individual fractions of compounds without caus-
ing any harm to the other fractions (Chew et al., 2017). All the successful
Fig. 2. – An overview of the maximum valorization of cyanobacterial biomass.
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past scenarios regarding the cyanobacterial biorefinery manifest that their
accomplishment was achieved by giving more considerations to the
multi-product recovery and culture conditions optimization (Ferreira da
Silva et al., 2020). The success of a cyanobacterial biorefinery will depend
upon the retrieval of maximum possible value-added products with less cost
and time (Chew et al., 2017). In the light of this understanding, we can ad-
join the fact that the feasibility of a biorefinery will depend upon the eco-
nomic practicability of products. Thus, the flaws in optimization of
culture conditions for the maximum accumulation of bioactive compounds
and the impracticability of recovering the maximum number of compounds
can be considered as a serious issue (Gifuni et al., 2019; Klok et al., 2013).
The following sessions briefly discuss strategies to overcome those bottle-
necks and subsequently construct an economically feasible cyanobacterial
biorefinery.
S. Prabha et al.
4. Cultivation of cyanobacteria: low cost approaches for feasibility
Nowadays, the main focus in achieving maximum accumulation of
value-added products from cyanobacteria is by the optimization of their
culture conditions. The main idea behind the optimization of culture condi-
tions is to increase the biomass of the micro-organism under consideration,
and thereby, increase the products extracted from them. Re-organization of
all the limiting parameters for the successful establishment of culture is also
important. Different cultivation strategies and cultivation systems can be
adopted for the mass cultivation of cyanobacteria.
4.1. Major cultivation strategies
The cultivation strategy of cyanobacteria is having a tremendous role in
the production of different compounds from them. There are four modes of
cultivation including, photoautotrophic, heterotrophic, mixotrophic and
photoheterotrophic. Cyanobacteria are conventionally cultivated photoau-
totrophically by providing carbon dioxide, sunlight and inorganic nutrients.
These cyanobacteria exhibited less biomass productivity due to the restric-
tions in light that affect the cell growth and thereby increase the overall pro-
duction cost (Corrêa et al., 2020). On the other hand, cyanobacteria can be
grown on either a mixotrophic or heterotrophic mode in the presence or ab-
sence of sunlight (Andrade and Costa, 2007). An organic carbon source can
act as nutrient for this and can increase the productivity accordingly
(Corrêa et al., 2020). Cyanobacteria are well known for their ability to uti-
lize organic substrates as an energy source even in the absence of light.
Hence, the heterotrophic mode of cultivation, along with the supplementa-
tion of exogenous carbon sources can be regarded as a suitable way for
cyanobacterial biomass productivity. The preferable usage of low-cost al-
ternatives, for organic carbon sources, such as starch hydrolyzes and cellu-
lose can decrease the production cost and thereby increase the economic
feasibility (Meireles dos Santos et al., 2017). Efficient production of intra-
cellular metabolites in cyanobacterium Phormidium sp. was achieved het-
erotrophically with the supplementation of maltodextrin and cassava
starch (Francisco et al., 2014). However, all species cannot grow under het-
erotrophic mode. For the accumulation of bioactive products along with
considerable growth, the best choice is mixotrophic cultivation as it can
combine the benefits of both autotrophic and heterotrophic cultivation
strategies (Patel et al., 2020; Zhan et al., 2017). The inorganic carbon
source can be glucose, acetate or glycerol to achieve mixotrophic growth
(Nagappan et al., 2020; Patel et al., 2020; Yu et al., 2009).
Mixotrophic cultivation is satisfied by providing inexpensive sugar and
organic acid as carbon and energy sources and can increase productivity by
balancing the production cost. The benefits from the mixotrophic strategy
includes, increased biomass production, higher lipid, carbohydrate and pig-
ment production, long-standing exponential growth phase, etc. (Kean et al.,
2020). There are plenty of reports available, demonstrating the higher bio-
mass productivity of different cyanobacteria under this mode of cultivation.
Nagappan et al. (2020) evaluated a five-fold increase in biomass productiv-
ity of Nostoc sp. MCC41 cultivated mixotrophically with the supplementa-
tion of 0.5% glucose as a carbon source when compared to the
phototrophic conditions. Another experiment conducted by Yu et al.
(2009) revealed a higher biomass concentration of 1.67 g L–1 in
mixotrophic cultivation which is 4.98 and 2.28 times more than the
phototrophic and heterotrophic mode respectively. Anabaena PCC 7120,
which was cultivated under the light in presence of exogenous glucose, ex-
hibited a maximum specific growth rate which is 1.6 fold higher than the
photoautotrophic cultivation (Zhan et al., 2017). However, cyanobacterial
growth for the high-value pigments, such as phycobiliproteins and caroten-
oids, on an industrial scale, usually prefer photoautotrophic cultivation
without organic substrates and contamination risks (Xie et al., 2015).
4.2. Cultivation systems
Identifying the most preferable culture strategy for raising
cyanobacteria should be the foremost objective for achieving maximum
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biomass productivity. The cultivation in open raceway ponds are cost-
efficient, on the other hand, choosing the photobioreactors for the high bio-
mass production will require higher capital investment (Tan et al., 2020).
The best choices for cultivation can be wisely made which may depend
upon the value of the bioactive products from the cyanobacteria. Bioreac-
tors with the higher surface to volume ratio could effectively use light en-
ergy, and efficiently increase the cell concentration, hence likely to be
ideal for the growth of photosynthetic microbes (Singh et al., 2016;
Venkata Mohan et al., 2020).
4.2.1. Photobioreactors
High quality biomass has to be preferred for the extraction of bio-active
compounds with pharmaceutical and nutraceutical applications. For that, it
is better to consider closed system cultivation strategies, using
photobioreactors. The benefits of closed systems can be reflected here
with the fair production of threefold or more biomass, as compared with
the open systems (Venkata Mohan et al., 2020). The idea of a bioreactor
is to assure more production, as well as avoid cross-contamination and
predator attacks. Through this method, we can easily raise those species,
which are highly sensitive to different culture parameters (Huang et al.,
2017; Johnson et al., 2018). The main drawbacks of the common
photobioreactors are the inefficiency in light management followed by
low biomass concentration (Gifuni et al., 2019). Alternatives for the cur-
rently available high cost, less productive photobioreactors are essential,
as the low concentrated biomass can highly affect the efficiency of the
downstream processing.
Various configurations of bioreactors are currently available, and most
of them are the applied versions of either bubble column, air-lift, tubular
or flat-panel reactors (Wang et al., 2012). It is suitable to design a bioreactor
according to the basic requirements of the species of cyanobacteria under
consideration, and also according to the nature of value-added products.
After choosing the cultivation system for the successful growth of
cyanobacteria, the economic feasibility of the system can be analyzed by
considering the Net Energy Ratio (NER). For an economically viable sys-
tem, NER will be ˃1. Most of the studies reveal that open raceway systems
mostly give NER ˃1 (De Bhowmick et al., 2019). But according to the bio-
mass production and control of contamination problems, photobioreactors
are mostly preferable. Hence, the solution will be a hybrid- design of an
open system coupled with a photobioreactor (Brennan and Owende,
2010). An ideal bioreactor design is unavailable, for the mass cultivation
of cyanobacteria, as most of the designs are less concerned with several lim-
iting factors which are contributing to the growth such as, light, tempera-
ture, aeration, hydrodynamics, etc. (Huang et al., 2017).
4.2.1.1. Photobioreactor - management strategies for higher efficiency. The idea
is to introduce an economically viable closed system, to be exact, a
photobioreactor, for the cultivation of cyanobacteria. The concept is little
bit challenging because the installation of a typical photobioreactor needs
much economic investment. (Huang et al., 2017). This challenging thought
can be made possible by implementing a few strategies for the effective uti-
lization of light, control of temperature, heat-reducing tactics and simulta-
neously enhancing the photosynthesis and thereby increasing the
production of cyanobacteria inside the photobioreactor (Johnson et al.,
2018).
By considering each factor, light is the most essential limiting factor for
the successful growth of cyanobacteria in a photobioreactor. Optimum
quantity and quality of light supplementation plays an important role in
photosynthesis, thereby increasing the productivity and pigment composi-
tion in cyanobacteria. The intensity and wavelength of light have consider-
able effects on cell growth and composition (Ho et al., 2015). Inefficient
light management problems always arise associated with almost all cultiva-
tion systems. During the growth of cyanobacteria, cell multiplication and
mutual shading of cells occur, creating a dark zone inside the inner part
of culture due to the light restriction. This would prevent photosynthesis
and further biomass productivity. This problem can be prevented by de-
creasing the optical path of the culture (Gifuni et al., 2019). A reduction
S. Prabha et al.
in optical path ≤0.01 m can be applied for increasing the photosynthetic
efficiency up to 5 to 8% (Janssen, 2016).
In comparison with the other artificial light sources, LED light with
more energy efficiency, has turned out to be an exemplary choice for the
growth of cyanobacteria (Park and Dinh, 2019). A comparative analysis
on the effect of different light sources on phycobiliprotein composition in
cyanobacteria Spirulina platensis has been done by Ho et al. (2018). C-PC
productivity of S.platensis was 40% higher under the white LED light than
the fluorescent lamp. A decrease in electrical consumption and increase in
C-PC production with a cost reduction from 288 US$ kg−1C-PC to 147 US
$ kg−1 C-PC was observed when the fluorescent lamp was replaced by
white LED light (Ho et al., 2018). Chlorophyll a and phycobiliproteins are
the major light-harvesting pigments present in cyanobacteria (Paswan
et al., 2016). LED light provides specific light sources and this specificity
may hinder the photosynthesis in cyanobacteria. Hence, to balance the
efficiency of energy uptake, cyanobacteria will actively synthesize
other pigments such as phycobiliproteins to cope with this situation.
Phycobiliproteins will actively absorb the light at the visible wavelength
range and will transfer it to the reaction centre consisting of chlorophyll a
(Hemlata and Fatma, 2009). The effect of different LED lights on the pig-
ment composition of cyanobacteria S. platensis was documented by Prates
et al. (2018) and a 2.7 times increase in the phycocyanin production was
observed under the presence of green LED light than the fluorescent light
as control. A similar experiment to detect the effect of different LED lights
on the biochemical composition of another cyanobacterium Synechococcus
nidulans was carried out using red, green and yellow LED lights. In that
study, all the LED light fails to evoke considerable effects on the pigment
composition. On the other hand, the cultures which were grown under
red and yellow LED light produce 50% more saturated fatty acids,
exhibiting the prospects for biodiesel production (Duarte et al., 2019).
The observed difference might have been the result of variations in cultiva-
tion setup and strain-specific light acclimatization responses.
The role of photoperiod is extremely important for the production of
phycobiliproteins. A partial photoperiod is always beneficial for the optimum
synthesis of these accessory pigment proteins. Continuous exposure to exces-
sive light may trigger the generation of free radicals and further leads to
photo-inhibition by the oxidization of several significant proteins, including
the D1 proteins within the photosynthetic apparatus (Khatoon et al., 2018;
Prates et al., 2018). Complementary chromatic adaptation (CCA) in
cyanobacteria facilitates the selective synthesis of different phycobiliproteins
in response to the availability of different wavelengths of light during varying
environmental conditions. CCA explains the tendency of a cyanobacterium to
produce phycoerythrin in the presence of green light and synthesis of phyco-
cyanin in response to the red light (Paliwal et al., 2017). CCA depends on
three pathways including; regulation by a photoreceptor, which is highly sen-
sitive to the red and green light; a complex signal transduction pathway,
which is comprised of a two-component system; and the last one is associated
with a redox state-based reaction occurring pathway. Similar studies Park
and Dinh (2019) revealed that the red and blue LED light favoured the
production of phycobiliproteins in Arthrospira maxima. It was also observed
that the combined supplementation of red and blue light enhanced the
pigment synthesis and biomass yield in this cyanobacterium.
The intensity of light can also influence lipid accumulation in
cyanobacteria. In contrast to the phycobiliproteins synthesis, the increase in
light intensity can progressively increase the lipid content in cyanobacteria.
The studies on the effects of light intensity and supplementation of carbon di-
oxide on the lipid productivity of cyanobacteria Synechococcus sp. PCC6803
reported that the lipid productivity was directly proportional to the intensity
of light provided (Cuellar-Bermudez et al., 2015a).
When, considering those photobioreactors which are solely depending on
solar radiation as the light source, solar panel associated with them could be
useful for the effective utilization of entire solar radiation for producing
chemical as well as electric energy (Moheimani and Parlevliet, 2013).
Along with light, proper mixing, aeration, pH, temperature, etc. should be
taken into consideration while handling photobioreactors for the growth of
cyanobacteria (Wang et al., 2012).The effective use of photons is important
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for the existence of a photobioreactor. Hence, the location and arrangement
of a bioreactor should ensure the efficient capture and utilization of photons.
It has been reported that the outdoor photobioreactors positioned vertically
in the east-west direction can efficiently harvest solar radiation (5% more)
than a photobioreactor placed in a North-South direction horizontally. More-
over, there are innumerable light management approaches for enhancing the
efficiency of photobioreactors. Solar tracking device associated bioreactors
are one of them. They are controllable devices, which always maintain the
growing culture towards solar radiation and thus increase exposure to sun-
light. Some researchers have been found that there is an increase in cell den-
sity with a decrease in the thickness of a photobioreactor (Nwoba et al.,
2019). But, it is not feasible to keep the culture in direct sunlight for the
whole day, because continuous exposure to the heavy solar radiation may
cause temperature rise, photo-inhibition and associated damaging effects.
Hence, these types of photobioreactors are always associated with tempera-
ture regulators, which will be pre-set with a particular temperature. When-
ever the temperature exceeds the set value, the device will control the
incident solar radiation in some way, like rotating the photobioreactor. But
these types of tracking device associated photobioreactors are not economi-
cally viable (Wang et al., 2012). To achieve economic feasibility, we can cou-
ple this type of photobioreactors with the technology of light filtration and
thus co-generation of electricity along with high biomass production can be
achieved. Similar approaches to rectify the shortcomings of conventional
sole goal photobioreactors will be promising (Nwoba et al., 2019)
Among the various photobioreactors (PBR) available, only a few can be
considered promising for the mass cultivation of cyanobacteria. Tubular
PBR, plastic PBR, column airlift PBR, flat-panel airlift PBR are some of
them (Kenekar, 2014; Johnson et al., 2018; Shastik et al., 2020; Wang
et al., 2012). The column airlift PBR and flat-panel airlift PBR do not con-
sume much power for their functioning and are efficient in mixing and
mass transfer, but they need much capital investment and cleaning costs.
When dealing with the economic feasibility, plastic bag PBR is the most at-
tractive (Cañedo and Lizárraga, 2016). But the main shortcomings of this
kind of photobioreactor are the poor mixing and short lifespan and lack
of temperature control tactics. Plastic bag bioreactors immersed in cool
water during summer can control the high temperature in a cost effective
manner. The plastic bag bioreactor can be maintained in association with
oceans and hence the tides can overcome the problems related to mass
transfer and bad mixing. The plastic bag bioreactors are comparatively
short-lived and more fragile. This problem can be solved by using quality
material for the making of this type of bioreactors. The main risk factor is
that the wide usage of this type of photobioreactors will generate a lot of
plastic wastes (De Bhowmick et al., 2019; Shastik et al., 2020)
The major bottlenecks associated with currently available culture sys-
tems are the inefficient light utilization and generation of dilute culture,
which can seriously affect the energy consumption during downstream pro-
cessing. Pre-concentration of the diluted culture is necessary, just before
centrifugation to decrease the energy cost. The introduction of thin culture
systems can solve these problems associated with dilute culture (Gifuni
et al., 2019). A flat plate bioreactor was introduced earlier, which aimed
to solve the problems associated with light penetration and less biomass
production. Later modified versions of flat plate PBRs, like ultra-thin flat
plate PBRs were introduced with a thickness of 3 mm, with effective light
utilization. These ultra-thin PBRs were successful in achieving a biomass
concentration of 24 g L–1, which is far higher than the biomass 3 g L–1 ex-
hibited by conventional PBRs (Gifuni et al., 2018). Apart from this, several
other shortcomings related to photobioreactors arise in association with the
volume of the culture. The state-of-art technologies of cultivation strategies
for overcoming those limitations are introduced, including immobilized
cultivation using porous substrate bioreactors (Podola et al., 2017).
4.3. Stress mediated accumulation of bioactive compounds
Cyanobacteria have higher degree of adaptation towards environmental
variations and constantly regulate their cellular mechanism accordingly.
These fluctuating environmental conditions can be regarded as stress
S. Prabha et al.
conditions and cyanobacteria are known to synthesize a variety of metabo-
lites with their varying cellular mechanisms to cope up with the stress
(Paliwal et al., 2017). Stress mediated accumulation of bioactive com-
pounds in cyanobacteria can be integrated into the pre-determined sustain-
able production of various compounds and steps towards an economically
viable cyanobacterial biorefinery (Anand and Arumugam, 2015). In detail,
the stress condition can act as a tool in which, single or multiple stress fac-
tors can be strategically designed to achieve a cyanobacterial biorefinery
for desired accumulation of one or more metabolites in an efficient way.
But, it is not possible to accumulate desired products without compromis-
ing or interfering in the pathway of other cellular mechanisms. For in-
stance, cyanobacterial cultivation under continuous stress conditions may
lead to the production of lipids and carbohydrates, but the growth will be
retarded to a great extent. Hence, to overcome this challenge, two-stage cul-
tivation of cyanobacteria can be adopted, where maximum biomass accu-
mulation was achieved in the first stage followed by stress conditions in
the second stage to induce the accumulation of desired products (Paliwal
et al., 2017). Major abiotic stress encountered by cyanobacteria are temper-
ature, salinity, light and nutrient stress.
Cyanobacteria exhibited varying biochemical compositions in response
to the stress of different incident light (Paliwal et al., 2017). Trabelsi et al.
(2009) reported the effect of different light intensities on the growth and
biochemical composition of the cells Arthrospira platensis. An increase in
growth rate along with the production of extracellular polymeric sub-
stances on exposure to high-intensity light was also observed. Several inves-
tigations were carried out in the past to detect the influence of light stress
on the accumulation of bioactive compounds in cyanobacteria. Khajepour
et al. (2015) analyzed the effect of light intensity and photoperiod on the
biomass production and composition of Nostoc calcicola. The results mani-
fest the preference of N. calcicola towards dim light and exhibited a signif-
icant increase in the phycobiliproteins accumulation when exposed to
low-intensity light. The carotenoids and carbohydrates are also observed
to be higher at higher light intensities and longer photoperiods. Thus, it is
possible to manipulate the biochemical composition in majority of the
cyanobacteria following the varying light regime.
Salt is essential for some of the metabolic reactions in cyanobacteria. Al-
though, at higher concentrations salinity can impart detrimental effects on
the growth of cyanobacteria, salinity induced stress strategies were widely
implemented for the enhanced production of desired products such as lipids
and pigments (Singh et al., 2014). Cyanobacteria is capable to adapt to the
majority of extreme situations, likewise, it can withstand salinity to a
greater extent (Bhadauriya et al., 2007; Singh, 2014). Cyanobacteria re-
spond to higher salt concentrations or stress by altering their secondary
metabolites production and accumulation. Induced synthesis of several me-
tabolites such as polyphenolics and flavonoids on different cyanobacteria
Hapalosiphon intricatus, Anabaena doliolum and Oscillatoria acuta in response
to different salt concentrations and the development of stress derived anti-
oxidant properties were reported earlier (Singh et al., 2014). Similarly, salt-
induced accumulation of carbohydrates is a main property exhibited by
cyanobacteria in response to salinity stress. The increase in carbohydrates
such as trehalose, sucrose, etc. can protect the cyanobacteria from salt stress
(Leema et al., 2010; Markou et al., 2012). The Spirulina maxima strains were
grown under salt stress for increasing the auto-fermentative ability and
product yield exhibited a 121 fold increase in ethanol after increasing the
salt concentration from 0.24 to 1.24 M NaCl (Carrieri et al., 2010). The ef-
fect of salt stress on growth and complementary chromatic adaptation in fil-
amentous cyanobacterium Fremyella diplosiphon was investigated by Singh
and Montgomery (2013) and it was found out that NaCl at 200 mM concen-
tration can inhibit the growth and reduce chlorophyll content, along with
the decreased accumulation of phycoerythrin and phycocyanin in the
green and red light respectively. The cell morphology was also affected
by NaCl concentrations, where the size of the cell increased with the in-
crease in salt concentration.
Chief metabolic activities like respiration and photosynthesis are
mainly dependent upon temperature and light, and hence these factors
can affect the growth and metabolite composition in cyanobacteria. Studies
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regarding the effect of temperature on the cyanobacterium Spirulina
platensis revealed the production of extra polymeric substance (EPS). The
three temperature variables considered were 30, 35 and 40 °C and the max-
imum growth was exhibited at 30 °C and the maximum production of EPS
was observed at 33 °C to 35 °C. The studies suggested that the increase in
EPS production just after the growth reduction might have been a response
to the oxidative stress confronted by the cyanobacterium (Trabelsi et al.,
2009). The effect of temperature on Spirulina platensis was evaluated by
Colla et al. (2007) and observed a negative effect on growth at 35 °C. How-
ever, at the same temperature an increase in the production of protein, phe-
nolic compounds and lipids was witnessed by (Colla et al., 2007).
Cyanobacterium Synechococcus sp PCC 7002 grown in medium containing
nitrogen as the only nutrient source, when exposed to 15 °C caused the
change of colouration of cells from blue-green to yellow-green, declined
the growth rate, and decreased the phycobiliproteins content as well as
the chlorophyll content. After transferring the same culture to 38 °C temper-
atures, it regained the inherent blue-green colour and normal growth
within 7 h of the exposure period. The change in pigment composition in
response to different ranges of temperature in a thermotolerant cyanobacte-
rium Oscillatoria sp. N9DM was investigated (Singh et al., 2012). It pro-
duced phycocyanin and phycoerythrin at 30 ± 2 °C and 55 ± 2 °C
respectively and phycoerythrocyanin was produced at an intermediate tem-
perature of 42 ± 2 °C. Here, a correlation between temperature and its ef-
fect on the complementary chromatic adaptation of cyanobacteria is
explicated.
The ability of cyanobacteria to accumulate various compounds in re-
sponse to the stress conditions due to the depletion or augmentation in nu-
trients is well investigated. The study on the influence of metabolic stress
condition by nutrient enrichment on Arthrospira platensis FACHB-314 was
examined by providing sodium glutamate and succinic acid as nitrogen
sources. The results demonstrated an increase in biomass productivity
and phycobiliproteins content at concentrations of 5 mM and 7.5 mM
glutamic acid and succinic acid respectively (Manirafasha et al., 2018). Ni-
trogen and phosphorous are the two essential nutrients, which are having
major roles in the growth and metabolism of cyanobacteria and hence, var-
iations in these nutrients can influence the biochemical composition of
cyanobacteria (Shanmugam et al., 2020). The four species of Microcystis
(M. aeruginosa, M. novacekii, M. protocystis and M. panniformis) was tested
for their response to various levels of nutrients especially nitrogen and
phosphorous. The results revealed that the Microcystis sp. grown on
nutrient-poor medium support cell growth and more lipid productivity
when compared to the control Cordeiro et al. (2017). During nitrogen star-
vation, acetyl Co-A synthesis and degradation of membrane lipid in to tri
acyl glycerol (TAG) will occur. Hence, a decrease in the nitrogen content
in growth medium can induce the lipid production in cyanobacterium
(Shanmugam et al., 2020). The outcome of this experiment gives a lead in
to the chances of a cost-effective strategy for cyanobacterial cultivation in
bio-fuel aspects. In the case of Anabaena NCCU-9, a well-studied nitrogen-
fixing cyanobacterium, maximum phycobiliprotein productivity was exhib-
ited, when cultured in the nitrogen-free medium along with 10 mM sodium
chloride (Hemlata and Fatma, 2009). In a nutrient stress analysis on
Spirulina platensis UTEX 1928, under nitrogen starvation conditions, the
biomass productivity of the cyanobacterium increased and total lipid con-
tent also elevated after a small gap period of initial decrease. Another
study on Spirulina platensis under nitrogen deprived condition has shown
limited growth and biomass production with increased carotenoid and
lipid content at both the optimum and higher concentrations of nitrogen
(El Baky et al., 2020).
5. Downstream processing: feasibility through cost-effective strategies
While comparing the economy of different stages of a biorefinery, the
cost of downstream processing is much greater (more than 50%) than the
cyanobacterial production cost (Mitra and Mishra, 2019). The cost of bio-
mass production and downstream processing can be mitigated by adopting
low-cost harvesting, cell disruption and extraction techniques and by
S. Prabha et al.
making use of the state-of-the-art technologies for multi-product recovery
along with the sequential extraction of different value-added products
(Costa et al., 2020; Mitra and Mishra, 2019).
5.1. Biomass harvesting: approaches for overcoming the challenges through
economic feasibility
Up to 30% of the total cost for the production of biomass is claimed to be
contributed by the techniques employed for harvesting/dewatering the bio-
mass. The harvested biomass is generally used for the extraction of several
products ranging from high valuable metabolites to low value compounds,
and hence the extraction procedure should be wisely chosen, depending on
the end product under consideration. Hence, an ideal technique to harvest
the biomass, which is further used for the extraction of a wide range of com-
pounds, is yet to be determined (Barros et al., 2015).
Albeit the wide range of applications, the key reason behind the failure
of a cyanobacterial biorefinery is the economic viability, especially for
the harvesting process. There are a lot of strategies that have been intro-
duced for the cost-effective harvesting of cyanobacteria (Dassey and
Theegala, 2013). It is important to check whether the selected harvesting
method allows the proper recycling of the medium (Barros et al., 2015).
Several methods have been introduced for the effective separation of
biomass from the growth medium. It includes flocculation (chemical floccu-
lation, bio-flocculation/auto-flocculation), flotation, gravity separation,
electrical-based separation, filtration and centrifugation are some of the
harvesting methods (Labeeuw et al., 2021). The large scale application of
any of these methods for the effective harvesting of biomass is always hin-
dered by the cost of production. The fully effective centrifugation method is
not at all feasible for harvesting biomass on an industrial scale (Tan et al.,
2020). A two-step method consisting of concentrating the biomass followed
by dewatering can be employed for the harvesting. Selection of the suitable
harvesting method with low operational cost and energy expenditure, for
the retrieval of a comparatively small amount of biomass from a large vol-
ume of the growth medium, is extremely important for the success of a
cyanobacterial biorefinery.
Flocculation techniques are the most common, effective and economi-
cally feasible technique, which can be used for the initial dewatering pro-
cess. Pre-concentration of cells by flocculation followed by removal of the
remaining water from concentrated biomass by centrifugation method is
acceptable, as it consumes a little energy and can reduce the cost of harvest-
ing (Vandamme et al., 2013). Various approaches have been introduced for
increasing the efficiency of flocculation methods to achieve maximum sep-
aration of biomass with minimum cost (Nguyen et al., 2019a). Flocculation
is achieved by the addition of a chemical compound, called flocculant,
which alters the chemical environment surrounding the cells, and thus
helps to overcome the repulsion between them, and consequently form ag-
gregates of cells within the medium (Schlesinger et al., 2012). An ideal floc-
culent should be less toxic, less expensive, and more effective which allows
the media to recycle again. Although there are a wide range of flocculants
proved to be effective for the harvesting of cyanobacteria, many of them
are having certain drawbacks which hinder their prospects of commercial
utilization. The classes of chemical flocculants include metal salts like mul-
tivalent iron and aluminium salts, which usually require higher doses to
flocculate the cyanobacterial biomass. Even though, these types of metal
ions as flocculating agents are fair enough to flocculate the biomass, traces
of them will remain within the medium, after flocculation and thus inter-
fere with the recycling process (Matter et al., 2019). The formation of
flocs induced by the increase in pH of the medium, with high flocculating
efficiency was demonstrated in different microalgal species (Wu et al.,
2012). The high pH in the growth medium can be induced by using several
cheap hydroxides, such as slaked lime, which evoke considerable effects
(Branyikova et al., 2018).
Bio-flocculating agents, and bio-polymers which are more effective with
less toxicity and environment friendly, had gained much attention nowa-
days. The market price of a non-toxic, bio-degradable polymer called
Gamma PGA (Poly glutamic acid) is 5 US$ kg−1, which can be used for
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the successful harvesting by flocculation of almost 45,000 L of microalgal
suspensions with 90% flocculating efficiency without the lysis of cells
(Zheng et al., 2012). Another positively charged biopolymer for neutraliz-
ing the negatively charged cyanobacterial cell surface for an effective floc-
culation process is chitosan. Even though this polymer is very effective, but
it is more expensive (Branyikova et al., 2018). Hence, an alternative to chi-
tosan is cationic starch, which is not only effective at lower doses but also
acts on a broader pH range than chitosan (Hansel et al., 2014). The cyano-
bacterium Microcystis aeruginosa has been known to be a bloom producing
prokaryote, which is found to be difficult to separate from the surrounding
medium. The introduction of a novel technique using human urine, to floc-
culate this cyanobacterium makes sense while dealing with the hard–to–
remove blooms. A large amount of urea present in the human urine can
be hydrolyzed into ammonia, by several microorganisms, during storage.
Ammonia is regarded as the best flocculant, which can be used for biomass
harvesting. On the other hand, to enhance the flocculation process, the ex-
tracellular polymeric substance (EPS) present in the walls of the microor-
ganisms, which hydrolyze the urea, can also help in the flocculation
process (Wang et al., 2018). Hence, this method can be regarded as a
good choice for the effective removal of cyanobacteria from the medium.
Reusability of culture medium, which is left behind, after the successful
harvesting of biomass would improve the sustainability of cyanobacterial
based biorefinery. Even after the recovery of biomass, the media used for
the cultivation would still contain a considerable amount of nutrients.
Thus, it is essential to retrieve the nutrients and recycle the medium to re-
duce water usage and nutrient cost (Johnson et al., 2018; Schlesinger
et al., 2012). The pH-induced flocculation strategies are much preferred
to ensure the reusability of the growth medium owing to the absence of in-
terference from the addition of flocculating agents. The nutrient cycling
process can be adopted for attaining economic feasibility for large scale
cyanobacterial cultivation. Wu and co-workers adopted the pH induced
flocculation by increasing the pH followed by reusing the growth medium,
neutralizing the pH and by supplementation of exhausted nutrients. The
second batches cultivated on the used medium exhibited growth pattern
similarity with the first batch cultivated in the fresh medium, indicating
the potential of recycling medium (Wu et al., 2012). Nitrogen and phospho-
rous are the major macronutrients used for the effective growth of
cyanobacteria and will be the main concern of media recycling idea
(Johnson et al., 2018). Andrade et al. (2019) carried out 4 cycles cultivation
of Spirulina sp. LEB 18 using recycled Zarrouk's medium in a raceway biore-
actor for 7 days. The cultivation was successful with high contents of carbo-
hydrate (58%), phycocyanin (2.47 mg mL–1) and saturated fatty acids
(60.53%), obtained in the 3rd, 4th and 1st cycles respectively. The semi-
continuous cultivation of Spirulina sp. LEB 18 by recycled media (using
zarrouk medium) with lower nutrients, without the renewal of medium,
were carried out by (Priscila et al., 2015). The cyanobacterium exhibited
a reasonable growth and biomass productivity (48.03 mg−1 L–1 d−1)
with high protein content (46.10 ± 2.59%) till 100 days from the day of in-
oculation and hence the cultivation reduces the production cost.
Auto-flocculation is caused by the extra polymeric substance secreted
by the cyanobacteria itself, and the bioflocculation involves the participa-
tion of other microorganisms for the flocculation of cells within the me-
dium (Barros et al., 2015). These chemical-free, low cost, low-energy,
non-toxic methods without the use of any flocculants can be utilized for
the effective flocculation and harvesting of cyanobacteria for the sustain-
able bio-fuel and bio-product applications (Iasimone et al., 2020). The
auto flocculation can be occurring naturally when the carbon dioxide in
the medium decreases, due to the active photosynthesis of cyanobacteria,
and thereby increasing the pH. The process of auto-flocculation can be in-
creased by the addition of low-cost products such as Sodium hydroxide
(NaOH). Bioflocculation which is induced by microorganisms has been suc-
cessfully used for wastewater treatment and thus separation of biomass.
Nevertheless the drawbacks associated with this type of flocculation is the
need for an additional substrate as growth requirements for microorgan-
isms and the competing growth of several other unwanted microorganisms
which can lead to the contamination of the biomass (Lee et al., 2009).
S. Prabha et al.
Mode of biomass mixing in cultivation can influence the flocculation of
filamentous cyanobacteria to a particular extent. Two cultivation systems
based on air-bubbling and shaking of biomass mixing has been monitored
for the floc formation and biomass settling properties and it was found
that both of them enhanced the two different modes of flocculation behav-
iours. Smaller and densely packed flocs, which indicate the fastest setting
biomass, were observed for air-bubbling based cultivation, whereas large
and week flocs, indicated the slowest settling cells, which were exhibited
by the shaking based biomass mixing cultivation system (Branyikova
et al., 2018; Iasimone et al., 2020). The major advantage of these types of
flocculation is medium recycling (Barros et al., 2015).
However, the success of medium recycling and cultivation strategies
would depend on a few variables, such as the production of several auto-
inhibitory organic compounds by different cyanobacteria and excessive
growth of several unwanted microorganisms such as bacteria and fungi in
the growth medium. Elimination of those compounds by proper steriliza-
tion can avoid these problems to a greater extent (Markou, 2014).
5.2. Cell disruption and extraction of value-added compounds
Most of the value-added products in cyanobacteria reside inside the
cells and thus efficient cell disruption techniques should be employed for
the effective and complete extraction of the desired bioactive compounds.
The efficacy of cell disruption methods cannot be compromised, since the
primary goal is the maximum retrieval of desired compounds (Shehadul
Islam et al., 2017).
Like other prokaryotes, the cell wall of cyanobacteria is made up of an
outer membrane rich in lipopolysaccharides and an inner layer of peptido-
glycan (Noreña-Caro and Benton, 2018). A basic understanding of the
cyanobacterial physiology and ultrastructure of cell wall components will
be helpful for the selection of suitable methods for the lysis of cells (Lee
et al., 2017b). The selection of a lysis method will depend upon the nature
of the desired bioactive compound. Hence, appropriate cell disruption tech-
niques, without compromising the stability of high-value end products
should be the prime consideration while choosing the lysis method (Mitra
and Mishra, 2019). Recent reports indicate that low-throughput methods
such as sonication, lyophilization of biomass followed by extraction of com-
pounds using suitable solvents, disruption of cells by mechanical means,
etc., is adequate to obtain effective lysis of cyanobacterial cell wall
(Mehta et al., 2015).
5.2.1. Cell disruption methods
There are several established methods for the lysis of cyanobacterial cells
for the effective extraction of various bioactive compounds. Lytic techniques
include both mechanical and non-mechanical methods, which can be done
either on a macro or micro scale. Mechanical or physical methods are usually
of higher cost and are used for large scale operations, while biological and
chemical methods, which involves the non-mechanical lysis methods, are em-
ployed for small scale purposes (Shehadul Islam et al., 2017).
Different lysis methods are preferred depending upon the targeted prod-
ucts under consideration. Depending on the cell wall nature of different
cyanobacteria, the techniques used for the lysis of their cells will be also dif-
ferent. Fig. 3 shows different techniques usually involved in the cell disrup-
tion and extraction of value added products from cyanobacteria. Most of the
metabolites resides inside the cell membrane, and they will be accessible by
breaking the cell wall. Although, mechanical methods such as freezing-
thawing, sonication, homogenization, bead milling etc. have low opera-
tional cost and facilitate efficient cell lysis, but certain drawbacks are also
associated with their practical application in large scale product recovery.
Mixing of the desired product with cell debris and other unwanted cell com-
ponents and the heating of sample can lead to the poor quality and degra-
dation of desired product (Manirafasha et al., 2016; Shehadul Islam et al.,
2017). Liquid extraction is mainly used for the extraction of bioactive com-
pounds from cyanobacteria and the choice for the solvent depends on the
polarity of target compound under consideration. The extraction of
phycobiliproteins were done using aqueous solvents and for bio-fuels and
10
Science of the Total Environment 814 (2022) 152795
many other bio-products, extraction is done using organic solvents
(Noreña-Caro and Benton, 2018).
The pulsed electric field technique (PEF) has been renowned as one of
the major fast mechanical lysis methods for the extraction of various metab-
olites such as lipids, proteins and carotenoids from cyanobacteria. The tech-
nique uses an electric field to increase the permeability of the cell
membrane with irreversible pore formation and subsequent release of com-
pounds from the cell (Lam, 2017). The technique becomes prominent, be-
cause of its low cost, high efficiency and less time consumption and eco-
friendly nature (Corrêa et al., 2020). Jaeschke et al. (2019) compared the
efficiency of two lysis methods (Bead milling and PEF treatment) for the ex-
traction of proteins from Arthrospira platensis. Although PEF and bead mill-
ing obtained the same amount of extracted phycobiliproteins, PEF was
observed to be more efficient, because of the better selectivity of this tech-
nique to obtain a more purified compound of interest.
Various chemicals were effectively used for the cyanobacterial cell lysis.
Chemical lysis can be either alkaline lysis or detergent lysis. Rather than al-
kaline lysis, detergent lysis is mostly used for the extraction of
cyanobacterial cell disruption. Sodium dodecyl sulfate (SDS), Triton X-
100, NP-40, Tween, Cetyl trimethyl ammonium bromide (CTAB) etc., are
the main detergents used for the lysis of cyanobacterial cells. They can
be, anionic, cationic, non-ionic or zwitter ionic. The problems associated
with the chemical lysis method are its low efficiency, incomplete lysis
and the difficulty in the removal of chemicals used for the extraction or
lysis (Shehadul Islam et al., 2017).
Different enzymes are also found to be effective for the successful dis-
ruption of cyanobacterial cells. The main advantages of enzymatic lysis
are their selectivity in action, lack of any byproduct formation, energy effi-
cient, stable and environment friendly. Thus, enzymatic cell lysis is less en-
ergy consuming and effective than many of the chemical and mechanical
means. The main drawback is the high cost of enzymes, which would
make this technique expensive (Demuez et al., 2015). The shortcomings
left behind by the complete chemical and enzymatic lysis can be solved to
a greater extent by an alternate chemical cocktail treatment designed espe-
cially for the lysis of cyanobacteria. A chemical cocktail consist of a deter-
gent; EDTA (increase the permeability of the membrane), Spermin and
DDT (decrease the strength of external layer) and lysozyme (for break
down the peptidoglycan layer) was considered and it was found to be effec-
tive and exhibit complete lysis of cyanobacterium Synechococcus sp. PCC
7942 without any damage to DNA, RNA and protein. The method was
found to be convenient, effective and reliable for the lysis of cyanobacte-
rium (Mehta et al., 2015).
Although there are innumerable choices for the cell disruption of
cyanobacterial cells, for the extraction of desired compounds, care should
be taken while selecting the most appropriate methods for the lysis after
considering the cell wall characteristics of cyanobacteria and the desired
product under consideration.
5.2.2. Extraction of bioactive compounds
Each cyanobacterium can produce a collection of bioactive compounds,
including several compounds which can act as alternatives to toxic
chemicals in food and pharmaceuticals, non-degradable plastics and
fossil-based fuels (Meixner et al., 2018). However, while checking the eco-
nomics of commercial production of any of the single products, one may
often find that the feasibility is still debatable due to the unbalancing be-
tween the cost of upstream and downstream processing against the market
value of an individual compound. Economic feasibility can be achieved by
decreasing the upstream and downstream costs. If we tend to concentrate
on the extraction of more than one value-added product at the same time
from the same harvested cyanobacterial biomass, the cost of production
can be successfully balanced against the economic value and thus increase
the economic feasibility (Chandra et al., 2019b). This can be achieved by re-
covering multiple products from a single cyanobacterium, and sequential
extraction of those products and development of an integrated downstream
process unit, which decrease the downstream processing steps and increase
the product recovery rate.
S. Prabha et al.
Fig. 3. – Techniques involved in the cell disruption and extraction of value added products from cyanobacteria.
5.2.3. Multiple product recovery with sequential extraction of products
It has been already discussed the strategies to enhance the production of
bioactive compounds along with the simultaneous accumulation of other
value-added compounds in cyanobacteria. Apart from its implementation,
successful recovery of maximum compounds from cyanobacteria, using
state-of-the-art technologies can considerably aid in the establishment of a
sustainable biorefinery (Chandra et al., 2019a). Most of the researches are fo-
cusing on single product recovery, which fails to valorize the biomass
completely (Mitra and Mishra, 2019). Most cyanobacterium contains an
11
Science of the Total Environment 814 (2022) 152795
abundant percentage of water-soluble membrane-bound protein pigments
called phycobiliproteins in their biomass. Hence, the primary target from a
cyanobacterial biorefinery would be the phycobiliproteins (Tavanandi and
Raghavarao, 2018). Recent researches have been engaged in the sequential
extraction of various products from cyanobacteria according to an order of re-
covery of high to low market value products. The mode of sequential extrac-
tion has been analyzed in detail in Spirulina platensis (Chaiklahan et al., 2018).
They experimented with several sequential extractions for the recovery of
lipids/Total fatty acids (TFA), polysaccharides and phycocyanin from
S. Prabha et al.
S. platensis and reported that the stepwise recovery of products in an order of
primary extraction of phycocyanin, followed by lipid/TFA and then polysac-
charides give an appreciable yield. While considering the economics of whole
steps involved in the extraction, it was revealed that the cost of recovery of
phycocyanin is less than the market value and hence, being a high-value
product, the individual recovery of phycocyanin itself is much feasible. Al-
though extraction of co-products can further add substantial value to the pig-
ment extracted residue, the commercial feasibility is contentious owing to the
high cost of extraction solvents and low product recovery. One of the solu-
tions to alleviate this problem is to concentrate on cost-effective strategies
for the higher production of cyanobacterial compounds. Table 2 shows differ-
ent metabolites produced by selected strains of cyanobacteria, solvents in-
volved in extraction and associated details. The data gives the detailed
information regarding the value-added products which can be possibly re-
trieved from some selected species of cyanobacteria. From the table, it is
clear that some of the cyanobacterium can produce multiple value-added
products, in a reasonable amount. Sequential extraction of products based
on their high to low market value can retrieve most of the value added metab-
olites. Even though, the economic feasibility can only be predicted by con-
ducting detailed feasibility studies among each cyanobacterium.
A detailed protocol for the sequential extraction of UV-protectant-
Mycosporine like amino acid (MAA) followed by lipids from Lyngbya sp.
was described by Chandra et al. (2019a) and they successfully extracted
lipid from the residual biomass after extracting MAA. They established an en-
hanced accumulation of lipid (PUFA- nutraceutical valuable) and saturated
lipids (application in biofuel field) as a result of the exposure to UV radiations
(UVA/UVB). They also reported a 50% increase in production of MAA in UVB
exposed cells than UVA treated cells as well as control. This can be demon-
strated as a practical approach for the accumulation of the desired compound
and its simultaneous recovery along with another high value compound.
The possibility of sequential extraction of phycocyanin followed by
chlorophyll pigment was investigated by Sintra et al. (2021) in Anabaena
cylindrica and Tavanandi and Raghavarao, in (2018). in Spirulina platensis.
From Anabaena cylindrica, Sintra and co-workers recovered 90% of C-PC
and 55% of the total chlorophyll using Na-Phosphate buffer and an aqueous
solution of surface-active compounds along with ethanol respectively. On
the other hand, Tavanandi and Raghavarao, (2018) proposed an optimized
extraction process for the separation of chlorophyll from the spent biomass
of Spirulina platensis, after the separation of phycocyanin, by using 100%
ethanol, which is a food-grade solvent.
In conclusion, to make a sequential extraction procedure to be a success,
further research should focus on comparing the lone individual extraction
of compounds with different combinations of sequential extractions. This
should be done along with the detailed analysis of cost during each combi-
nation and thus finally identifying the most economically feasible step. Ide-
ally, compounds which are having good market value and less stability,
such as phycobiliproteins can be primarily recovered from the
cyanobacterial biomass followed by the MAA, lipid and carotenoids and
polyhydroxylalkanoates (PHA), etc.
6. Advancements of nanotechnology in cyanobacterial biorefinery
Recent works have demonstrated the successful applications of nano-
technology in various fields of biology including clinical diagnostics, drug
delivery, therapeutics and so on. The anti-microbial activities of nanoparti-
cles, particularly helped in achieving this outputs. (Pathak et al., 2019).
Also, metal nanoparticles can be used for upstream and downstream pro-
cessing in cyanobacterial biorefinery. The following sections details a
closed-loop approach for the synthesis and utilization of cyanobacteria
based nanoparticles. Application of different nanoparticles in the down-
stream processing involved in cyanobacterial biorefinery is also discussed.
6.1. Role of nanoparticles in cyanobacterial biorefinery- upstream to downstream
Novel strategies and technologies for cost-effective harvesting and
downstream processing are particularly important for the success of a
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Science of the Total Environment 814 (2022) 152795
cyanobacterial biorefinery. Different metal nanoparticles have proved
their practicability in the cyanobacterial harvesting processes, accumula-
tion of bioactive compounds and cell disruption. The major advantages in-
cludes, best performance, cost-effectiveness, fast nature, environmental
friendly approach and the recyclability (Nguyen et al., 2019b).
Several reports came up in the last decade on the action of nanoparticles
on the enhancement of growth and accumulation of bioactive compounds
in cyanobacteria. The nanoparticles in the culture medium induced
oxidative stress in the cyanobacterial metabolism, which resulted the inter-
ferences in signaling cascades subsequently leading to selective accumula-
tion of bioactive compounds to alleviate the metabolic stress. This
mechanism of defense induce the formation of anti-oxidant compounds
along with the enhancement in the growth of cyanobacteria (Miazek
et al., 2015; Nguyen et al., 2019b). Padrova and co-workers reported the
positive effect of zero-valent iron nanoparticles on the growth of cyanobac-
terium Arthrospira maxima (Pádrová et al., 2015). An optimum concentra-
tion of 5.1 mg L–1 iron nanoparticle in zehnder medium increased the
growth, along with an enhancement in lipid production. However, it de-
creased the production of saturated fatty acids and boosted the accumula-
tion of polyunsaturated fatty acids in cyanobacterium Arthrospira and a
few green algae. Similarly, the role of nanoparticles in light management
in photobioreactors was investigated by Ooms et al. (2015) in which they
have introduced a plasmonic nano-patterned reflective surface equipped
with plasmonic nanodisks in the photobioreactor to address the challenges
in light management. Their results revealed a significant increase in the
growth rate of cyanobacterium Synechococcus elongatus cultivated in the
modified photobioreactor as compared to the photobioreactor equipped
with untreated glass.
The interaction between nanoparticle and cells is a very critical precon-
dition for the harvesting and dewatering of cyanobacteria. The harvesting
efficiency of cyanobacteria Microcystis aeruginosa was enhanced by using
polyethylenimine (PEI) coated magnetic nanoparticles (Yang et al., 2018).
The interaction between iron nanoparticle coated with PEI was studied
and the harvesting efficiency of cyanobacterium with and without PEI
coats were compared. It was found that coated iron nanoparticles showed
much better harvesting efficiency.
An economically viable integrated downstream strategy by simulta-
neous recovery and harvesting of microalgae using a tri-functional biopoly-
mer nano-composite and a chitosan –TiO2 conjugated particle was
proposed by Dineshkumar et al. (2017). The optimum concentrations of
chitosan, chitosan-TiO2 conjugated particles and magnetic nano-particles
needed for the process were identified as 0.11, 0.09 and 0.07 g g−1 respec-
tively, and the harvesting efficiency was found to be nearby 98%. TiO2
hampered photo-catalysis mechanism was in operation when exposed to
UV radiation and was used for the disruption of cells. Nearly 97% of the in-
tracellular lipid and leutin were successfully recovered by this method and
was compared with the ultra-sonication method as control. Another inte-
grated approach for simultaneous harvesting and cell disruption has been
demonstrated by Seo et al. (2016). Fe3O4 nanoparticle decorated with cat-
ionic surfactant (CTAB) was successfully used for integrating two crucial
steps in downstream processing. The quarternary heads of the cationic sur-
factants not only help the negatively charged microalgal cells to flocculate
but also weaken the cell wall, to cause easy disintegration. The speciality of
iron oxide nanoparticles is that they can be recycled and can be further used
in the next downstream processing.
6.2. Cyanobacterial factories for the production of nanoparticles
Currently, there are nearly 1000 nanotechnology-based commercial
products available in the global market, which has been tremendously
increased during the past few years and is expected to reach up to three
trillion US $ annually (Pathak et al., 2019). As the demand for
nanotechnology-based products is increasing, the production may not be
limited and as a result, the chemical methods used for the synthesis of nano-
particles will discharge several harmful byproducts into the environment
(Hamouda et al., 2019). Moreover, available physical methods for the
S. Prabha et al. Science of the Total Environment 814 (2022) 152795

Table 2
Value added metabolites produced by several selected strains of cyanobacteria; solvents involved in extraction and associated details.
Cyanobacterial strain Value-added product Yield Extraction solvent Remarks References

Synechocystis sp. PCC poly-ß-hydroxybutyrate 38% of dry cell Chloroform, acetone, With Phosphorous deficiency (Panda and Mallick,
6803 weight (DCW) diethyl ether 2007)
Lipid 14% of DCW Chloroform, methanol (Cuellar-Bermudez
et al., 2015a)
Synechocystis sps Lipid 24.3% DCW Diethyl ether methanol (Ashokkumar et al.,
2019)
Lipid 14 mg g−1 Chloroform, methanol Extracted from the spent biomass
poly 6.0% DCW Chloroform, ethanol
(3-hydroxybutyrate)
Chlorophyll 0.33–2.45 mg g−1
Synechocystis salina Acetone, methanol, (Meixner et al., 2018)
Carotenoids 0.26–1.87 mg g−1 Extraction carried out in dark at 4 °C
ethanol
Phycocyanin 123 mg g−1
Proteins 242 mg g−1 Extracted from the spent biomass
Carbohydrates 6.1 mg g–1 Phenol-sulphuric acid
Carbohydrate 27% w w−1 Sulphuric acid
(Ashokkumar et al.,
Synechocystis sps Bioethanol 0.18 g g −1 Sulphuric acid. Yeast
2019)
solution
Synechococcus nidulans Chlorophyll a 5.5 μg mL–1 Acetone, methanol, (Duarte et al., 2019)
LEB 115 Carotenoid 2.0 μg mL–1 ethanol
Lipid 13.62% DCW Obtained under green light
Isobutanol 450 mg L–1
Synechococcus Isobutyraldehyde 1100 mg L–1 (Lau et al., 2015)
elongatus PCC 7942 1 butanol 29.9 mg L–1
Ethanol 230 mg L–1 (Lau et al., 2015)
Lyngbya sps MAA⁎ 17.40 mg mL–1 Chloroform, methanol Obtained after exposure to UV-B (Chandra et al., 2019a)
Lyngbya sp. CU2555 MAA (Rastogi and
Incharoensakdi, 2014)
Phycoerythrin 27.47 mg mL–1 Concurrent fractionation
Potassium phosphate
Lyngbya sp. A09DM Phycocyanin 20.43 mg mL–1 Concurrent fractionation (Sonani et al., 2014)
buffer
Allophycocyanin 6.55 mg mL–1 Concurrent fractionation
Lyngbya sps Lipid 30.40% DCW Chloroform, methanol Extracted From the spent biomass (Chandra et al., 2019a)
Obtained after exposure to UV-A
Lyngbya sp. CU2555 Scytonemin Methanol, ethyl ace-tate (Rastogi and
Incharoensakdi, 2014)
Oscillatoria C-Phycocyanin 2 mg g −1 Tris-cl buffer Obtained after anion exchange chromatography (Soni et al., 2006)
quadripunctulata
C-Phycocyanin 62.54 mg mL−1
With 0.3 M of glycine and 5 mM of sodium glutamate
C-Phycoerythrin 2.7 g−1 Phosphate buffer
Oscillatoria sp. supplementation in culture
C-Allophycocyanin 224.02 mg L–1 (Tiwari et al., 2019)
BTA170
Lipid 22.31% DCW
Carbohydrate 32.25% DCW
Carbohydrate 38% DCW Sulphuric acid Culture grown in BG11, pH 8, temp 32 °C
Lipid 43% DCW Chloroform, methanol Culture grown on synthetic waste water with NaHCO3
Plectonema terebrans
(0.6 g/L),
BERC10 (Shahid et al., 2021)
Phycocyanin 154.9 mg g−1 Culture grown on synthetic waste water
Allophycocyanin 37.1 mg g−1 Phosphate buffer Culture grown on synthetic waste water
Phycoerythrin 9.1 mg g−1 Grown on BG-11 medium
Nostoc muscorum Polyhydroxybutyrate 6.44% w w−1 Methanol and acetone (Khajepour et al.,
NCCU-442 2015)
Nostoc sp. Strain Phycoerythrin 0.193 mg mL–1 Phosphate buffer Gel filtration chromatography (Kannaujiya and Sinha,
HKAR-11 Phycocyanin 0.100 mg mL–1 Gel filtration chromatography 2016)
Nostoc sp. NK (KCTC Phycocyanin 57.43 mg L–1 d–1 Na-phosphate Under the red lamp condition. (Lee et al., 2017a)
12772 BP)
−1
Nostoc flagelliforme Carotenoid 1.84 mg g Grown under purple light (400 nm) (Han et al., 2017)
Nostoc Linckia 0.17 mg g−1 Culture exposed to 0.01–1 μM IBA (Mansouri and
Talebizadeh, 2017)
ß-carotene 225.44 μg g−1
Lutein 117.56 μg g−1
Zeaxanthin 88.46 μg g−1
Ethyl acetate, methanol (Rodrigues et al.,
Echinenone 79.07 μg g−1
2015)
Myxoxanthophyll 18.83 μg g−1
Canthaxanthin 1.89 μg g−1
P.autumnale C-Phycocyanin 2.05 × 105 μg g−1 Phosphate buffer
0.322 mg mL–1 Tris-cl buffer After hydrophobic interaction chromatography (Soni et al., 2008)
Chlorophyll a 2.700 μg g−1 Acetone, petroleum ether, (Rodrigues et al.,
diethyl ether 2015)
SFA⁎ 45.20% DCW Chloroform (Siqueira et al., 2018)
MUFA⁎ 34.70% DCW Chloroform
PUFA⁎ 19.90% DCW
Anabaena variabilis Phycoerythrin 400 μg mL–1 Acetate buffer After anion-exchange chromatography (Chakdar and Pabbi,
CCC421 2012)
Total lipid (TL) 46.9% DCW
Anabaena variabilis. SFA⁎ 50.97% of TL Chloroform, methanol Upon ultrasonic treatment (Han et al., 2016)
MUFA⁎ 33.79% of TL

(continued on next page)

13
S. Prabha et al.
Table 2 (continued)
Cyanobacterial strain Value-added product Yield Extraction solvent
PUFA⁎ 15.23% of TL
Anabaena doliolum Chlorophyll a 7.9 mg g−1
Anabaena doliolum Carotenoid 52 mg g−1
Anabaena cylindrica Hydrogen 2.1 μmol mg−1
IAMM-1
⁎ SFA- Saturated fatty acid; MUFA- Mono unsaturated fatty acid; PUFA- Poly unsaturated fatty acids; MAA- Mycosporine like amino acid.
production of nanoparticles are very expensive. At this point, alternate
methods for the synthesis of nanoparticles, with the aid of green chemistry,
is essential for the production of safe, effective, bio-degradable,
environment-friendly biogenic nanoparticles. Although, several plant-
derived compounds were found to be exhibit the capability of reducing dif-
ferent metals for the formation of nanoparticles, prospects of utilizing mi-
crobial metabolites on an industrial scale have been suggested only
recently(Husain et al., 2015). In this context, cyanobacteria can be regarded
as the most effective biological system for the synthesis of nanoparticles,
due to the presence of several compounds like polysaccharides and
phycobiliproteins that are capable of the reduction of metal ions, for the
synthesis of respective metal nanoparticles (El-Naggar et al., 2017).
6.2.1. Cyanobacteria mediated green synthesis of nanoparticles
The reduction of several metallic ions into nanoparticles by
cyanobacteria is the key point behind the green synthesis of nanoparticles
here. This phenomenon is explained by the activity of several reducing
agents such as Nicotinamide adenine dinucleotide phosphate (NADPH) or
NADPH dependent reductase, which are getting recycled by taking part in
several reactions like photosynthetic electron transport chain, mitochon-
drial electron transport chain, several redox reactions in the thylakoids
and cell membrane in the cytoplasm, etc. (Sharma et al., 2016).
Table 3 shows cyanobacteria mediated nanoparticle synthesis and asso-
ciated applications. Different cyanobacteria were reported to mediate the
green synthesis of several metal ion based nanoparticles with varying size
and shape. These cyanobacteria derived nanoparticles exhibited a plethora
of applications including antifungal, antibacterial, anticancer, wound
healing, catalytic activity as well as several other biomedical applications
(Sharma et al., 2016). Biosynthesis of the nanoparticle becomes economi-
cally more feasibile when the synthesis occurs rapidly in the reaction vessel.
In this regard, Mubarak Ali et al., synthesized silver nanoparticles using the
cyanobacterium Oscillatoria willei which reduced the silver ions in the silver
nitrate solution provided and stabilized the nanoparticles by using a se-
creted protein of its own (Mubarak Ali et al., 2011). The AgNO3 being a
toxic reagent, would kill the cyanobacteria and further Ag+ ions get re-
duced to nanoparticles and will be released from the dead cells of
cyanobacteria. The dead cyanobacteria also release proteins and other bio-
active compounds, which reduce silver ions outside the cells, or in the
solution.
Also the bioaccumulation of gold by cyanobacterium Plectonema
boryanum UTEX 485 was studied (Lengke et al., 2006). The mechanism be-
hind the bioaccumulation of nanoparticles is similar to the biosynthesis of
the nanoparticle by the reduction process. Cyanobacteria on interaction
with the gold (III) chloride initiate the formation of gold nanoparticles
and ultimately deposit the metallic gold in the form of octahedral platelets
in the solution and the walls of cyanobacteria. In general, the conventional
chemical methods adopted for the retrieval of metals from ore needs more
capital investments. Green technologies for the recovery of such metals are
environment friendly as well as inexpensive in nature. Similar studies
regarding the cyanobacteria mediated synthesis of gold nanorods and nano-
particles has been reported earlier, with Nostoc ellipsosporum, Synechocystis
sp. PCC 6803 and Anabaena sp. (Rösken et al., 2014;Parial et al., 2012b;
Focsan et al., 2011).
Bactericidal efficacy analysis of gold (Au) nanoparticle synthesized
through protein-mediated reduction using Spirulina platensis has showed
substantial antagonistic activity against two pathogenic microbes Bacillus
14
Science of the Total Environment 814 (2022) 152795
Remarks References
Culture grown in Molybdenum deficient media
(Lau et al., 2015)
subtilis and Staphylococcus aureus. The spherical gold nanoparticles with a
size of approx. 5 nm were enough to penetrate the peptidoglycan layer of
the bacteria to cause death (Uma Suganya et al., 2015). The silver nanopar-
ticles synthesized from Oscillatoria limnetica were analyzed for its anti-
bacterial activities against multidrug-resistant bacteria (Escherichia coli
and Bacillus cereus), and its anti-tumour activities against human breast
(MCF-7) and human colon cancer (HCT-116) cell lines. Similarly, Lengke
and co-workers reported the production of spherical nanoparticles, derived
from palladium, synthesized by using cyanobacterium Plectonema
boryanum, with aqueous solutions and minimize the waste byproducts
and associated pollution(Lengke et al., 2007). Green synthesis of silver
nanoparticles was reported by San Keskin et al. (2016), in which they ana-
lyzed the antibacterial activity and found that the nanoparticle inhibited
the growth of Bacillus subtilis ATCC-6653, Escherichia coli ATCC-10536
and Staphylococcus aureus ATCC-25923. Along with that, the photo-
catalytic ability of the nanoparticle was also examined, which revealed an
18% degradation of organic dye (Methylene blue) by photo-catalysis pro-
cess. Screening of different cyanobacteria, such as Anabaena sp.,
Aphanizomenon sp., Cylindrospermospsis sp., Lyngbya sp., Limnothrix sp.,
Synechocystis sp.,and Synechococcus sp. for the green synthesis of nanoparti-
cles was carried out by using their biomass as well as the cell-free medium
in which they grew. Results revealed the presence of nanoparticles in both
the test samples (using biomass and cell-free medium), which reveals the
involvement of a polysaccharide like an extracellular compound in the for-
mation of silver nanoparticles (Patel et al., 2015).
Anti-cancerous, anti-hemolytic and cytotoxic activities of nano-particles
derived from cyanobacteria were extensively studied (Hamouda et al.,
2019). Further, phycobiliprotein mediated synthesis of nanoparticles
gained more interest. Phycoerythrin extracted from the cyanobacterium
Nostoc carneum and phycocyanin from Nostoc linckia were considered for re-
ducing the silver for the fabrication of silver nanoparticles, which were fur-
ther examined for in-vitro anti-cancerous activity in mammary gland breast
cancer cell lines (MCF 7), human lung fibroblast (WI 38) and human am-
nion (WISH) and for in-vivo activity in Ehrlich ascites carcinoma bearing
mice. The results threw light in to the anti-tumour, anti-hemolytic nature
of silver nanoparticles which can have applications in pharmaceutical, cos-
metic, therapeutic and medical fields (El-Naggar et al., 2017, 2018). Re-
ports suggested that cyanobacteria can act as ‘nano factories’ for the
green synthesis of different metal-based nanoparticles, which is of great
utility in several disciplines of physics, chemistry and biology (Lengke
et al., 2007).
6.2.2. Cyanobacterial bloom to boon – a nanotechnological approach
Uncontrolled growth of cyanobacteria due to the nutrient enrichment in
water bodies create serious problems in water quality and the release of
cyanotoxins, leading to the deterioration of water bodies (Xu et al.,
2014). Effective management strategies should be initiated to deal with
the cyanobacterial bloom to prevent the damage of water bodies. However,
the approaches in managing cyanobacterial blooms all over the world pose
a serious challenge (Rosen et al., 2017). The following section discuss the
effective management of this crucial situation in the light of nano-
biotechnology.
There are several chemical and physical methods for the removal of the
cyanobacterial biomass from the affected water source. From the available
reports, it is evident that the flocculation based methods are the effective
and less expensive method for the successful removal of cyanobacterial
S. Prabha et al. Science of the Total Environment 814 (2022) 152795

Table 3
Cyanobacterial mediated synthesis of nanoparticles and their applications.
Cyanobacteria Nano-particle Size Shape Application References

Oscillatoria Silver 3.30–17.97 nm. Spherical Strong antibacterial activity against multidrug-resistant bacteria (Hamouda
limnetica (Escherichia coli and Bacillus cereus) et al., 2019)
Cytotoxic effects against both human breast (MCF-7) cell line and
human colon cancer (HCT-116) cell line
Spirulina platensis Gold ~5 nm Irregular Anti-bacterial activity against gram positive (Uma Suganya
Bacillus subtilis and Staphylococcus aureus. et al., 2015)
Oscillatoria willei Silver 100 nm to Spherical (Mubarak Ali
NTDM01 200 nm et al., 2011)
Synechococcus Silver ~140 nm. Spherical Anti-bacterial against B. subtilis, E. coli and S. aureus (San Keskin
sp. et al., 2016)
Nostoc carneum Silver 7.1–26.68 nm. Spherical Antibacterial, antihemolytic, in vitro and in vivo cytotoxic (El-Naggar
activities. et al., 2018)
Nostoc linckia Silver 9.39 to Spherical Antibacterial activity against pathogenic Gram-positive (El-Naggar
25.89 nm. (Staphylococcus aureus), Gram-negative bacteria (Pseudomonas et al., 2017)
aeruginosa, E. coli and Klebsiella pneumonia).cytotoxic activity
against MCF-7 breast cancer cell line
Anabaena sp. Silver 24.13 nm Irregular Antibacterial activity against (Patel et al.,
66–2 Bacillus megatarium (ATCC-13402), Escherichia coli (ATCC-10836), 2015)
Bacillus subtilis (ATCC-19162), Staphylococcus aureus (ATCC-29213),
Pseudomonas aeruginosa (ATCC-39324),
Micrococcus luteus (ATCC)
Synechocystis sp. Silver 14.64 nm Irregular Antibacterial activity against (Patel et al.,
48–3 Bacillus megatarium (ATCC-13402), Escherichia coli (ATCC-10836), 2015)
Bacillus
subtilis (ATCC-19162), Staphylococcus aureus (ATCC-29213),
Pseudomonas aeruginosa (ATCC-39324),
Micrococcus luteus (ATCC)
(Microchaete Silver 60 and 80 nm spherical, polydisperessed Azo dye decolorization ability (Husain et al.,
NCCU-342) 2019)
Phormidium tenue CdS 5 nm Spherical Pigments labelled CdS nanoparticles can be applied as bio-labels. (Mubarakali
NTDM05 et al., 2012)
Anabaena Gold 1–50 nm Spherical,Triangular,Irregular,Hexagonal Not mentioned (Roychoudhury
sphaerica et al., 2016a)
Plectonema Gold ∼10 nm to Octahedral platelets Can be used in bio-recovery of gold from gold(III)-chloride (Lengke et al.,
boryanum 6 μm solutions. 2006)
Lyngbya Gold – Silver ~5–25 nm. Spherical Might have biomedical applications (Roychoudhury
majuscula alloy np et al., 2016b)
Lyngbya Gold <20-nm-sized Spherical Bio-recovery of Gold (Chakraborty
majuscula et al., 2009)
Cylindrospermum Silver 38–88 nm Pentagonal Extracellular cell free biosynthesis (Husain et al.,
stagnale 2015)
NCCU-104
Microcoleus sp. Silver 44–79 nm Spherical Corynebacterium sp. (MTCC 3080), Proteus vulgaris (MTCC 426), (Sudha et al.,
Bacillus subtilis (MTCC 2393), S.aureus (MTCC 3160) and E.coli 2013)
(MTCC 4604).
Phormidium Gold 7.92–25 Spherical, triangular, hexagonal Applications in medical field (Parial et al.,
valderianum 2012a)
Nostoc Gold 20–40 nm DecahedralIcosahedral Novel technique for exclusively gold nanorods with an (Parial et al.,
ellipsosporum approximately uniform distribution of aspect ratio. 2012b)
Lyngbya Gold 2 nm −25 nm Spherical, Hexagonal Photomining of gold by live cyanobacterial biomass (Chakraborty
majuscula et al., 2009)

biomass (Branyikova et al., 2018; Eroglu et al., 2015; Lee et al., 2009; Salim
et al., 2011; Vandamme et al., 2013; Zheng et al., 2012). For the effective
recovery of cyanobacterial biomass in less time and with more specificity,
nanotechnology-based harvesting techniques can be employed
(Dineshkumar et al., 2017; Yang et al., 2018). A mixture of two amino
clay-based nano system was introduced by Ji et al. (2016), for the effica-
cious harvesting of cyanobacteria from the bloom. A well-organized net-
work of different sized magnesium and cerium amino clay-based nano-
system bridges between the cyanobacterial cells resulted in 100% harvest-
ing efficiency within one hour time. The recycling ability of this kind of
nano-systems increases its feasibility.
The harvested cyanobacterial biomass from the cyanobacterial bloom
can be either discarded or further utilized. The safe disposal of harmful
blooms is a global issue and should require more attention. Presently, the
advancements in different cyanobacteria derived nano-materials which
are exhibiting several applications in bio-medicine, and other applied sci-
ences is something that the researchers are looking forward with hope.
Lee et al. (2015), introduced a method for the safe removal of
cyanobacterial biomass by giving equal priority to environmental
sustainability as well as economic viability. They succeeded in the develop-
ment of a green carbon nano-tag (G-tag) from biomass, aiming at the devel-
opment of anti-cancer therapy. 100 g of nanotags were developed from
100 kg cyanobacterial biomass using an eco-friendly approach. The high
stability, solubility and low toxicity of the tag paved the way for the
development of a multi-functional T-tag conjugated with Doxorubicin and
can be further used as a fluorescent tag delivery system for sensing the
action of the Doxorubicin along with imparting enhanced anti-cancerous
activity to it.
By understanding the bright side of cyanobacterial blooms, MacÁrio
et al., (2021) proposed an innovative idea for the effective management
of cyanobacterial bloom. Most of the value-added products from
cyanobacteria possess high market value. Hence, from the perspective of
cyanobacterial biorefinery, the harmful cyanobacterial blooms can be effi-
ciently valorized to retrieve economically valuable products, which can
be marketed. The economic viability of the proposed idea has been vali-
dated, which revealed the potential for a positive return. On a logical
view, the income generated can be spent for the control and prevention
of further blooms. Fig. 4 shows the diagramtic representation of the

15
S. Prabha et al.
Fig. 4. – Diagrammatic representation of harvesting and valorization of cyanobacterial biomass from cyanobacterial blooms.
harvesting and valorization of cyanobacterial biomass from cyanobacterial
blooms.
7. Integrated approaches to increase the feasibility of multiple
product recovery
The maximum valorization of cyanobacterial biomass can be achieved
by the recovery of multiple products and thereby increase the economic fea-
sibility of the biorefinery. A conventional multi-product biorefinery is not
feasible due to the high expensive techniques involved in the production
and extraction. From the earlier reports, it is evident that cultivation to
downstream processing is expensive due to the lack of simplified technolo-
gies in processes for efficient harvesting, cell disruption and extraction. A
holistic approach towards cultivation and downstream processing and
thereby, decreasing the number of steps involved in the recovery of
targeted products in low cost without compromising the quality and recov-
ery efficiency are being investigated nowadays (Lam et al., 2018; Sataloff
et al., 2020; Van den Berg et al., 2019).
7.1. Integrated cultivation systems
Development of integrated cyanobacterial cultivation systems with
low production cost can be a breakthrough to increase the profit from
a biorefinery. An approach put forward by Harun et al. (2011) explains
the possibility of the integration of methane production with biodiesel
production to yield economic feasibility to the bio-diesel biorefinery
from microalgae. They propose the idea that methane produced by the
anaerobic digestion of microalgae can be used to generate electricity,
which can satisfy the energy requirement for further cultivation and
downstream processes for biodiesel production and substantially de-
crease the production cost by 33%. Several other groups demonstrated
the co-generation of electricity from integrated cultivation systems of
microalgae/cyanobacteria, thereby increasing the economic feasibility
of biorefineries. Nwoba et al. (2019) reported the possibility of an
16
Science of the Total Environment 814 (2022) 152795
integrated photo bioreactor associated with an operation unit for the ef-
fective utilization of solar energy to co-produce electricity, which can be
further used for running the production operations and thereby de-
crease the biomass production cost. Cultivation systems integrated
with wastewater treatment plants developed as a common strategy to
reduce the production cost for energy-based low-value products such
as bio hydrogen, biodiesel, bioethanol, etc. (Chavan and Mutnuri,
2019; Venkata Mohan et al., 2019). Hence, the techniques used for
reusing the nutrients (mainly nitrogen & phosphorous), in wastewater,
especially from the agro-industrial sectors, for the cultivation of
cyanobacteria may bring about a circular bio-economy. Cyanobacteria
can effectively utilize inorganic pollutants such as nitrogen and phos-
phorous from wastewater for their growth and thus increasing biomass
production. Hence, the removal of inorganic pollutants, which need
more expensive physico-chemical treatments, can be satisfied by the
cultivation of fast-growing cyanobacteria in wastewater, and thus an in-
teroperate approach that pave way to a circular bio-economy can be ful-
filled (Beyl et al., 2019).For the successful growth of specific
cyanobacteria in wastewater, the peculiarity and optimized growth con-
ditions and tolerance level of selected fast-growing cyanobacteria is pri-
marily identified before the pilot-scale cultivation (Markou and
Georgakakis, 2011).
Cultivation of different cyanobacteria in synthetic, municipal, agro-
industrial waste water has been proved effective and exhibited promising
results. The growth of Synechococcus sp. in anaerobic digestion effluent
(AD effluent) diluted with sea water was reported by Beyl et al. (2019).
The work demonstrated that the supplemention of MgSO4, to the effluent
can bring about a cost-effective, cultivation media for the effective growth
and biomass production of cyanobacteria (Beyl et al., 2019).
In this era of cyano-remediation, initiated with the aid of state-of-the-art
technologies, it is possible to extract value-added products from
cyanobacteria cultivated in waste water and thus can lead towards a cost ef-
fective cyanobacterial biorefinery. Table 4 shows some selected strains of
cyanobacteria grown in wastewater from different origins and associated
S. Prabha et al.
Table 4
List of selected species of cyanobacteria cultivated in waste water from different origins and value –added products formed.
Waste/Waste water Cyanobacteria grown
Domestic waste water Cyanobacterium sp. IPPAS B-1200
Desertifilum sp. IPPAS B-1220
Cyanobacterium aponinum IPPAS B-1201
Waste water from Waste stabilization pond Merismopedia
CENA106, Limnothrix
CENA110), Leptolyngbya CENA103, (Synechococcus CENA108,
Pulp-and-paper waste Phormidium insigne
Fish processing wastewater Aphanothece microscopica Nägeli
Synthetic waste water Plectonema terebrans BERC10
Municipal waste water Synechocystis PCC6803
Shrimp wastewater Synechocystis sp. PCC 6803
Dairy wastewater Leptolyngbya
Lymnothrix
Winery and raisin wastewaters Leptolyngbya
Lymnothrix
Urban wastewater and liquid digestate, Chroococcidiopsis sp.,
Aphanocapsa sp.
Aquaculture waste water Aulosira fertilissima
Textile waste water Anabaena ambigua,
Swine waste water Synechocystis sp.
value-added products formed during the cultivation. Some of these
cyanobacteria are effectively utilized for nutrient removal from wastewa-
ter. These wastewater raised cyanobacteria is found to be an excellent can-
didate for biofuel production. They can be used for the production of lipids
(for biodiesel), bio‑hydrogen, carbohydrates, isoprenoids, hydrocarbons,
etc. (Singh et al., 2019). Cyanobacteria like Anabaena variabilis, Anabaena
cylindrica, Nostoc commune, Synechococcus, Cylindrospermum, Aphanocapsa
montana, Gloeobacter sp., Gloeocapsa alpicola, Microcystis sp., etc were re-
ported to produce molecular hydrogen (Singh et al., 2019). Cost-effective
production of bio-plastics from cyanobacteria by using wastewater as an or-
ganic and inorganic nutrient source is one of the most sensible approaches.
Even though the proteins extracted from several strains of cyanobacteria
are found to be not toxic to mammals, it is impracticable to predetermine
the exact chemical composition of waste water from several origins,
which may alter day by day.
Apart from cost-effective production of cyanobacterial value-added
products, CO2 mitigation, bioremediation of industrial, municipal,
and agrochemical wastes, efficient nutrient cycling are possible.
Polyhydroxyalkanoates (PHA) currently known as bio-plastics, are truly
regarded as the neutral lipids stored as carbon and energy source inside
the cell of cyanobacteria (Gomes Gradíssimo et al., 2020). Cyanobacteria
mostly accumulate these bio plastics in the form of Polyhydroxybutyrate
(PHB) and some other derivatives of PHA. Several studies revealed that
the efficacy of wastewater-growing cyanobacteria to accumulate PHB is
in the same range of cyanobacteria that grow in the chemical medium
and in some cases, even higher proportion of accumulation was observed
(Arias et al., 2020; Bengtsson et al., 2008). Effective utilization of wastewa-
ter discharge from the shrimp cultivation system for the growth of cyano-
bacterium Synechocystis PCC 6803 in a photobioreactor, led to the
production of Poly-β-hydroxybutryate (Ashokkumar et al., 2019). Arias
et al. (2018) validated the effects of several nutritional and photoperiodic
conditions for the effective production of PHAs in cyanobacteria. It was
found that the accumulation of PHAs and carbohydrates are more in the
wastewater-borne cyanobacteria in the presence of limited amount of
nutrients.
Bioethanol production integrated with microalgal and cyanobacterial
cultivation for successful biomass production was evaluated by Sydney
et al. (2019). Alkalinized vinasse with NaOH supplementation in a bubble
column reactor exhibited a higher carbon transfer rate along with a reduc-
tion in turbidity, chemical oxygen demand and biological oxygen demand,
which supported the growth of cyanobacteria. Maximum biomass produc-
tivity was achieved with supplementation of 70% and 100% concentration
of vinasse in the medium, which in turn demonstrated a successful inte-
grated biorefinery approach.
17
Science of the Total Environment 814 (2022) 152795
Metabolite produced References
Biodiesel (Bolatkhan et al., 2020)
Biodiesel (Bolatkhan et al., 2020)
Biodiesel (Bolatkhan et al., 2020)
(Furtado et al., 2009)
(Kirkwood et al., 2005)
Single cell oil production/biodiesel (Queiroz et al., 2011)
Lipid, phycobiliproteins, carbohydrates (Shahid et al., 2021)
Biodiesel (Ashokkumar et al., 2019)
poly-β-hydroxybutyrate (Krasaesueb et al., 2019)
Biodiesel (Tsolcha et al., 2018)
Biodiesel (Tsolcha et al., 2018)
Polyhydroxybutyrates and carbohydrates (Arias et al., 2018)
Poly-ß-Hydroxybutyrate (Samantaray et al., 2011)
Lipid (Brar et al., 2019)
Lipid (Cheng et al., 2020)
7.2. Integrated harvesting and downstream strategies
The main goal of the cultivation of cyanobacteria is the final recovery of
purified targeted products. However, harvesting to retrieve the final prod-
uct involves numerous steps, which are expensive, time consuming, and it
also compromise the recovery efficiency of the final product. Individual op-
erations in the whole downstream processing such as harvesting, cell disin-
tegration, and extraction processes can be successfully integrated and this
will simplify the whole process. Biomass harvesting is an important step be-
fore the cell disruption, for the extraction of valuable compounds from bio-
mass. Kim et al. (2015) realized the possibility of an integrated harvesting
and cell disruption method for the simplification of downstream process-
ing. They successfully utilized FeSO4 and FeCl3 as a flocculant for the har-
vesting process and as a catalyst for the lysis of microalgal cells. It was
found that the quality of the oil extracted from the microalgal biomass de-
pend upon FeSO4 and FeCl3, and better quality oil exhibited by the FeCl3
utilized an integrated system. The emerging technologies utilized inte-
grated systems for efficient harvesting and cell disruptions. The application
of thermo-responsive polymers in simultaneous harvesting and effective
cell disintegration processes has been reported (Zheng et al., 2015, 2016).
Thermo-responsive polymers such as poly-(N-isopropylacrylimide-co-
allylamine) were introduced as a novel harvesting approach by heating
the mixture of polymers and algae above the critical solution temperature
of the polymers, which separate the biomass from the solution. These poly-
mers were found to be reusable and can be recovered by gentle washing just
after the successful separation of cyanobacteria and can be further used in
the next culture. These polymers were also found to be effective for the
cell wall disruption for lipid and sugar recovery. Here the process of har-
vesting and cell disintegration can be integrated into a simple operation
by the efficacious usage of these thermo-responsive polymers. Another re-
usable poly-ampholytic flocculant has been introduced for the effective
dewatering and harvesting of microalgal biomass. These flocculants with
switchable molecular charges, depending on the pH, exhibited 99% floccu-
lation efficiency, and can be successfully retrieved from the culture after the
dewatering of biomass (Morrissey et al., 2015). Alkali enhanced extraction
followed by direct trans-esterification of Chlorella vulgaris opens up a simple
approach for combining crucial steps in downstream processing. Direct pro-
duction of fatty acid methyl esters was achieved by the addition of NaOH in
excess methanol to the water containing biomass. This can be an efficient
attempt for the direct extraction of fatty acid methyl esters from the wet
biomass of microalgae/cyanobacteria (Salam et al., 2016).
One of the major high value-products from a cyanobacterial bio-
refinery is phycobiliproteins. Increase in purification steps results in the
loss of proteins during each step and thus reduce the overall yield.
S. Prabha et al.
Simultaneous separation and concentration of proteins in a single step
using aqueous two-phase systems can be an alternative to the multiple pro-
cessing steps usually used for the concentration and purification of proteins,
with higher yield (Wang et al., 2011). The major benefit of aqueous two-
phase extraction is its low processing time, energy and high yield. A differ-
ential partitioning technique is employed here for the selective partitioning
of phycobiliproteins from the other contaminant unwanted proteins using
predetermined quantities of polymers such as polyethylene glycol (PEG)
and salts (Chethana et al., 2015). PEG and other salts are inexpensive and
can be used for large scale operations for the purification and concentration
of proteins (Kannaujiya et al., 2017b). Narayan and Raghavarao (2007)
carried out aqueous two-phase extraction (ATPE) using direct homogenate
of Spirulina platensis for the downstream processing of one of the
phycobiliproteins, C-Phycocyanin. The authors reported that the ATPE
technique integrated the individual processes of cell removal, extraction
and protein concentration into a single unit operation (Teixeira, 2012) re-
ported an energy-efficient technique to deconstruct wet microalgal cells
using ionic liquids to release all the hydrophobic compounds in the pres-
ence of high temperatures. This one-step direct extraction without using
any other catalysts, bases or acids is a simple, efficient and scalable process,
which is not limited to biodiesel and lipid. Fig. 5 represents an overview of
integrated cultivation, harvesting and extraction units possible in sustain-
able cyanobacterial biorefineries.
8. Possibility of uplifting cyanobacterial strains for minimum waste
biorefineries
Owing to its numerous applications, including high nutraceutical and
pharmaceutical value, Spirulina platensis can be regarded as the most
Fig. 5. – An overview of integrated cultivation, harvesting and extraction units possible in a sustainable cyanobacterial biorefinery.
18
Science of the Total Environment 814 (2022) 152795
industrially explored and exploited cyanobacteria (Mitra and Mishra,
2019). Numerous of research works have been taking place all over the
world to explore the potential of cyanobacteria in different areas of science
(Singh et al., 2017). A retrospective analysis of the past researches by
reconsidering proficient strains of cyanobacteria manifest their possibility
to act as efficient candidates for multiple products proffering minimum
waste biorefinery. It is conceivable that the future researches will be able
to uplift those cyanobacterial strains to the next level.
8.1. Minimum waste cyanobacterial biorefinery models for a circular bio-economy
It is possible to uplift several cyanobacterial strains to the next level of
economically feasible integrated cyanobacterial biorefineries. As men-
tioned earlier, most of the biorefineries are economically unfeasible, be-
cause of the higher investment behind the production of biomass and
expensive downstream strategies. Selection of efficient strain, alternative
cost-effective technologies for the higher production of biomass and low-
cost harvesting techniques are important for the success of a biorefinery
on a commercial scale. The following section of the review analyzes some
possibilities of uplifting cyanobacteria for future biorefineries, with a few
examples.
A recent report unveiled the ability of a new strain of cyanobacterium
Plectonema terebrans to attain maximum growth (140 mg mg L–1 d–1
(dried mass)) in municipal wastewater and convert the wastewater nutri-
ents into multiple value-added products, along with a potential of auto-
flocculation (Shahid et al., 2021). Despite the higher content of pigment
proteins, it is possible to extract high-quality lipids (for biodiesel applica-
tions) and carbohydrates, from the spent biomass, manifesting the potential
of this particular strain to act as a multiple product proffering, zero-waste
S. Prabha et al.
biorefinery. Further research on large scale production of biomass and
other optimization studies are required for fortifying this strain to a success-
ful candidate for biorefinery.
Another cyanobacterium Synechocystis is a widely explored one, for the
production of several bioactive compounds. A Synechocystis based, low-cost
biorefinery for sustainable bioenergy production was proposed by
Ashokkumar et al. (2019). The large scale cultivation of Synechocystis
PCC6803 was carried out in open raceway ponds in municipal wastewater
in a semi-continuous mode and achieved a growth rate of 0.21 g L–1 d−1
and estimated to produce 94.5 tones dry biomass ha−1 year−1. This
Synechocystis based integrated biorefinery achieved a 32–35% reduction
in the production cost by using the municipal wastewater as the growth me-
dium of cyanobacterium. They adopted a cheap method for harvesting the
biomass by a combined use of iron (III) chloride and biopolymer and recov-
ered almost 98% of the biomass. The cost of lipid extraction is also reduced
by accepting direct trans-esterification using tungstate zirconia as the cata-
lyst and obtaining a 90% yield. Moreover, the lipid extracted biomass was
considered for the extraction of bio-ethanol which inturn increased the fea-
sibility of this minimum waste biorefinery. Another low-cost approach for
the growth of Synechocystis salina was proposed by Meixner et al. (2016)
by raising the cyanobacterium in digestate supernatant as a low-cost nutri-
ent media. They successfully demonstrated a comparatively higher produc-
tion of biodegradable poly-3-hydroxybutyrate(PHB) by Synechocystis salina
in the diluted digestate than the BG- 11 media. In the same cyanobacterium,
they reported the efficient extraction of poly-3-hydroxybutyrate from the
pigment extracted biomass and further analysis of other value-added prod-
ucts like proteins, carbohydrates and lipids in the residual biomass, can be
considered as a feed or fertilizer (Meixner et al., 2018). The cultivation of
Synechocystis sp. PCC 6803 in shrimp wastewater was accomplished with
a biomass yield of 500 mg L–1 after 14 days and poly-β-hydroxybutyrate,
similar to the commercially available PHB is produced. The PHB content
was 32.48% (w/w) DW, with a maximum productivity of 12.73 mg L–1
d−1 (Ashokkumar et al., 2019; Panda and Mallick, 2007). Synechocystis
PCC 6803, which is lacking a phosphate regulator SphU, cultivated in
shrimp wastewater, attained a biomass yield of 500 mg mg L–1 after
14 days of cultivation and exhibited high nutrient removal efficiency
(Krasaesueb et al., 2019).
The above-discussed models can be improved to economically feasible
self-sustainable minimum to zero waste discharging cyanobacterial
biorefineries. A self-sustainable biorefinery model would be able to in-
crease the effective utilization and valorization of resources with minimum
or zero waste discharge and hence cause less impact on the environment.
Thus, a fully successful feasible cyanobacterial biorefinery model will not
only be economically viable but also environmentally sustainable. The con-
version of cyanobacterial bloom to boon with the help of nano-technology
has uncovered a better path to environmental sustainability.
9. Promising solutions and future perspectives
The major challenges for the industrial-scale success of biorefineries are
the high cost of production and expensive downstream processing tech-
niques. Hence, from the perspective of economic feasibility, several strate-
gies can be implemented for decreasing the cost of production, harvesting
and downstream processing. The integrated cultivation systems looped
with wastewater plants for the production of cyanobacterial biomass can
come up as a sustainable system for the production of biofuel and other
co-products. Before moving on to the large scale cultivation using wastewa-
ter, it is essential to optimize the culture conditions of the respective
cyanobacteria using the wastewater on a lab scale. For those value-added
products which are already having high economic value, the cyanobacterial
biomass needs to be from non-contaminated sources. Also, the reusability
of culture medium can be adopted to achieve sustainability of the
biorefinery. Maximum valorization of biomass can be attained by imple-
menting the strategies for co-accumulation and stress-mediated accumula-
tion of products. Solar panels associated with cultivation systems, which
are using solar light as the sole energy source can co-generate electricity,
19
Science of the Total Environment 814 (2022) 152795
which inturn can satisfy the energy requirements in all stages of production,
extraction and purification processes in cyanobacterial biorefineries.
The biomass retrieval from the culture medium involves a series of high
energy-requiring, harvesting and dewatering processes, and further extrac-
tion needs much capital investment. Hence, in this milieu, the energy-based
systems are forced to shift their focus from low-value energy compounds to
high-value non-energy based products. As cyanobacteria are primarily
exploited for high value- non- energy-based products, such as pigments, se-
quential extraction of high-value compounds followed by low-value co-
products can be employed. One of the benefits of cyanobacteria is that
their primary extraction of phycobiliproteins will not interfere with the sec-
ondary extraction of other pigments and lipids, since the previous utlizes
aqueous extraction and later involves organic solvents. A low volume-
high-value bioactive product strategy can be employed for minimizing
the production cost to a certain extent.
Apart from phycobiliproteins, cyanobacteria are a pool of several other
valuable bioactive compounds such as carotenoids, chlorophylls, UV- pro-
tectants and different bio-active compounds with anti-biotic and anti-
cancer activities. In order to achieve maximum valorization of biomass,
maximum products from a cyanobacterial biorefinery can be retrieved
and further purified. But from the perspective of economic feasibility, for
the most appropriate viable co-products from the biorefinery can be se-
lected and retrieved. It is important to analyze the nature of cyanobacteria
considered, the possible value-added products which can be retrieved and
identify the techno-economic feasibility of the system. For this, the capital
investment of production, extraction and purification of compounds along
with the market value of main products and co-products should be studied
in detail and the plea should be made accordingly. Unfortunately, it is not
possible to consider all the co-products for the extraction, since, the capital
investment for their extraction cannot counterweigh the market value of
products. Hence, when the extraction solvents and associated techniques
become expensive than the targeted products, new alternative methods
for the feasible extraction of multi-products from a biorefinery has to to
be developed and introduced in future. If the multi-product recovery is pos-
sible, then the sequential extraction of compounds in the order of high to
low-value product extraction is to be considered. The spent biomass after
the sequential extraction can be considered as fertilizer or animal feed de-
pending upon the presence of compounds in the residual biomass.
One of the major drawbacks is that the economic feasibility studies are
limited to a few cyanobacterial species and scarce attention is given to the
new species. Techno-economic feasibility studies regarding novel strains
are necessary to uplift them for the future. Researches should be expanded
to develop novel approaches that can optimize the culture conditions for
the mass production of novel cyanobacterial strains. Developing an axenic
culture of cyanobacteria and further maintenance of culture and optimiza-
tion of media composition for higher productivity still require immense ef-
fort and labour. Those basic level works are essential for unveiling efficient
novel strains for the future. The work in this line can uplift the efficient
strains from laboratory to industrial scale with more unique applications
which can address the limitations of existing strains. The developments in
harvesting, extraction and purification technologies will make notable
changes in the profitability and economic success of cyanobacterial
biorefineries. It is conceivable, genetic engineering as the most successful
solution for generating feasible strains for industries. Genetic and metabolic
engineering can develop prominent strains with fascinating attributes,
aided with their easy production, easiness in harvesting and extraction pro-
cesses. However, the techniques involved in engineering novel strains usu-
ally involve expensive technologies and hence practicability would take
more time.
10. Conclusion
Cyanobacteria are wonderful blue-green factories for the production of
several value-added products, which are having immense market value.
The major fascinating qualities include their short life cycle, higher photo-
synthetic efficiency and higher biomass production. They are the
S. Prabha et al.
storehouse of several bioactive compounds with immense market value and
can be considered as a biorefinery for the production and retrieval of value
added products. Multiple products proffering cyanobacterial biorefinery as-
sociated with cost-effective techniques can increase the economic feasibil-
ity of biorefinery to a certain extent. Low-cost cultivation strategies
coupled with co-accumulation and stress-mediated accumulation of various
bioactive compounds can decrease the investment. Unlike microalgae,
cyanobacteria are more responsive to the incident light by the mechanism
of complementary chromatic adaptation, unique to them. This point out
the fact that the light management strategies have a substantial effect on
the production of metabolites in cyanobacteria. Moreover, the develop-
ment of holistic- integrated- harvesting and downstream strategies impart
immense effect on the feasibility of cyanobacteria based biorefineries. Inte-
grated cultivation-harvesting-extraction systems can bring forth economic
viability without consuming much time and energy. Cultivation systems in-
tegrated with wastewater treatment plants can reduce the water and nutri-
ent requirement for cyanobacterial production to a certain extent. Also the
possibility to uplift several proficient strains of cyanobacteria other than
Spirulina platensis with the help of state-of-the-art research cannot be over
ruled. The nanotechnological applications in cyanobacterial biorefineries
generate results that were not foreseen. The valorization of cyanobacterial
bloom with the aid of nanotechnology and further retrieval of value-added
products will lead to economic as well as environmental sustainability. The
innovations in cultivation and optimization techniques and the develop-
ments in extraction and purification techniques will open up a better future
to cyanobacterial biorefineries.
CRediT authorship contribution statement
Syama Prabha: Conceptualization, Data curation, Visualization,
Writing – original draft. Aravind K. Vijay: Data curation, Formal analysis,
Writing – review & editing. Rony Rajan Paul: Data curation, Writing –
review & editing. Basil George: Conceptualization, Supervision, Funding
acquisition, Writing – review & editing.
Declaration of competing interest
There are no known conflicts in financial interests or personal relation-
ships that could have developed to influence the research work.
Acknowledgements
The work was supported by the grant received under the extra-mural
research scheme (SERB-EMR/2016/001016) funded by Science and
Engineering Research Board, DST, Government of India.
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