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Cyanobacteria as an eco‐friendly resource for biofuel production: A Critical

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DOI: 10.1002/btpr.2835

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Received: 17 January 2019 Revised: 7 April 2019 Accepted: 24 April 2019
DOI: 10.1002/btpr.2835

REVIEW

Cyanobacteria as an eco-friendly resource for biofuel

production: A critical review

Parisa Farrokh1,2 | Mojgan Sheikhpour3,4 | Alibakhsh Kasaeian5 | Hassan Asadi3 |

Roya Bavandi6

1
Department of cell and molecular biology,
School of Biology, Damghan University, Abstract
Damghan, Iran Cyanobacteria are photosynthetic microorganisms which can be found in various
2
Institute of Biological Sciences, Damghan
environmental habitats. These photosynthetic bacteria are considered as promising
University, Damghan, Iran
3
Department of Mycobacteriology and feedstock for the production of the third- and the fourth-generation biofuels. The
Pulmonary Research, Pasteur Institute of Iran, main subject of this review is highlighting the significant aspects of the biofuel pro-
Tehran, Iran
4
duction from cyanobacteria. The most recent investigations about the extraction or
Microbiology Research Center (MRC), Pasteur
Institute of Iran, Tehran, Iran separation of the bio-oil from cyanobacteria are also adduced in the present review.
5
Faculty of New Science and Technologies, Moreover, the genetic engineering of cyanobacteria for improving biofuel production
University of Tehran, Tehran, Iran
6
and the impact of bioinformatics studies on the designing better-engineered strains
Branch-Marine Science and Technology
Faculty, Islamic Azad University North Tehran, are mentioned. The large-scale biofuel production is challenging, so the economic
Tehran, Iran considerations to provide inexpensive biofuels are also cited. It seems that the future
Correspondence of biofuels is strongly dependent to the following items; understanding the metabolic
Mojgan Sheikhpour, Department of pathways of the cyanobacterial species, progression in the construction of the
Mycobacteriology and Pulmonary Research,
Pasteur Institute of Iran, Tehran, Iran. engineered cyanobacteria, and inexpensive large-scale cultivation of them.
Email: m_sheikhpour@pasteur.ac.ir;
mshaikhpoor@gmail.com KEYWORDS
biofuel, biomass, cultivation, cyanobacteria, genetic engineering

1 | I N T RO D UC T I O N
Fossil fuels prepare about 85% of the worldwide energy, so that, without
1
finding alternative energy resources, they will be drained in the future.
The emission of CO2 from combustion of classical fuels is another draw-
back, which causes global warming and climate changes.2 Other polluting
components from fossil-fuel combustion include SO2, NO, NO2, black
carbon, and polycyclic aromatic hydrocarbons.3 The environmental
Abbreviations: AAR, acyl–ACP reductase; AAS, acyl–acyl carrier protein synthetase; ACP,
acyl carrier protein; AdhA, alcohol dehydrogenase; ADO, aldehyde-deformylating oxygenase;
CASOP, computational approach for strain optimization aiming at high productivity; CCRD,
central composite rotatable design; cMCSs, constrained Minimal Cut Sets; CT, calliterpenone;
FAME, fatty acid methyl esters; FARM, farm-level Algae Risk Model; Fas, farnesene synthase;
FFAs, free fatty acids; GM, genetically modified; HDN, hydrodenitrogenation; HDO,
hydrodeoxygenation; HFM, hollow fiber membrane; LED, light emitting diodes; LIMS,
limonene synthase; MCDA, multi-criteria decision analyses; MEP/DXP, 2-C-methyl-D-
erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate; PBP2, penicillin-binding protein;
PEF, pulsed electric fields; PHB, polyhydroxybutyrate; RSM, response surface methodology;
TGRP, tail gas reactive pyrolysis.
concerns about greenhouse gases will impact the quality of the human
life.4 Also, the dependence of the world's energy production on fossil
fuels has become as a great challenge nowadays.5 The alternative natural
resources include sunlight, wind, geothermal power, and biomass.1
Besides the energy supply, the solar energy could be applied as a proper
source for the pollutants removal in the environment.6 Also, the usage of
solar energy and capturing CO2 through photosynthesis seems to be a
promising substitute for fossil fuel during last decades.7,8 Biofuel includes
liquid, gas, and solid fuels which are generally produced from biomass.9
Biomass is a renewable resource of energy and biorefinery is referred to
the processes which can be used for the conversion of biomass to
biofuels and the other forms of energies.5 Biofuels include bioalcohols,
biodiesel, bio-oil, biogas, bio-char, and biohydrogen.9
Recently, the basic aim for the supply of fuel and energy is to dis-
cover the potentials of producing the biofuels from the recycling of
the available, cheap, and eco-friendly waste materials. The various

Biotechnology Progress. 2019;e2835. wileyonlinelibrary.com/journal/btpr © 2019 American Institute of Chemical Engineers 1 of 16

https://doi.org/10.1002/btpr.2835
2 of 16
methods are in use to produce biofuels including industrial fermenta-
tion as a new and widespread technique. However, this new tech-
nique stands out among other strategies due to its choice of raw
materials and processing. Complex chemical reactions take place in
the product resulting from biofuel, for example, methane and CO2
10,11
with traces of H2S, H2, and NH3. Therefore, biosensing systems
for monitoring of these parameters in bioreactors and optimizing of
12
biofuel production in controlled systems are remarkable. Even the
use of several species together with widespread sources of biologi-
cally active compounds, for raising the amount of the product, could
be beneficial.13,14
Several generations of bio-based fuels have been made during last
years. The first-generation biofuels are derived from oil, sugar, or the
starch of food crops. However, the biofuels obtained in this way need
fertile land, water, pesticides, and compete with food supply. The
second-generation biofuels are produced from the nonedible cellu-
losic and lignocellulosic agricultural materials. Although this kind of
biofuel does not effect on the food supply, the low yield and the need
for land area limit their applications.2,15-17 Furthermore, because of
the wider culture of the suitable plants, the first and second genera-
tions of biofuel have unfavorable effects on the ecosystem.1 The
third-generation biofuels are derived from eukaryotic microalgae and
prokaryotic cyanobacteria. Algae is capable to become the most
important biofuel source in the near future.18 Microalgae and cyano-
bacteria are accounted as the most promising sources for biodiesel
production, because of their short cultivation time and high oil con-
tent.19 These photosynthetic microorganisms have significant poten-
tial for production of lipids, as a biofuel feedstock.2,15-17 The fourth-
generation biofuels are based on the third-generation biofuels with
2,15
the combination of genetic and metabolic engineering. The effi-
ciency of the photosynthesis and biomass production in cyanobacteria
is up to 10%, which is higher than that of plants (around 1%) and algae
(5%).20,21
Cyanobacteria or blue-green algae are photosynthetic Gram-
negative bacteria.20,22-24 According to the fossil information, cyano-
bacteria are old organisms which were originated on the earth from
21-23,25,26
3.5 billion years ago. The morphology of the cyanobacteria is
diverse, which contains unicellular, colony forming, and filamentous
forms.20,22,23 They are found in different geothermally heated regions
of the Earth and considered as biotechnological treasures for high-
temperature processes. They are utilized for CO2 sequestration, bio-
fuel production, and bioremediation such as bioremoval of phenolic
compounds.27,28 Various kinds of chemical compounds are produced
by the wild-type and engineered cyanobacteria; which include the
alcohols, diols, hydrocarbons, fatty metabolites, proteins, carbohy-
drates, carboxylic acids, terpenes, isoprenes, toxins, antioxidants, pig-
1,2,25,29
ments, vitamins, hydrogen, and methane. These metabolic
components have different applications in the biofuel, food, pharma-
22,25
ceutical, and cosmetic industries. Therefore, metabolic engineer-
ing of these microorganisms creates new hope for desired products.
In comparison to other photosynthetic organisms, the biofuel pro-
duction based on cyanobacteria has some advantages like the high
growth rate with more feedstock productivity,1,2,15 less water and
FARROKH ET AL.
land demand,1,15,23 simple and cheap nutritional requirements,1,2,15
and the easy genetic manipulation of many cyanobacteria through the
genetic engineering tools.2,15,17,23 In addition to these benefits, the
biofuels obtained from cyanobacteria are a mixture of alkanes, fatty
acids, and fatty alcohols similar to fossil fuels.30 Therefore, the biofuel
seems to be the best substitute for the fuels of transport vehicles
without conducting great changes in their engines.1
In spite of all advantages, there are some environmental concerns
for the application of genetically modified (GM) cyanobacteria for the
biofuel production. The horizontal gene transfer and competition
between the GM cyanobacteria and other microorganisms could
affect the natural ecosystems.31,32 Nevertheless, the large-scale culti-
vation of cyanobacteria and preparing them for biofuel production
has not been economical, so far.2,33 The advent of the first application
of biofuel in a car backs to the usage of peanut oil in the engine in
1900.15 However, the studies on the cultivation of algae and using
the waste material for their growth were started in 1950.34 For the
first time, ethanol was produced by an engineered Synechococcus
elongatus PCC 7942, containing alcohol dehydrogenase II from
Zymomona mobilis, in 1999.2 The Algenol (Fort Myers, FL) was
founded in 2006, which was the first successful company for produc-
ing biofuels from engineered cyanobacteria.22,31
The high contribution (65%) of CO2 emission from burning the
fossil fuels and limitation of their sources are important factors for
discovering the new energies.35 During past decades, several attempts
have been made for using photosynthetic organisms as living tools for
conversion of solar energy to renewable energy. The fourth-
generation biofuels is more environmentally friendly since the con-
sumption of CO2 by microorganisms is being increased through
genetic engineering methods. Therefore, the fourth-generation
biofuels by absorbing more CO2 reduces the greenhouse gases emis-
sion.36 In the past decade, cyanobacteria have received special atten-
tion as the cell factories for biofuel generation. Here, we mention
some recent reviews which were focused on the importance of cya-
nobacteria for biofuel production. Quintana et al.1 in 2011 studied the
advantages of the third-generation biofuels, which were produced by
cyanobacteria, in comparison with the former biofuels. Furthermore,
they clarified carbohydrate, lipid, and amino acid metabolic pathways
in cyanobacteria, which could be used for making engineered strains.
Balasubramanian et al.37 described the usage of industrial wastewa-
ters for the cultivation of cyanobacteria. This way of wastewater
usage is a cost-effective procedure for biofuel production from bio-
mass. Rosgaard et al.16 in 2012 introduced the genetic engineering of
cyanobacteria and plants for biofuels and biochemical production.
They specifically focused on the improvement of CO2 fixation through
the engineering methods. Gupta et al.38 in 2013, explained classifica-
tion of cyanobacteria and their beneficial compounds. They also listed
the cyanobacterial strains with higher content of lipid for biofuel gen-
eration. Sarsekeyeva et al.15 in 2015 explained the potential of cyano-
bacteria for biofuel production (cyanofuel) and summarized the
engineering and nonengineering methods for the improvement of cya-
nofuels production. Mazard et al.22 in 2016 surveyed the advantages
of using marine cyanobacteria for the production of valuable
FARROKH ET AL.
metabolite and indicated their biotechnological applications. Ghosh
et al.39 described main factors about isolation and identification of
microalgae and cyanobacteria and also explained the diversity of the
species. In addition, they presented tools which are used for the
genetic manipulation of the microalgae and cyanobacteria in order to
improve their lipid metabolism and photosynthetic efficiency. Silva
et al.31 focused on the production of bioethanol by microalgae and
cyanobacteria. They presented three possible ways for this purpose;
the fermentation of lysate biomass with yeast, the use of dark fermen-
tation metabolic pathways, and the use of engineered cyanobacteria
2
for modifying the preexisting pathways. Rajneesh et al. in 2017 dis-
cussed the simultaneous production of biofuels and secondary metab-
olite in cyanobacteria for overcoming to the economic limitation of
bioenergy production.
Although many reviews introduced cyanobacteria and cyanofuels,
each of them only focused on some special aspects in this area. Due to
the application of cyanobacteria in the generation of future biofuels, the
extension of our knowledge about these kinds of bacteria is necessary.
Therefore, we present a comprehensive review of the important features
of cyanobacteria in the biofuel production. In the present review, first of
all, general consideration of some cyanobacterial strains with the capabil-
ity to produce biofuel feedstock is introduced. We highlight the essential
factors for the cultivation of cyanobacteria including the media and
waste materials which could be used as a feed supply and the physical
parameters. Furthermore, the downstream processes for the extraction
and separation of bio-oil, the genetic engineering of cyanobacteria for
the production of biofuel, in silico studies, and the economic aspect of
biofuels production are discussed here.
2 | COMPARISON BETWEEN VARIETIES OF
CYANOBACTERIA
Because of the old origin of cyanobacteria, they can survive in various
ranges of environment including deserts, hot springs, freshwater to marine
aquatic habitats, and other extreme conditions.20,22,24,26 Among various
cyanobacteria, the thermotolerance are noticeable for their tolerance in
the large-scale outdoor cultivation and biofuel production. Four
cyanobacterial strains (Chroococcidiopsis thermalis, Microcoleus chtho-
noplastes, Mastigocladus laminosus, and Oscillatoria sancta) were isolated
from geothermal spring of Roman Baths, UK. Among these isolates, only
M. chthonoplastes under nitrogen starvation or at temperature of 40
C
produced notable unsaturated esters (C18:1, C18:2, and C18:3) in addi-
tion to saturated ones (C16:0 and C18:0) for biodiesel synthesis.40 Aboim
et al. compared the fatty acid and biodiesel qualities of eight different
Amazon cyanobacterial strains in ASM-1 and BG-11 media. The results
indicated that two strains, Synechocystis sp. CACIAM05 and Microcystis
aeruginosa CACIAM03, had better fatty acid profile and biodiesel quality
in BG-11 medium and could be candidate for the synthesis of biodiesel.
Under this experimental condition, the lipid extract of both strains con-
tained more than 75% of C6:0.41 Da Rós et al. studied five cyanobacterial
isolates belonging to Synechococcus, Trichormus, Microcystis, Leptolyngbya,
and Chlorogloea genera. Synechococcus sp. PCC7942, Microcystis
3 of 16
aeruginosa NPCD-1, and Trichormus sp. CENA77 had better fatty acids
content (higher percent of C16:0) and biomass productivities (more than
30 mg L−1 day−1) than the others. Therefore, those isolates seem to be
proper feedstock for biodiesel generation.42 In another study, 11 filamen-
tous nitrogen-fixing cyanobacteria were compared based on their biomass
and lipid productivities (Table 1) and biodiesel properties. Finally, Ana-
baena sphaerica MBDU 105 was the most suitable strain for biodiesel pro-
duction which was identified by a multi-criteria decision analyses (MCDA).
The fatty acid composition of A. sphaerica MBDU 105 contained both sat-
urated and unsaturated fatty acids with higher percent of C16:0.43 Dalavai
et al.44 investigated fatty acid methyl ester contents of four marine
cyanobacterial isolates including Oscillatoria sps. BDU 142191, Pho-
rmidium tenue BDU 141753, Lyngbya sps. BDU 90901, and Synechoccus
elongatus BDHKU 10201. Gas chromatography and mass spectroscopy
analysis showed that unsaturated fatty acids (phthalic acid and squalene)
were the most component of their lipids which are beneficial feedstock
for biodiesel production. Sen et al.45 found that two cyanobacteria,
Limnococcus limneticus and Leptolyngbya subtilis, isolated from East Kol-
kata Wetland, simultaneously had the capability of both removing chro-
mium contamination and biofuel production. The biomass and lipid
productivity of these cyanobacteria increased when the wastewater con-
taining chromium Cr(VI) was used instead of BG-11 medium and the best
yield was observed in 15 mg/L Cr(VI) (Figure 1). Mashayekhi et al.46 iso-
lated a freshwater cyanobacterium, Synechococcus elongatus, from the
Ramsar hot springs, Iran. The culture of the isolate in BG-11 medium
under the pH of 9, 72 mL/min flow rate, and 2% CO2 produced the
highest growth rate. Figure 2 shows the dry cell weight and biomass pro-
ductivity of the strain during 16 days of cultivation. The gas chromatogra-
phy analysis of the fatty acid content of the isolate showed that more
than 37% of its total fatty acids contained C18 isomers, and it was suitable
for biodiesel production. Anahas et al.19 characterized 15 heterocystous
cyanobacterial strains which were isolated form freshwater habitats of
India or symbiotic form with Azolla and Cycas. The production of the fatty
acids suitable for biodiesel production, such as palmitic (C16:0) and stearic
(C18:0) acids were noticeable in these isolates. Among them, Nostoc cal-
cicola MBDU 602, Anabaena variabilis MBDU 013, and Desmonostoc
muscorum MBDU 105 had higher lipid and biomass productivity.
In summary, various morphologically different cyanobacteria from
distinct habitats could have potential to produce lipids as a biofuel
feedstock. The chain length of fatty acids and saturation/unsaturation
state of them influence the biofuel properties, which are obtained
from cyanobacteria. In most of the mentioned applicable lipid profiles,
palmitic and stearic acid isomers are predominant. In spite of many
well-studied cyanobacteria, the screening and comparing of other iso-
lates are the important and ongoing issues to reach appropriate strains
for the commercial applications.
3 | C U L T I V A T I O N P R O C ES S
Cyanobacteria produce organic compounds by using water, CO2, and
sunlight. The most significant factors which influence their culture are
the light intensity, temperature, CO2 concentration, pH, and the
4 of 16 FARROKH ET AL.

T A B L E 1 Cyanobacterial strains used by Anahas et al. with their biomass productivity, lipid content, and lipid productivity [reprinted with
permission from Elsevier]43

Cyanobacterial GenBank Biomass productivity Lipid content Lipid productivity

S. no. strains accession no. Source of isolation (mg L−1 day−1) (%wt) (mg L−1 day−1)
1. Camptylonemopsis minor KC971096 Rice field, Thiruverumbur, 14.13 ± 0.001 7.910 ± 0.218 1.202 ± 0.017
MBDU 013 Tiruchirappalli 10
480 1800 N,
78
410 700 E
2. Calothrix marchica MBDU KC971090 Rice field, Budalur, Thanjavur 17.33 ± 0.001 6.774 ± 0.140 1.083 ± 0.022
602 10
790 6700 N, 78
970 600 E
3. Calothrix sp. MBDU 013 KC971094 Freshwater pond, 20.67 ± 0.000 11.221 ± 0.137 2.358 ± 0.141
Thiruverumbur, Tiruchirappalli
10
480 1800 N, 78
410 700 E
4. Nostoc sp. MBDU 009 KP096229 Rice field, Mathur, Tiruchirappalli 16.00 ± 0.001 7.903 ± 0.305 1.340 ± 0.275
10
720 7000 N, 78
580 500 E
5. Nostoc sp. MBDU 013 JN542385 Freshwater pond, 20.00 ± 0.001 6.749 ± 0.131 1.419 ± 0.095
Thiruverumbur, Tiruchirappalli
10
480 1800 N, 78
410 700 E
6. Anabaena sphaerica KP096231 Rice field, Poondi, Thanjavur 10
9.33 ± 0.000 18.651 ± 0.243 1.681 ± 0.208
MBDU 105 850 5100 N, 78
940 900 E
7. Calothrix dolichomeres KP096227 Azolla sp. Thiruverumbur, 11.40 ± 0.001 10.382 ± 0.208 1.048 ± 0.010
MBDU 013 Tiruchirappalli 10
480 1800 N,
78
410 700 E
8. Calothrix linearis MBDU KP096228 Azolla sp. Kallanai, Thanjavur 10
18.33 ± 0.000 6.426 ± 0.223 1.126 ± 0.071
005 830 2100 N, 78
810 700 E
9. Nostoc piscinale MBDU KP096230 Azolla sp. Thiruverumbur, 17.33 ± 0.003 4.682 ± 0.996 0.645 ± 0.092
013 Tiruchirappalli 10
480 1800 N,
78
410 700 E
10. Anabaena sp. MBDU 006 KC971092 Azolla sp. Kollidam river, 16.33 ± 0.001 8.620 ± 0.246 1.463 ± 0.044
Tiruchirappalli 10
870 0000 N,
78
690 900 E
11. Nostoc sp. MBDU 007 KP096232 Cycas circinalis, Gundur, 14.00 ± 0.002 9.577 ± 1.988 1.492 ± 0.128
Tiruchirappalli 10
730 5100 N,
78
730 0600 E

F I G U R E 1 (a) Dry biomass, (b) lipid content of a consortium of Limnococcus limneticus and Leptolyngbya subtilis at different initial metal
concentrations (10–25 mg/L) with time [reprinted with permission from Elsevier]45
FARROKH ET AL. 5 of 16

carbonate concentrations. The maximum lipid synthesis was gained at

150 μmol m−2 s−1 light intensity and 1.2 g/L Na2CO3 (Figure 3). Under
the experimental conditions, unsaturated fatty acids were increased and
the lipid feedstock was converted to biodiesel, enzymatically, by Novo-
zym® 435.
Patel et al.54 showed that the addition of calliterpenone (CT), from a
plant called Callicarpa macrophylla, into Synechocystis PCC 6803 culture
medium enhanced its growth rate. Also, this addition could enhance the
biomass amount and the production rate of carbohydrate and lipid. The
optimum condition was obtained at 0.01 mM of CT in BG-11+ at the pH
of 7.5, after 10 days. The optimization of carbon/nitrogen ratio in the cul-
tivation of Phormidium autumnale was investigated by Siqueira et al55 By
using synthetic BG-11 medium and exogenous sucrose source with the
C/N ratio of 40, the cyanobacterial oil composition had desirable proper-
F I G U R E 2 Dry cell weight and biomass productivity of the
ties for biodiesel production. Anabaena sp. PCC 7120, a filamentous cya-
Synechococcus elongatus during 16 days of cultivation [reprinted with
permission from Elsevier]46 nobacterium, produces biofuel feedstock under minimal nutritional
conditions.
Johnson et al.56 cultivated this strain in BG-11 basal medium with
components of the culture medium (carbon, nitrogen, phosphorus, and
various nitrogen sources (NaNO3, N2 gas, NH4Cl, and CH4N2O).
trace elements).37,47
Ammonium chloride and sodium nitrate similarly provided the best
conditions for the maximum growth rate of Anabaena. Since NaNO3
3.1 | BG-11-based medium has a negative effect on the environment and human health, NH4Cl
seems to be a notable source for the large-scale cultivation process of
The optimization of medium component concentrations is an essential
Anabaena. In another study, the addition of bicarbonate, tannery efflu-
factor for the cultivation of cyanobacteria in order to generate
ent, coir pith, and light stress was examined on the biomass and lipid
biofuels. Karatay et al.48 proposed three candidate thermophilic cya-
production of Synechocystis sp. NN. The highest amount of lipid and
nobacteria as Synechococcus sp., Cyanobacterium aponinum, and Pho-
biomass yield was obtained in the BG-11 medium, supplemented with
rmidium sp., for preparing lipid extract to produce biodiesel. Between
4 mL/L tannery effluent and 0.6 g/L coir pith.57
these cyanobacteria, the highest lipid concentration and saturated
compounds were observed in Synechococcus sp. in BG-11 medium
containing 0.25 g/L NaNO3 at pH 7. In another study, the lipid and 3.2 | Other media for cultivation of cyanobacteria
biomass productivity of cyanobacteria was examined under the pho-
Ohtaguchi et al.58 cultivated Synechococcus leopoliensis in photo-
toautotrophic and mixotrophic/heterotrophic modes. Cyanosarcina
bioreactor containing modified Detmer medium. The bioconversion of
sp. MIC-BG4, Phormidium sp. MIC-BG1, Nostoc sp. MIC-BG2, and
S. leopoliensis biomass to ethanol was carried out with Saccharomyces
Anabaena were four cyanobacterial strains investigated under these
sake under anaerobic condition. The ability of lipid production of
nutritional conditions in BG-11 medium. The lipid content and its
quality in all of the strains were higher in the mixotrophic cultivation
by adding 2% glucose.49 Kaiwan-arporn et al.50 obtained the highest
biomass production of Synechocystis aquatilis TISTR8612 in continu-
ous culture condition for an airlift photobioreactor. The BG-11
medium was used as the culture medium, and the optimum conditions
were obtained at 10% of CO2 concentration. Kotelev et al.51 cultured
two thermophilic cyanobacteria, Mastigocladus laminosus and Ana-
baena variabilis, in photobioreactor containing BG-11 medium with
2% CO2 injection. Under this condition, their biomass accumulation
was increased, which could be used for lipid extraction.
Simultaneous production of two biofuel feedstock (glycogen and
lipids) and polyhydroxybutyrate (PHB) as a biodegradable plastic was
optimized in Synechocystis sp. PCC 6803. The highest productivity was
observed at the heterophototrophic condition in BG-11 medium with
0.4% glucose, nitrogen elimination and light intensity of 200 μmol pho-
F I G U R E 3 The optimum region of cell productivity and lipid
tons m−2 s−1 after 12 days.52 Silva et al.53 cultured Synechococcus content as a function of light intensity and Na2CO3 concentration
sp. PCC 7942 in BG-11 medium at different light intensities and [reprinted with permission from Elsevier]53
6 of 16
Microcystis aeruginosa strain NPCD-1was examined by Da Rós et al59
The lipid analysis of M. aeruginosa after cultivation in an ASM-1
medium under 25
C and 109 μmol photon m−2 s−1 showed that its
lipid had an appropriate quality for biofuel production (Figure 4). Fur-
®
thermore, the transesterification of the obtained oil by Novozym
435 generated a noticeable ethyl ester concentration.
Two strains of A. platensis, rsemsu 1/02-P, and rsemsu 1/02-T,
were also cultured in another study under stress conditions in order
to induce lipid production. The strains were cultured in the Zarrouk
medium with suboptimal temperatures, nitrogen and phosphoric star-
vation, and 2% of CO2. Neutral lipids contents were increased by the
stressors in both of the strains, which could be valuable for the lipid-
based biodiesel generation.60 The effect of CO2 and nitrogen sources
on the growth and lipid synthesis of Synechococcus subsalsus, a fast-
growing cyanobacterium, was investigated by Setta et al 61
The cyano-
bacterium was cultured in seawater, enriched with Conway medium.
They found that the S. subsalsus is not a notable candidate for biofuel
generation, due to its low lipid accumulation.61 The conversion of gly-
cogen to ethanol, using saccharification and fermentation methods,
causes special attention to this primary feedstock. Synechococcus
sp. PCC 7002 is a marine euryhaline cyanobacterium, which was
selected for optimization of glycogen production by Aikawa et al62
The highest amount of glycogen was produced after 7 days in a modi-
−2 −1
fied medium, under 4% CO2, 600 μmol photons m s and nitrogen
starvation in brackish water. The result showed that Synechococcus
sp. PCC 7002 could be a favorable strain for biofuel production.
3.3 | Using waste material as a feed supply
Approximately, 71% of the earth's surface has been covered by water,
which is being polluted by human beings by different industrial activi-
ties and pouring wastes into rivers and coasts.63 The conversion of
wastewaters into biomass and biodiesel by cyanobacteria is an appro-
priate method for energy harvesting from organic compounds. The
F I G U R E 4 Comparison between the profile of fatty acids in
M. aeruginosa NPCD-1 and palm oil [reprinted with permission from
Elsevier]59
FARROKH ET AL.
waste material and by-products are concerned as cheap organic
sources for biofuel production. The culture of Oscillatoria annae in
NPK medium with the addition of molasses was optimized through
the response surface methodology (RSM) and central composite rotat-
able design (CCRD). Under this condition, biomass and lipid produc-
tion in O. annae were improved for biodiesel generation.64 In another
study, olive mill wastewater, treated by a membrane pilot plant sys-
tem, was used as culture media for the biomass production of
Arthrospira platensis. The membrane technology seems to be an
appropriate method for purifying of olive mill wastewater.65
Maroneze et al.66 applied cattle-slaughterhouse wastewater in bubble
column bioreactor containing Phormidium sp. for biodiesel production.
Francisco et al.67 used cassava wastewater as a culture medium for
Phormidium sp. to produce bio-oil. At a carbon/nitrogen ratio of
68 and 30
C in a continuous bioreactor, the maximum oil product
with 99.98% ester content was detected.
Neves et al.68 also applied the wastewater of cassava processing
for the fed-batch cultivation of filamentous cyanobacterium, Pho-
rmidium autumnale. This wastewater was rich in organic compounds
without any inhibitory effect on microalga and cyanobacteria growth.
The production of biomass and lipids in P. autumnale increased under
this condition. Another research group found that the mixed culture
of three cyanobacteria (Nostoc muscorum, Anabaena oryzae, and Spiru-
lina platesis) grew optimally in the 50% concentration of olive mill
wastewater. Besides the phycoremediation of the wastewater, the
biomass production of cyanobacteria has a potential application in
biodiesel production.69 Gismondi et al.70 designed a flow lane incuba-
tor for the biofilm growth of three cyanobacteria, identified as Ana-
baena augstumalis VRUC163, Calothrix sp. VRUC166, and Nostoc
sp. VRUC167. In this study, an artificial wastewater sample was used
as a substrate. These strains were capable of remove phosphorus
from wastewater and produce lipids which were beneficial for biofuel
production. Dineshbabu et al.71 showed that a filamentous marine
cyanobacterium, Phormidium valderianum BDU 20041, could use flue
gas (coal burnt) and ossein effluent for its growth and biomass pro-
duction. The quality of the cyanobacterial lipid content, obtained
under these conditions, was suitable for biodiesel production.
In conclusion, preparing the optimum medium for the cultivation
of cyanobacteria is critical due to its direct effect on the lipid and bio-
mass productions. Among the conducted studies, the BG-11-based
mediums are more frequent than the other media. Furthermore, occa-
sionally, some researchers added some nitrogen and carbon sources in
the BG-11 medium for better output. In addition, the application of
various waste materials for the feed supply of cyanobacteria seems
interesting, since they are cheap, and their removal from the environ-
ment is beneficial.
4 | EFFECTIVE PHYSICAL PARAMETERS
The commercial production of biofuel from cyanobacteria depends on
designing of the cultivation systems for better CO2 delivery and the light
exposure. Furthermore, some researchers have attempted to develop
FARROKH ET AL.
the vessels and bioreactors for the closed culture methods, while some
have worked on the open outdoor culture systems.21 Noel et al.72 pres-
ented a membrane-mediated system for liquid-based CO2 delivery to
the cyanobacterial culture in a photobioreactor. This method with pro-
viding lower energy and cost seems to be beneficial for the commercial
production of biofuel. Kalontarov et al.73 designed a hollow fiber mem-
brane (HFM) for CO2 delivery in a photobioreactor. The cultivation of
Synechococcus elongatus using HFM technology increased the
cyanobacterial growth and biomass accumulation. This system also
seems to be applicable for biofuel generation from cyanobacteria. Kim
et al.74 examined the effect of light intensity, CO2 concentration, and its
bubble size on the lipid synthesis of Arthrospira platensis KMMCC CY-
007 in a rectangular flask. The best condition was observed at 7.5 Can-
dela and 1% fine bubble CO2, after 7 days in the modified Schlösser
medium. Therefore, A. platensis seems to be a cyanobacterium, having
the potential of biodiesel production. In another study, the cultivation of
diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142, under
mixotrophic and CO2 gas, sparging conditions improved its metabolism
rhythm and biomass production, which could be useful for biofuels pro-
duction.75 Bruno et al.76 studied three biofilm-forming cyanobacteria,
which were isolated previously from Italy. They compared the effect of
the batch system and semicontinuous flow-lane incubator for the pro-
duction of cyanobacterial biomass. The results showed that biomass and
lipid production was higher in the flow-lane incubator.
Selvan et al.77 examined the biodiesel production of two marine cya-
nobacteria (Lyngbya sp. and Synechococcus sp.) in various media, for
example, ASNIII, BG-11, and seawater enrichment medium. The culture
of cyanobacteria in the sea water enrichment medium within tubular
light emitting diodes (LED) photobioreactor had a better effect on the
biomass production. Biodiesel, obtained from the transesterification of
extracted lipids in Synechococcus sp., was higher than Lyngbya sp. Singh
et al.78 obtained the high amount of biomass (2.01 g−2day−1), by the cul-
tivation of Leptolyngbya sp. ISTCY101 in BG-11 medium supplemented
with 50 mM NaHCO3 in a semi-continuous mesh incubator. The mesh
incubator supported the biofilm development of Leptolyngbya sp. with
easy biomass harvesting for the biofuel production.
Economou et al. 79
applied an attached-growth photobioreactor
for culturing Limnothrix sp., as a filamentous cyanobacterium. This bio-
reactor provided better biomass and lipid production, in comparison
to the suspended-growth cultivation methods; also, it could be more
economical. Jackson et al.80 designed some vessels for the culture of
Synechococcus sp. PCC 7002 as an appropriate candidate for biofuels
production. The higher growth rate of Synechococcus 7002 was
obtained under strong light intensity. Johnson et al.81 cultured Ana-
baena sp. PCC 7120 in the BG-11 medium with 0.5 g/L NaHCO3 as a
carbon source in some sealed test tubes. This method avoids from
volatilizing of useful chemical compounds (e.g., limonene), produced
by cyanobacteria, and may provide mutants with high tolerance to
chemical components.
The scale-up biomass production of photosynthetic microorganisms
is another essential issue for large-scale production of biofuels. Schoepp
et al.82 presented a system for the kilogram-scale outdoor cultivation of
cyanobacteria and microalgae. Two examined cyanobacteria, Arthrospira
7 of 16
platensis and Anabaena sp. PCC 7120, were successfully cultivated in this
system. Regarding the physical parameters, which affect the lipid and
biomass productivity during the cyanobacteria cultivation, many efforts
are needed for further improvement of the cultivation systems. The
operation and controlling of the physical parameters in closed photo-
bioreactors are easier than that of the open systems. However, the
required cost and energy for the development of bioreactors are higher
than the open ponds.21 The desirable criteria in the designing of vessels
for culturing cyanobacteria are including the rapid growth rate and easy
biomass harvesting with lower cost.
5 | EXTRACTION/SEPARATION METHODS
Harvesting cyanobacterial cells and cell disruption are serious steps in
biofuel production. Several methods including centrifugation, floccula-
tion, foam fractionation, membrane filtration, and ultrasonic separa-
tion have been used frequently for cell separation.18 Cell disruption
can be performed by physical, chemical, and enzymatic methods.21,83
Sheng et al.84 investigated various solvents and methods for the
extraction of lipids from Synechocystis PCC 6803. The using of chloro-
form and methanol, based on the Folch and Bligh & Dyer methods,
produced the highest diacylglycerols yield (Figure 5). This research
group also examined seven physical methods including; the bead beat-
ing, autoclaving, French press, freeze drying, microwave, ultrasound,
and pulsed electric fields (PEF) for the intracellular lipids recovery of
Synechocystis PCC 6803. Among those, the microwave and PEF were
economic for the large-scale lipid extraction from Synechocystis.83
The in situ transesterification of the extracted lipids, for example, tri-
glycerides from cyanobacteria was effective for the high yield generation
of biodiesels like glycerol and fatty acid alkyl ester.85 In another study,
the anaerobic digestion of Gloethece membranacea in a semicontinuous
digester at 25
C produced the noticeable amount of methane, which
could be used as biofuel.86 Zeng et al.86 used silica-supported nickel
phosphide for the catalytic pyrolysis of cyanobacteria, isolated from
water bloom of the Taihu Lake in China, to produce bio-oil. The obtained
bio-oil was rich of long-chained alkanes and had an acceptable quality for
biofuel generation. Levine et al.87 applied hydrothermal carbonization
strategy to convert bi-culture biomass of Nannochloris (a microalga) and
Synechocystis (a cyanobacterium) to the primary component for biodiesel
production. In this system, some organic materials which were released
from the microorganisms in the aqueous phase were used again by them
as nutrients.
Another research group proposed that the enzymatic hydrolysis of
the freshwater cyanobacterial cells, following supercritical fluid pre-
treatment, efficiently enhanced the ethanol production. They showed
that a mixture of three enzymes, α-amylase, glucoamylase, and cellu-
lase, had the better effect on the conversion of starch into ferment-
able monosaccharides.88 Enzymatic hydrolysis of Synechococcus
sp. PCC 7002 was performed by Möllers et al.89 to produce a sub-
strate for bioethanol production through yeast fermentation. Proper
degradation of Synechococcus was achieved by lysozyme and two
alpha-glucanases (Liquozyme® SC DS and Spirizyme® Fuel).
8 of 16 FARROKH ET AL.

F I G U R E 5 Percent of fatty acid methyl esters

(FAME) after the lipid extraction of Synechocystis
PCC 6803 biomass using different
solvents/solvent combinations [reprinted with
permission from Elsevier]84

An approach called “cell milking” seems to be useful for the extrac-

tion of intracellular biofuels components of cyanobacteria. In this
method, long-chain alkanes, for example, n-decane, create holes within
the cell membrane by damaging lipid-bilayer construction, and the
biofuels components pass through the cells.90 Moreti et al.91 applied
the coagulation/flocculation/dissolved air flotation (C/F/DAF) process
with Moringa oleifera seed powder as a coagulant for production of bio-
oil products from Anabaena flos-aquae. The obtained sludge from
A. flos-aquae had a high quantity of oleic acid, and it could be used as a
substrate for biodiesel production. Chagas et al.92 showed that the tail
gas reactive pyrolysis (TGRP) was an effective process for converting
Spirulina biomass to bio-oil compounds. The obtained bio-oil contained
aromatic hydrocarbons and nitrogen compounds; these were thermo-
stable, which could be distilled for fuel production. Nevertheless, the
biomass of cyanobacteria is valuable for biofuel production, the high
F I G U R E 6 The effect of final pyrolysis temperature on the
viscosity and O and N heteroatomic of their contents (protein and lipid)
product yields [reprinted with permission from Elsevier]95
encounter their bio-oil production with some difficulties. Performing
the TGRP technology with the combination of hydrodeoxygenation heating rate of 10 K/min and Mg/Al ratio of 4. The thermochemical
and hydrodenitrogenation (HDO/HDN) and final extraction with liquefaction of cyanobacterial biomass is another method for bio-oil
hydrochloric acid would be operative for the generation of clean hydro- extraction. Meng et al.96 extracted highest bio-oil from a collection of
carbon from Spirulina biomass.93 cyanobacteria (from Dianchi Lake, China) at 320
C for 30 min with
Velmurugan et al.94 utilized different metal oxides (Al2O3, CaO, ethanol–water mixture (4.6 v/v) and the biomass-liquid ratio of
Fe2O3, MgO, and ZnO) in individual, binary, and ternary forms for the 1 g/15 mL.
extraction of glycogen and lipid contents from Synechocystis sp. PCC Generally, the cell wall of cyanobacteria should be disrupted to
6803. The CaO–MgO duplex form showed a maximum recovery yield extract the lipid feedstock. The physical, chemical, and enzymatic pro-
of glycogen and lipid, which were suitable for biodiesel production. cedures or combination of them could be used for this purpose.
The high nitrogen compounds of cyanobacterial water bloom limited Although enzymatic ways performed in mild conditions in comparison
the direct usage of its bio-oil. Gao et al.95 studied the catalytic pyroly- to the physical and chemical methods, the physical techniques are
sis of cyanobacterial biomass over LDO/ZSM-5 composites with vari- more cost-effective for the large-scale operation. Providing the
ous Mg/Al molar ratios. The higher yield of bio-oil with less nitrogen highest yield of biofuels with lower cost is considerable factors in this
content was obtained at the temperature of 823 K (Figure 6), with the context.
FARROKH ET AL. 9 of 16

6 | G E N E T I C A L LY E N G I N E E R E D temperature to 46
C, the cytoplasmic membrane was hydrolyzed and

CYANOBACTERIA FFAs was released. This genetic method is called thermorecovery,
which could be used for the biofuel feedstock collection. Other thermo-
The genetic manipulation tools have successfully been used in the con- stable lipases102-104 seem to be appropriate candidates for making
39
struction of engineered cyanobacteria. Synechococcus elongatus PCC engineered cyanobacteria with the capability to release FFA.
7942 and Synechocystis sp. PCC 6803 is the model cyanobacteria, which A lytic Synechocystis sp. PCC 6803, genetically engineered by
1
are usually applied in experiments. Generally, most of the studies focus Miyake et al.,105 will facilitate the recovery of biofuel and related
on the overexpression of important precursors, the deletion of pathways compounds. Two genes from T4 bacteriophage which are encoding
that are in competition with these precursors, and also engineered holin and endolysin were introduced to Synechocystis sp. under the
strains with the capability to release bio-oils.97 Figure 7 illustrates the control of green-light regulated promoter. The expression of genes
general overview of a metabolic pathway for the production of free fatty encoding acyl-ACP thioesterase and acetyl-CoA carboxylase from a
acids and other chemicals in cyanobacteria. green alga, Chlamydomonas reinhardtii CC-503, into Synechococcus
elongates PCC 7942 increased the free fatty acid production. These
enzymes were involved in the fatty acid biosynthesis and release.
6.1 | Modified strains with enhanced production and
excretion of free fatty acids However, the improvement of FFA biosynthesis in the engineered
cyanobacterium reduced its growth rate and the FFA excretion, due
Lipids are valuable precursors for hydrocarbon-based biofuel produc- to the negative physiological effects.106 Voshol et al.107 reported that
tion. The conversion of cyanobacteria to cell factories, by producing
the overexpression of era gene in Synechococcus elongates PCC 7942
lipids in large scale through metabolic engineering methods, can be
increased its total fatty acid production. Era encodes a GTP-binding
favorable for next generation of fuel production.98 Using cell-free
protein and involved in fatty acid and hydrocarbon metabolism. It
fatty acids (FFAs) is a beneficial method, because of omitting the time
seems that era gene could be further manipulated for the biofuel
and cost-consuming steps of recovery of the cells from the medium or
the extraction of the biofuels from the cells. Therefore, many efforts
have been made for the enhancement of FFA production and secre-
tion in cyanobacteria.
The inactivation of acyl–acyl carrier protein synthetase (Aas) by
Kaczmarzyk et al.,99 increased the FFA production in Synechocystis
sp. PCC 6803 and Synechococcus elongates PCC 7942. Aas catalyzes
the esterification of FFA to acyl carrier protein (ACP) and recycles the
released fatty acids as the components for the synthesis of complex
lipids. Liu et al.100 made several genetically engineered Synechocystis
sp. PCC 6803 (SD100) which increased FFA-secretion yields signifi-
cantly in comparison to the wild-type. The methods include the exoge-
nous expression of E. coli tesA gene in Aas-deficient, the deletion of
PHB synthesis genes (slr1993 and slr11994), the overexpression of acc
genes encoding acetyl-CoA carboxylase and the deletion of penicillin-
binding protein (PBP2). Liu et al.101 inserted a thermostable lipase gene
F I G U R E 8 The construction of thermorecovery strains. The
from Fervidobacterium nodosum Rt17-B1 into Synechocystis sp. PCC
Synechocystis PCC 6803 were transformed with a suicide vector
6803 genome, through the double-crossing over method (Figure 8). containing the thermostable lipase gene. The cassette was inserted in
The lipase was produced in the engineered strain; by increasing the the cmp gene region [reprinted with permission from Elsevier]101

F I G U R E 7 The schematic pathway for the

production of free fatty acids and valuable
chemicals in cyanobacteria [reprinted with
permission from Elsevier]29
10 of 16
synthesis. Kato et al.108 showed that the equilibrium between the
FFA production and its exportation is an important factor for the rate
of FFA productivity in cyanobacteria. They manipulated Syn-
echococcus elongatus mutant (dAS1T) by inactivation of wzt gene.
Since wzt had an effective role in the exportation of O-antigen in the
outer membrane of cyanobacteria, the wzt inactivation caused remov-
ing the O-antigen layer from the surface of the dAS1T cells; therefore,
the FFA secretion was increased. In another study, the lipid produc-
tion of Synechocystis sp. PCC 6803 was improved by the expression
of GPD1 from Saccharomyces serevisiae and DGAT from Rhodococcus
opacus. These genes, which encode glycerol-3-phosphate dehydroge-
nase and diacylglycerol acyltransferase, are the key ones in the lipid
metabolism of cyanobacteria.98
6.2 | Manipulation of cyanobacteria for production
of chemicals
The production of many chemical compounds such as aliphatic and
aromatic hydrocarbons, isobutyraldehyde, ethylene, and isopropanol
has been reported via genetic engineering of cyanobacteria. Recombi-
nant Synechococcus elongates PCC 7942 was introduced by Atsumi
et al.109 which was capable of converting CO2 into isobutyraldehyde
phototrophically. The recombinant S. elongates was further improved
by overexpression of Rubisco enzyme (rbcLS) from related S. elongates
PCC 6301 which increased CO2 fixation through Calvin cycle.
Isobutyraldehyde transforms easily to many hydrocarbons, for exam-
ple, isobutanol can be used as a replacement for gasoline fuel.
The capability of cyanobacteria in the biosynthesis of alka(e)nes
has been focused on making GM strains to produce biofuel in large-
scale. Gao et al.110 overexpressed acyl-ACP synthetase in an aas
knockout mutant of Synechocystis sp. PCC 6803, which contained a
fatty acyl-CoA reductase gene from jojoba. The fatty acyl-CoA reduc-
tase converted fatty acyl-CoA to fatty alcohols, which were the bene-
ficial substitution to the petroleum fuels. Wang et al.111 made various
mutants from Synechocystis sp. PCC 6803 to improve its alka(e)nes
biosynthesis capability. The overexpression of aar and ado genes dou-
bled the alka(e)ne production in this strain. Aar encodes an acyl-ACP
reductase (AAR) reduced acyl-ACP into aldehyde which can be further
oxidized to alkane or alkene by aldehyde-deformylating oxygenase
(ADO). The expression of foreign alkane biosynthetic genes from two
other cyanobacteria: Synechococcus elongates PCC 7942 (orf1593 and
orf1594) and Nostoc punctiforme PCC 73102 (npun1710 and
npun1711) had the significant effect on the productivity alka(e)ne in
Synechocystis sp. PCC 6803. Another mutant of Synechocystis
increased the alka(e)ne production through the overexpression of
111
acyl-ACP reductase (aar) which diverted carbon flux to acyl-ACP.
Ethylene is a chemical compound, which is widely used in various
plastic industries and can be applied to produce gasoline by polymeriza-
tion technology. The expression of optimized efe encoding ethylene-
forming enzyme from Pseudomonas syringae pv. Phaseolicola into
Synechocystis sp. PCC 6803 produced ethylene from CO2 photosyntheti-
cally. The importance of EFE is related to the fact that the ethylene biosyn-
thesis in P. syringae is carried out during one step from α-ketoglutarate,
FARROKH ET AL.
as a Krebs cycle intermediate.112 Anabaena sp. PCC 7120 is a filamentous
cyanobacterium, engineered successfully with the exogenous expression
of codon optimized farnesene synthase (Fas) gene from the Norway
spruce. The Fas has a role in the conversion of farnesyl pyrophosphate to
the farnesene (C15H24).113 Halfmann et al.114 engineered filamentous
Anabaena sp. PCC 7120 to produce limonene (C10H16), a cyclic hydrocar-
bon, which could be used for the generation of fuel for jet and other diesel
engines. In their research, the expression of limonene synthase gene (lims)
from Sitka spruce into Anabaena sp. produced limonene in secreted form
through the MEP/DXP pathway (2-C-methyl-D-erythritol 4-phosphate/
1-deoxy-D-xylulose 5-phosphate). Limonene biosynthesis was further
increased in this strain by overexpression of dxs, ipphp, and gpps genes,
encoding three MEP/DXP pathway enzymes, by changing carbon flux
from the Calvin cycle.114 The photosynthetic production of isopropanol
was reported by Hirokawa et al.115 in engineered Synechococcus elongatus
PCC 7942. The expression of five genes as thl and adc from Clostridium
acetobutylicum, atoAD from E. coli K-12, sadh from C. beijerinckii, and pta
from E. coli into the S. elongates provided essential enzymes for iso-
propanol production through photosynthesis.
Another concern for biodiesel production by cyanobacteria is the
yield and cost. Glycerol is an available and inexpensive by-product,
which can be used as a carbon source for chemical synthesis. By the
expression of the glycerol respiratory pathway genes of E. coli in Syn-
echococcus elongatus PCC 7942, Kanno et al.116 produced
2,3-butanediol with the utilization of glycerol. Alcohol dehydrogenase
(AdhA) enhances the tolerance of Synechocystis sp. PCC 6803 to
external ethanol; therefore, it is valuable for biofuel generation by
cyanobacteria. The heterologous expression of pyruvate decarboxyl-
ase from Zymomonas mobilis along with the overexpression of endog-
enous AdhA in Synechocystis increased both ethanol production and
strain survival.117
In summary, the wild-type cyanobacteria usually do not have all of
the requirements for biofuel generation; therefore, the improvement
of their potential is necessary. The manipulation of the cyanobacteria
could be made through overexpression or inactivation of endogenous
genes or expression of foreign genes. The Calvin cycle is one of the
metabolic pathways which have an important role in the production
of pyruvate and acetyl-CoA as intermediates. Therefore, the enzymes
which are involved in these pathways are interesting for metabolic
engineering. A list of the articles, which show the improvement of the
potential of cyanobacteria for biofuel production, is presented in
Table 2.
7 | BIOINFORMATIC STUDIES
The computational methods provide useful approaches for under-
standing metabolic network in microorganism and by use of them the
targeting modification of cyanobacteria for biofuel production will be
more powerful.119 Cyanobacterial metabolism has got special atten-
tion for metabolic engineering to improve their biofuel production.
Several metabolic reconstructions are available for Synechocystis
sp. PCC 6803 and different researchers also presented new updates.
FARROKH ET AL. 11 of 16

TABLE 2 A summary of the genetic engineering studies which were focused on the improvement of cyanobacterial strains for biofuel
production

Cyanobacterial strain Modification Result of genetic engineering References

Modified strains with enhanced production and excretion of free fatty acids
99
Synechocystis sp. PCC 6803Synechococcus Inactivation aas Increase of FFA production
elongates PCC 7942
100
Synechocystis sp. PCC 6803 Exogenous expression of tesA deletion of PHB Increase of FFA secretion
synthesis genes overexpression of acc deletion
of PBP2
101
Synechocystis sp. PCC 6803 Insertion a lipase gene Better release of FFA
105
Synechocystis sp. PCC 6803 Insertion of holin and endolysin genes Facilitate the recovery of biofuel
106
Synechococcus elongates PCC 7942 Exogenous expression of acyl-ACP thioesterase Enhancement of FFA production
and acetyl-CoA carboxylase genes
107
Synechococcus elongates PCC 7942 Overexpression of era Increase of total fatty acid
production
108
Synechococcus elongatus mutant (dAS1T) Inactivation of wzt Enhancement of FFA secretion
98
Synechocystis sp. PCC 6803 Exogenous expression of GPD1and DGAT Improved lipid production
Manipulation of cyanobacteria for production of chemicals
109
Recombinant Synechococcus elongates Overexpression of rbcLS Increase of CO2 fixation
PCC 7942
110
Synechocystis sp. PCC 6803 Overexpression of acyl-ACP synthetase Production of fatty alcohols
111
Synechocystis sp. PCC 6803 Overexpression of aar and ado exogenous Improvement of alka(e)nes
expression of alkane biosynthetic genes biosynthesis
overexpression of aar
112
Synechocystis sp. PCC 6803 Exogenous expression of efe Ethylene biosynthesis
118
Anabaena sp. PCC 7120 Exogenous expression of fas Production of farnesene
114
Anabaena sp. PCC 7120 Exogenous expression of lims overexpression of Production of limonene
dxs, ipphp, and gpps
115
Synechococcus elongatus PCC 7942 Exogenous expression of thl, adc, atoAD, sadh, and Isopropanol biosynthesis
pta
116
Synechococcus elongatus PCC 7942 Exogenous expression of the respiratory pathway Production of 2,3-butanediol
genes
117
Synechocystis sp. PCC 6803 Exogenous expression of pyruvate decarboxylase Ethanol biosynthesis
and alcohol dehydrogenase

A group of researchers reconstructs and compare the genome-scale
models of two related cyanobacteria: Cyanothece sp. ATCC 51142
and Synechocystis sp. PCC 6803. The metabolic differences of these
models indicated that they had various potential for biofuel produc-
tion.120 Knoop et al.121 made a genome-scale reconstruction for Syn-
echocystis sp. PCC6803, based on tricarboxylic acid cycle, an alleged
glyoxylate shunt and role of photorespiration in cellular growth. In
another study, genome-scale network model of Synechocystis sp. PCC
6803, Computational Approach for Strain Optimization aiming at high
Productivity (CASOP) and constrained Minimal Cut Sets (cMCSs) were
used for designing a strain to produce ethanol and isobutanol. The
results showed that mild enhancing ATP turnover increased biofuel
122 119
production. Mohammadi et al. made a Java tool for reconstruc-
tion of metabolic network of Synechocystis sp. PCC 6803. Three avail-
able metabolic networks of this cyanobacterium was merged and a
new model for biofuels production created. The Flux Balance Analysis
revealed increased production of ethanol, isobutanol, 3-methyl-
1-butanol, 2-methyl-1-butanol, and propanol based on new model.
Shabestary et al.123 applied genome-scale models and algorithms for
the simultaneous increase of growth rate with biofuel synthesis in
Synechocystis sp. PCC 6803 model iJN678. The results proposed that
the reduction of ATP/NADPH ratio could enhance fatty acid and ter-
pene derived fuels and the gene knockout of NADH-utilizing reac-
tions could increase the production of alcohols (e.g., butanol). Because
of the importance of large-scale FFA production in cyanobacteria, an
in silico screening method based on proteome prediction was intro-
duced to accelerate the identification of favorable candidate strains.
The FFASC software was the first in silico tool for screening of cyano-
bacteria, which was presented by Motwalli et al124
The reconstruction of genome-scale metabolic networks through
bioinformatics tools provides an applicable approach for the improve-
ment of biofuel production. These methods identify some of the pro-
duction bottlenecks and propose appropriate modifications for
removing these barriers.125
12 of 16
8 | E C O N O M I C A L P O I N T OF V I E W
The economic aspects of biofuel production, including; resource
requirements (CO2, nutrients, water), land requirement, installation
and operation costs, and needs of energy are important factors in
32
profitability of the third- and fourth-generation biofuel production.
Among all advantages, one of the main problems of biofuels is the
cost of production.126,127 Some efforts have been made during last
years for lowering the production cost, and it seems that half of the
total energy will be provided by biofuels in developing countries up to
2050.9 Vieira et al.128 used the biomass of cyanobacteria and micro-
algae obtained from the effluents of lagoon ponds as the biofuel feed-
stock. The extracted lipids from biomass had similar quality to the
traditional methods and it was applicable for biodiesel synthesis. The
utilization of water bloom for energy generation is economical and
also has environmental benefits. In another study, the cyanobacterial
water bloom of Taihu Lake, China was used as biofuel feedstock. The
catalytic pyrolysis of the water bloom with higher aromatic content
and lower nitrogenous and oxygenic compounds provided high-
quality components for bio-oil production.129
El-Mekkawi et al.130 compared the economic feasibility of Micro-
cystis aeruginosa cultivation in the flat plate and bubble column photo-
bioreactors. Although bubble column photobioreactor was beneficial
for biomass production in lab-scale, flat plate photobioreactor was
applicable for the large-scale cultivation. The economic possibility of
limonene production by genetically manipulated Anabaena sp. PCC
7120 was evaluated theoretically in 10 years through Farm-level
Algae Risk Model (FARM) for two scenarios. In the first and second
scenarios, respectively, engineered Anabaena sp. PCC 7120 has been
used for real limonene production and assumption of 100-fold
increase in productivity. Table 3 summarizes the operating cost of lim-
onene production at year 5 of the 10 years simulation. A total cost of
$108,075,868 is needed for production of 3,243,300 L/yr limonene in
TABLE 3 Summary of expenses for the theoretical limonene
production
Expense Cost ($)
CO2 0 engineered strains. In addition, the possibility of the use of waste
Nutrients 39,442
Labor and overhead 395,740
Workman's comp and unemployment tax 31,659
Water 407,134
Utilities 646,023
Electricity 25,747,929
Maintenance costs 764,698
Insurance 3,103
Harvesting chemicals 80,040,140
Total cost 108,075,868
Facility at Year 5 (for first and second scenarios). The limonene
productivity was 32,433 L/year and 3,243,300 L/year for the first and
second scenarios, respectively [reprinted with permission from
Elsevier].131
FARROKH ET AL.
the second scenario.131 In comparison with the traditional methods,
$2,926,000 are required for the production of 800 tonnes/year limo-
nene from citrus waste.132 Therefore, the using of engineered cyano-
bacteria would have economic potential for the biofuel generation in
the future.131 Flocculation is an economical method for biomass
recovery; however, usually, one method does not have a similar yield
for different microalgae and cyanobacteria. Therefore, flocculant dos-
age is an important factor in the selection of the candidate
cyanobacterial isolate to produce biofuel.133 Beside direct application
of cyanobacteria for the biofuel production, they can be used as a
feed supply for eukaryotic microalgae which can produce biodiesel
feedstock. Nascimento et al.134 indicated that the biomass of N-
fertilizer cyanobacterium, Nostoc sp. strain M2, provided a nitrogen
source for microalgae culture. Whereas the commercial production of
biofuels from microalgae is expensive, this approach prepares cost-
effective nutrient source.
In summary, although microalgae and cyanobacteria could be used
for biofuel generation, their large-scale biomass production is expen-
sive and we need to overcome this limitation.
9 | C O N CL U S I O N
Cyanobacteria are ancient photosynthetic microorganisms which are
widespread in nature. These bacteria have received special attention
as an alternative source for the biofuel generation. The most advan-
tages of cyanobacteria rather than the traditional sources are the min-
imal requirement for their culture, the short generation time, and the
ease of genetic manipulation. The success of the application of cyano-
bacteria in the biofuel synthesis depends on the genetic engineering
of them. By the expression of the various foreign genes in cyano-
bacteria, the production of many valuable chemicals is possible
through the redirecting of metabolic pathways. Although at present
most of the studies have been restricted on the model cyanobacterial
species, genetic manipulation of the others with different metabolic
potential seems to be possible at futures. Definitely, bioinformatics
tools have also a significant role in the construction of new
materials for the culturing of cyanobacteria is interesting. The waste
effluents are the cheap source and their elimination has environmen-
tal positive effects.
As discussed in this review, CO2 delivery and light exposure sys-
tems in the designing of photobioreactors and the providing condition
for biofilm formation are important factors for obtaining higher
cyanobacterial biomass. The extraction of bio-oil from the biomass is
another challenging step. The results achieved from the previous stud-
ies show that the physical methods such as pulsed electric fields and
microwave are economic for large-scale lipid extraction.
Taken together, our planet really needs clean energies for future
and cell factories of cyanobacteria are appropriate candidates for this
purpose. We believe that by the recent researches and progressions
have been made for the production of biofuel from the engineered
FARROKH ET AL.
cyanobacteria, most of the future efforts should be focused on the
economical commercial cultivation of cyanobacteria.
ORCID
Mojgan Sheikhpour https://orcid.org/0000-0002-1927-6555
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How to cite this article: Farrokh P, Sheikhpour M,
Kasaeian A, Asadi H, Bavandi R. Cyanobacteria as an eco-
friendly resource for biofuel production: A critical review.
Biotechnol Progress. 2019;e2835. https://doi.org/10.1002/
btpr.2835