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Abiotic stresses as tools for metabolites in microalgae

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Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Abiotic stresses as tools for metabolites in microalgae

Chetan Paliwal a,b, Madhusree Mitra a,b, Khushbu Bhayani a, S.V. Vamsi Bharadwaj a,b, Tonmoy Ghosh a,b,
Sonam Dubey a, Sandhya Mishra a,b,⇑
a
Salt and Marine Chemicals Division, CSIR-Central Salt & Marine Chemicals Research Institute, Gijubhai Badheka Marg, Bhavnagar 364002, Gujarat, India
b
Academy of Scientific and Innovative Research, AcSIR-CSMCRI, Gijubhai Badheka Marg, Bhavnagar 364002, Gujarat, 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

 Abiotic stresses are important tools

for metabolites in microalgae.
 Temperature, nutrient starvation,
salinity and light influence PUFAs.
 Nitrogen and light stress influence
phycobiliproteins.
 Salinity, light and nutrients influence
carotenoids.

a r t i c l e i n f o a b s t r a c t

Article history: Microalgae, due to various environmental stresses, constantly tune their cellular mechanisms to cope
Received 31 March 2017 with them. The accumulation of the stress metabolites is closely related to the changes occurring in their
Received in revised form 8 May 2017 metabolic pathways. The biosynthesis of metabolites can be triggered by a number of abiotic stresses like
Accepted 10 May 2017
temperature, salinity, UV- radiation and nutrient deprivation. Although, microalgae have been considered
Available online 15 May 2017
as an alternative sustainable source for nutraceutical products like pigments and omega-3 polyunsatu-
rated fatty acids (PUFAs) to cater the requirement of emerging human population but inadequate bio-
Keywords:
mass generation makes the process economically impractical. The stress metabolism for carotenoid
Abiotic stress
Microalgae
regulation in green algae is a 2-step metabolism. There are a few major stresses which can influence
Metabolites the formation of phycobiliprotein in cyanobacteria. This review would primarily focus on the cellular
Fatty acids level changes under stress conditions and their corresponding effects on lipids (including omega-3
Biofuels PUFAs), pigments and polymers.
Ó 2017 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1217
2. Effect of different abiotic factors on metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1218
2.1. Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1218
2.1.1. Light irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1218

⇑ Corresponding author at: Salt and Marine Chemicals Division, CSIR-Central Salt & Marine Chemicals Research Institute, Gijubhai Badheka Marg, Bhavnagar 364002,
Gujarat, India.
E-mail address: smishra@csmcri.res.in (S. Mishra).

http://dx.doi.org/10.1016/j.biortech.2017.05.058
0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
 C. Paliwal et al. / Bioresource Technology 244 (2017) 1216–1226 1217

2.1.2. Temperature stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1220

2.1.3. Salinity stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1220
2.1.4. Nutrient starvation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1220
2.1.5. The effect of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221
2.1.6. UV- radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221
2.2. Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221
2.2.1. Phycobiliproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221
2.2.2. Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223
2.3. Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223
2.3.1. Polyhydroxyalkanoates (PHA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223
2.3.2. Exopolysaccharides (EPS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223
3. Future prospective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1224
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1224
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1224

1. Introduction
The growth conditions for any organism isolated from its habi-
tat are species/strain specific, but under extreme circumstances
they produce certain metabolites to overcome and acclimatize
the stress conditions, which could be biotic or abiotic. Microalgae
shows great adaptability towards the abiotic stress factors and pro-
duce valuable metabolites. Such unique characteristic of microal-
gae can be exploited for the production of desired metabolites
through the abiotic stress as a tool integrated with microalgal
biorefinery for its sustainable development. Microalgae have
evolved from being a simple food source to the modern age high
value products and energy feedstock. Microalgae left a footprint
3.5 billion years ago, when the oldest known prokaryote, belonging
to cyanobacteria, fossilized in the Archean rocks of western Aus-
tralia (Woese et al., 1990). Eukaryotic microalgae, with their
defined organelles for specific cellular roles, have gradually
evolved from the prokaryotes (Archibald, 2015). Cyanobacteria
were the first life capable of oxygenic photosynthesis on earth cre-
ating oxygen in the atmosphere for us, regulating our biosphere
and with the urge to survive under any diverse extreme conditions
they might have gradually developed several unique means of
acclimatization with either accumulating or releasing i.e. intracel-
lular or extracellular compounds. These intracellular and extracel-
lular compounds, due to its potential high value applications are
sought globally for which strategy for efficient exploitation of the
microalgae is being developed where abiotic stresses play a role
of a very sharp tool in the biorefinery machinery. One can develop
the microalgal machinery of biorefinery based on the need, avail-
ability, natural environment and economics of the country where
it is to be exploited. The stress tool according to the geographical
location of its set up can be designed strategically, for which either
a stress factor or combination of multiple stress factors can be sta-
tistically optimized to achieve sustainability. Cultivation of
microalgae under continuous stress conditions might enhance
the lipid or carbohydrate accumulation but, one has to compromise
with the growth rates, thus reducing their overall productivities,
which ultimately increase the biofuel production cost (Pancha
et al., 2015a, 2015b, 2015c).
Most widely used strategy to overcome this problem is the two-
stage cultivation of microalgae in which cells are first grown in
nutrient-sufficient conditions to achieve maximum biomass pro-
ductivity and the culture conditions are then altered to induce
the stress conditions, thereby stimulating the accumulations of
lipid and carbohydrate in the second stage (Chen et al., 2011).
Today microalgae represent a viable alternative source for high-
value products along with the biofuel. The species Chlorella pro-
tothecoides (Cp), has been widely studied providing a high amount
of lutein and fatty acids (FA) profile ideal for biodiesel production
(Campenni et al., 2013). Nutrient deficiency or nutrient stress has
been well documented to increase TAG accumulation in microal-
gae specifically, nitrogen or phosphate limitation (Yeesang and
Cheirsilp, 2011; Rodolfi et al., 2009; Hu et al., 2008; Li et al.,
2008). Lipid productivity is strain-specific function of physiological
responses to many factors, including incident light intensity and
cultivation temperature (Griffiths and Harrison, 2009) and salinity
(Gouveia and Oliveira, 2009). It was also proven that light dilution
results in a significant enhancement in productivity of biomass
(Fernández-Ábalos et al., 1998). This is due to the radiation on cul-
ture surface, which increases the frequency of the dark cycles of
the cells, providing a higher rate of light impulses per reaction cen-
tre (Fernández-Ábalos et al., 1998).
Polyunsaturated fatty acids (PUFAs) are the essential compo-
nents for the growth of higher eukaryotes. Among them, eicos-
apentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA,
C22:6) are the most important fatty acids owing to enormous
nutraceutical and pharmaceutical applications. Microalgae are
the primary producer of PUFAs in the marine food chain and more-
over it can grow under autotrophic, mixotrophic and heterotrophic
culture conditions (Li et al., 2009), fix atmospheric carbon dioxide
(CO2) (Schenk et al., 2008), can convert light energy and CO2 into
biomass rich in carbohydrates, proteins and lipids, have shorter
harvesting time than plants, and have less chemical contamination
than fish oil.
Under abiotic stress condition, microalgae produce a large array
of compounds (including PUFAs) to survive in the extreme envi-
ronmental conditions through adaptation. Marine microalgal cells
typically contain 10–50% (w/w) oil and can produce high percent-
age of total lipid (up to 30–70% of the dry weight) (Ward and Singh,
2005). There exists a close relation between the growth phases of
microalgae and the accumulation of fatty acids, as, during cell divi-
sion and unfavourable growth conditions (where salinity, temper-
ature, light, pH, and nutrients etc. may be suboptimal), fatty acids
play an important role as energy stockpile. Therefore, the high-
energy content of omega-3 PUFAs and its ability to maintain mem-
brane fluidity leads to the accumulation of PUFAs (mainly omega-3
fatty acids) during stress condition (Cohen et al., 2000).
To date, microalgae belonging to the genera Phaedactylum, Nan-
nochloropsis, Thraustochytrium and Schizochytrium are known to
contain EPA and/or DHA. Among them, Phaeodactylum tricornutum
(Yongmanitchai and Ward, 1991) and Nannochloropsis sp. (Sukenik,
1991) reported to contain EPA up to 39% of the total fatty acids,
while DHA percentage of 30–40% of the total fatty acids was
observed in strains Thraustochytrium (Burja et al., 2006) and
Schizochytrium limacinum (Zhu et al., 2007). Under optimized stress
conditions (i.e. optimal carbon, nitrogen and phosphorus concen-
1218 C. Paliwal et al. / Bioresource Technology 244 (2017) 1216–1226
trations, different salinity regime and light intensity (Mitra et al.,
2015a; Pal et al., 2011; Jiang et al., 1999), UV radiation (Sharma
and Schenk, 2015; Liang et al., 2006) and controlled pH and tem-
perature (Mitra et al., 2015b; Jiang and Gao, 2004; Tatsuzawa
and Takizawa, 1995a, 1995b) conditions, high biomass productiv-
ity and commercially acceptable EPA and DHA productivities are
achieved with microalgae.
Apart from external stress conditions, researchers have focused
on the development of high lipid and PUFA containing microalgal
strains using metabolic engineering. Metabolic engineering with
genetic engineering is a promising approach, in targeted biosyn-
thetic pathway for enzymes involved as the key players towards
achieving the enhanced productivities. Only few genes encoding
the enzymes involved in the fatty acid biosynthesis have been
identified in some microalgal species till date (Chi et al., 2008;
Tonon et al., 2005; Domergue et al., 2003). Although, lack of infor-
mation about the mechanisms involved in the fatty acid biosynthe-
sis pathway and the role of enzymes, urge for extensive research
studies on algal metabolism.
Plants are rather complex in their organization and their life
cycle is much dependent on the environment, the studies with
their genomes are much complicated in comparison to bacteria.
Therefore, studies on the molecular mechanisms of stress
responses are not easy in higher plants. Cyanobacteria exhibit a
close phylogenetic relationship with plant chloroplast and there-
fore, are regarded as most appropriate model system for studying
plant responses to various stresses. It is much easier to carry out
such kind of research on cyanobacteria. It should be noted that at
present cyanobacteria, similarly to Escherichia coli, became one of
the most actively studied groups of living organisms after the com-
plete nucleotide sequence of the genome of Synechocystis sp. PCC
6803 was determined in 1996 (Kaneko and Tabata, 1997). Any
changes (temperature, salinity, pH, etc.) around the cell are first
sensed by the specialized sensory proteins, or sensors changing
their properties under the stress. Sensors transfer the signal about
the changes to other polypeptides – the transducers, which in their
turn regulates expression of the stress responsive genes. Trans-
ducer proteins may recognize the special regions of DNA directly,
interact with them, and regulate transcription. Finally, protection
proteins and/or metabolites are synthesized that help the cells
and organisms to adapt or acclimate to new environment. Stress
treatments may also directly affect the structure of DNA (chromo-
some packing) and cause alterations in transcription of many
genes.
Mostly, cells recognize the abiotic stress not always but when it
exceeds a certain critical level. However, cellular sensory systems
are always necessary for cells to recognize even minor changes
in the environment to produce an adequate response to these
changes. Cyanobacteria can regulate their tetrapyrrole content
and composition in response to signals, such as nutrient availabil-
ity, light intensity, light wavelength and temperature (Prassana
et al., 2004).
To tolerate salinity stress, microalgae respond by accumulation
of compatible solutes, also salinity stress has been reported to
upregulate carotenoid synthesis genes such as canthaxanthin and
astaxanthin. This may be because salinity stress generates reactive
oxygen species, hence algae respond by increasing synthesis of car-
otenoids (Li et al., 2009). Another mechanism by which algae sur-
vive under salinity stress is by changing membrane composition by
upregulating ion transporters, and aquaporins. High salinity toler-
ating algae such as Dunaliella salina produce sucrose, glycerol, pro-
line, glycine, betaine, etc as a compatible solute (osmoprotectant)
under salinity stress.
There is a need to understand the underlying molecular mech-
anism of stress responses. These would help us identify the regula-
tory elements involved and also identify bottlenecks in the
production of biofuel precursors. This knowledge would help us
engineer algae to be more resilient to stress and produce more bio-
fuel precursors under non-stress conditions. This data can also be
incorporated into the metabolic models of algae which would help
us develop in silico models of algae with desirable properties. Cur-
rently our understanding of microalgal and cyanobacterial stress
responses is limited to model organisms and prediction of gene
function by homolog identification. Availability of genetic tools is
limited and not well developed. However, by the use of transgenic
microalgae, adapted to stressful conditions, we can grow them
under conditions which inhibit growth of contaminating/ compet-
ing microalgae.
2. Effect of different abiotic factors on metabolites
2.1. Lipids
Microalgal lipids generally categorised into structural lipids
(polar lipids mainly polyunsaturated fatty acids or, PUFAs) and
storage lipids (non-polar lipids including saturated fatty acids
(SFAs) and monounsaturated fatty acids (MUFAs)). Storage lipids
mainly stored in the form of triacylglycerols (TAGs) are transester-
ified to produce biodiesel and supply energy to the metabolic pro-
cesses. PUFAs are known to maintain membrane functions and
contribute as the matrix for numerous metabolic processes. Gener-
ally, TAGs are accumulated in the cytosolic lipid bodies in presence
of light while during dark hours participated in the polar lipid syn-
thesis (Thompson, 1996). Some microalgal species, for example
Nannochloropsis sp., Pavlova lutheri, P. tricornutum etc. are known
to accumulate PUFA in TAG. A sudden change in the growth condi-
tions ameliorates microalgal lipid content. To combat with the
growth limiting stress conditions (including light intensity, tem-
perature, salinity, nutrients, pH and UV- radiation), microalgae
started accumulating lipids and/or starch as a crucial part of their
survival mechanism. Under varying abiotic stress conditions, total
lipid along with fatty acids profile undergo extreme variation
(Table 1a and 1b).
2.1.1. Light irradiation
When stressed with various light intensities, remarkable
changes in the biomass and nutrient profile of microalgae have
been reported. High light intensity increased the neutral lipid con-
tent (mainly TAG) with a simultaneous decrease of polar lipids
owing to the oxidative damage of PUFAs (He et al., 2015; Breuer
et al., 2013; Carvalho and Malcata, 2005). Exposure to high light
increased the level of TAG in many algal species including Chlorella
sp. (He et al., 2015), Monoraphidium sp. (He et al., 2015), Scenedes-
mus obliquus (Breuer et al., 2013), Pavlova lutheri (Carvalho and
Malcata, 2005), Nannochloropsis gaditana (Mitra et al., 2015a).
Although light irradiance act as a stimulant in the production of
microalgal biodiesel however the %accumulation of TAG will vary
for different species.
Several research studies with EPA producing microalgae includ-
ing Nannochloropsis sp. (Sukenik et al., 1989), N. salina (Van
wagenen et al., 2012), N. gaditana (Mitra et al., 2015a) confirmed
that PUFA (especially EPA and/or DHA) content of microalgae is
inversely related to light irradiation. This phenomenon may be
attributed to the Reactive Oxygen Species (ROS) (which formed
under unfavourable growth conditions) quenching activity of PUFA
(Sukenik et al., 1989, 2009). Moreover, PUFAs may involve in the
functioning of thylakoid membrane and are essential for the pho-
tosynthesis (Cohen et al., 1988; Kates and Volcani, 1966). Under
the effect of high irradiance, the photosynthetic potential of algae
becomes less, suggesting the requirement of relatively less thy-
lakoid membranes. Owing to this phenomenon, high light accli-
 C. Paliwal et al. / Bioresource Technology 244 (2017) 1216–1226 1219

Table 1a
Effect of abiotic stress factors on neutral lipid (TAG) content of microalgae.

Microalgae Neutral lipid Abiotic stress factors applied Major observations References
(TAG)
Chlorella sp. TAG High light intensity Increase in TAG He et al. (2015)
Monoraphidinium sp. TAG High light intensity Increase in TAG He et al. (2015)
Scenedesmus obliquus TAG High light intensity Increase in TAG Breuer et al. (2013)
Pavlova lutheri TAG High light intensity Increase in TAG Carvalho and Malcata (2005)
Acutodesmus TAG Temperature stress at 35 °C Increased accumulation of neutral lipid Chokshi et al. (2015)
dimorphus
Dunaliella tertiolecta TAG Increasing NaCl concentration (0.5 M to Increase in total lipid content and TAG Takagi and Yoshida (2006)
2.0 M)
Scenedesmus sp. TAG Salinity stress of 400 mM Increase accumulation of neutral lipid Pancha et al. (2015b)
Chlorella sp. TAG Nitrogen starvation Increased accumulation of lipids (mainly TAG) Praveenkumar et al. (2012)
Chlorella vulgaris TAG Nitrogen starvation Increased accumulation of lipids (mainly TAG) Yeh and Chang (2011)
Scenedesmus sp. TAG Nitrogen starvation Increased accumulation of lipids (mainly TAG) Pancha et al. (2014)
Phaeodactylum TAG Phosphorus limitation Increased accumulation of TAG Reitan et al. (1994)
tricornutum
Isochrysis galbana TAG Phosphorus limitation Increased accumulation of TAG Reitan et al. (1994)
Chaetoceros sp. TAG Phosphorus limitation Increased accumulation of TAG Reitan et al. (1994)
Pavlova lutheri TAG Phosphorus limitation Increased accumulation of TAG Reitan et al. (1994)
Monodus subterraneus TAG and PUFA Phosphorus limitation  6-fold increase in TAG and decrease in EPA Khozin-Goldberg and Cohen
content (2006)
Scenedesmus sp. TAG Nitrate starvation Increased accumulation of neutral lipid Pancha et al. (2015a,b,c)
Monoraphidinium SFA Optimum carbon source Increase in SFA content Patidar et al. (2014)
minutum
Chlorella sp. TAG Alkaline pH stress Increased accumulation of TAG Guckert and Cooksey (1990)
Tetraselmis sp. SFA and MUFA UV-B radiation Overall increase in the SFA and MUFA content Goes et al. (1995)
Chaetoceros mualleri MUFA Combined effect of UV-A and UV-B Increase in the MUFA content Liang et al. (2006)
Pavlova lutheri TAG UV radiation Increase in TAG with decrease in EPA and DHA Guihéneuf et al. (2009)

Table 1b
Effect of abiotic stress factors on PUFA content of microalgae.

Microalgae PUFA Abiotic stress factors applied Major observations References

Chaetoceros brevis EPA and Low temperature and low irradiance Enhanced production of EPA and DHA Boelen et al. (2013)
DHA
Thalassiosira weissflogii EPA and Low temperature and low irradiance Enhanced production of EPA and DHA Boelen et al. (2013)
DHA
Pyramimonas sp. EPA and Low temperature and low irradiance Enhanced production of EPA and DHA Boelen et al. (2013)
DHA
Emiliania huxleyi EPA and Low temperature and low irradiance Enhanced production of EPA and DHA Boelen et al. (2013)
DHA
Fibrocapsa japonica EPA and Low temperature and low irradiance Enhanced production of EPA and DHA Boelen et al. (2013)
DHA
Isochrysis galbana ALA and Decrease in temperature 25 °C to 10 °C Significant increase in ALA and DHA Zhu et al. (1997)
DHA content
Pavlova lutheri EPA Decrease in temperature (10 °C) Increase in EPA content Tatsuzawa and Takizawa
(1995a,b)
Phaeodactylum EPA Decrease in temperature 25 °C to 10 °C Increase in EPA content Jiang and Gao (2004)
tricornutum
Nannochloropsis sp. EPA Combined effect of low temperature and low 3.4-fold increase in the EPA content Mitra et al. (2015b)
irradiance
Nannochloropsis EPA Increase in salinity Decrease in EPA productivity Mitra et al. (2015a)
gaditana
1
Crypthecodinium cohnii DHA 9 g L NaCl Increase in DHA content Jiang et al. (1999)
Phaeodactylum EPA Nitrate stress Increased accumulation of EPA Yongmanitchai and Ward
tricornutum (1991)
Nitzschia laevis EPA Urea used as a nitrogen source Increase in EPA productivity Yongmanitchai and Ward
(1991)
Chlorella kessleri PUFA Phosphorus starvation Increase in unsaturated fatty acid El-Sheek and Rady (1995)
content
Phaeodactylum EPA High phosphorus supplementation in the media Increase in EPA content Yongmanitchai and Ward
tricornutum (1991)
Crypthecodinium cohnii DHA Utilization of glucose as carbon source Increase in DHA content Jiang et al. (1999)
Nitzschia laevis EPA Silicate limitation Increased accumulation of EPA Wen and Chen (2000)
Pinguiococcus PUFA pH 7 Increase in PUFA content including EPA Sang et al. (2012)
pyrenoidosus
Nannohloropsis oculata PUFA UV-A radiation Increased accumulation of PUFA Srinivas and Ochs (2012)
Phaeocystis antarctica PUFA Low UV-B radiation Increased accumulation of PUFA Skerratt et al. (1998)
Tetraselmis sp. PUFA UV-B radiation 50% decrease in the PUFA content Goes et al. (1995)
Phaeodactylum EPA UV radiation Increase in EPA content Liang et al. (2006)
tricornutum
1220 C. Paliwal et al. / Bioresource Technology 244 (2017) 1216–1226
mated algae resulted in the accumulation of less PUFA (mainly EPA
and/or DHA) (Patil et al., 2005).
2.1.2. Temperature stress
Research studies till date have projected a general trend
between the lipid profile of microalgae and temperature. It has
been observed in most of the microalgae that polar lipid content
increased with decreasing temperature whereas increased temper-
ature leads to the higher accumulation of nonpolar lipids (TAG)
(Renaud et al., 2002). Temperature stress at 35 °C led to an eleva-
tion in the lipid content (22.7%) and increased accumulation of
neutral lipid (59% of total lipids) in Acutodesmus dimorphus
(Chokshi et al., 2015).
Long chain PUFAs (LC-PUFAs, mainly EPA and DHA) play a cru-
cial role in maintaining cell membrane fluidity (Nishida and
Murata, 1996). Thus, the fatty acid profile of microalgae grown in
relatively low temperature conditions must be composed of a good
amount of PUFA (mainly EPA and DHA), suggesting the require-
ment of those LC-PUFAs in the metabolic profile to survive in the
unfavourable conditions. In a recent study with five polar and
cold-temperate microalgae, enhanced EPA and DHA production
was achieved under low temperature and irradiance conditions
(Boelen et al., 2013). With a decrease in temperature from 25 °C
to 10 °C, a significant increase in the ALA and DHA content was
observed in Isochrysis galbana (Zhu et al., 1997). EPA content of
Pavlova lutheri augmented from 20.3 to 30.3% when temperature
was reduced to 15 °C (Tatsuzawa and Takizawa, 1995a,b). Simi-
larly, when the culture temperature was shifted from 25 °C to
10 °C for 12 h, significant increase in the EPA content was observed
in Phaeodactylum tricornutum (Jiang and Gao, 2004). Mitra et al.
(2015b) observed a 3.4-fold augmentation in the EPA content (%)
of Nannochloropsis sp., when cultivated in a two-stage cultivation
process under the combined effect of low temperature and irradi-
ance. Algae adapt to low temperatures by increasing the produc-
tion of unsaturated fatty acids such as PUFAs which maintain
membrane fluidity at low temperatures.
2.1.3. Salinity stress
Salinity may also tune the lipid biosynthesis pathway in
microalgae, although not in an unwavering mode. Microalgae that
can tolerate high salt concentration are most suitable candidate for
salt tolerance study. Total lipid content along with a higher per-
centage of TAG was observed in Dunaliella tertiolecta while stressed
with increasing NaCl concentration (0.5 M–2.0 M) (Takagi and
Yoshida, 2006). In a two-stage process, salinity stress of 400 mM
was resulted in 24.77% lipid (containing 74.87% neutral lipid) in
Scenedesmus sp. (Pancha et al., 2015a,b,c). An increase in the salin-
ity (30–40 g L 1) reported to reduce the biomass productivity
together with lipid and EPA productivities in Nannochloropsis gadi-
tana (Mitra et al., 2015a). When grown in a medium containing
9 g L 1 NaCl, the DHA content of Crythecodinium cohnii ATCC
30556 escalated up to 56.9% of the total fatty acids (Jiang et al.,
1999). Jiang and Chen (1999) reported highest DHA yield of
131.55 mg L 1 for C. cohnii ATCC 30556 at 9 g L 1 NaCl while sup-
plementation of 5 g L 1 NaCl showed highest DHA yield of
128.83 mg L 1 in C. cohnii RJH.
2.1.4. Nutrient starvation
Microalgal growth and their lipid and fatty acid composition are
closely related to the nutrient content of their cultivation medium.
Nutrient starvation being one the majorly applied lipid induction
strategies, has been reported to regulate the percentage of polar
and nonpolar lipid although their effect differs within different
species (Rodolfi et al., 2009). For example, enhanced accumulation
of TAG with a dwindling PUFA content was reported for diatom
Stephanodiscus minutulus, when cultivated under silicon, nitrogen
and phosphorus limiting conditions (Lynn et al., 2000).
2.1.4.1. Nitrogen stress. Nitrogen is the most applied nutrient stress
factor towards the optimisation of lipid metabolism process of
microalgae. In response to nitrogen starvation, accumulation of
lipids (mainly TAG) was detected in numerous microalgal strains
including Chlorella vulgaris (Yeh and Chang, 2011), Chlorella sp.,
(Praveenkumar et al., 2012), Scenedesmus sp. (Pancha et al.,
2014). Increased accumulation of EPA in P. tricornutum under
nitrate stress condition was reported by Yongmanitchai and
Ward (1991), whereas EPA productivity was maximum in N. laevis
when urea was supplemented in the medium as the nitrogen
source. However, utilization of ammonia as the sole nitrogen
source in diatom cultivation, was found to be detrimental for
growth and PUFA production. This negative correlation between
PUFA content and nitrogen starvation may be related to the fact
that when algal cells were exposed to nitrogen limiting condition,
cells escalated TAG synthesis (consist of SFAs and MUFAs) pathway
at the expense of polar lipids (mainly PUFAs) (Cohen, 1999).
2.1.4.2. Phosphorus limiting condition. Phosphorus being an essen-
tial nutrient in the algal media, plays a major role in microalgal
growth and also involved in metabolic processes such as energy
transfer in cells, nucleic acid biosynthesis, phospholipid biosynthe-
sis and membrane development. Phosphorus limitation occasioned
increased accumulation of TAG in P. tricornutum, Isochrysis galbana,
Chaetoceros sp., Pavlova lutheri, though decrease lipid content in
Tetraselmis sp. and Nannochloris atomus (Reitan et al., 1994). In
Monodus subterraneus, 6-fold increase in the TGA percentage
was observed with a gradually decreasing EPA content, when sub-
jected to phosphorus limiting stress condition (Khozin-Goldberg
and Cohen, 2006). On the contrary, an elevated level of unsaturated
fatty acids was observed in phosphorus starved cells of Chlorella
kessleri (El-Sheek and Rady, 1995). Given the abundance of PUFAs
in the phospholipid fraction, the concentration of phosphorous in
the growth media can be directly involved in the overall yield of
PUFAs (Guschina and Harwood, 2009). When grown in a growth
medium supplemented with high phosphorus, enhanced produc-
tion of EPA was observed in P. tricornutum (Yongmanitchai and
Ward, 1991).
2.1.4.3. Carbon source. Of late, microalgae are gaining interest for
their ability of carbon sequestration and also to evaluate the
changes occurred in their fatty acid profile when stressed with
organic and inorganic carbon source. In a study carried out by
Patidar et al. (2014) on Monoraphidium minutum the effect of opti-
mum glucose, fructose and microalgae biodiesel waste residue and
sodium acetate were found to increase the saturated fatty acid con-
tent of microalga. When stressed with intermittent supply of
sodium hydrogen carbonate at different pH level, drastic changes
in the fatty acid profile of Chlorella variabilis ATCC 12198 was
observed by Patidar et al. (2016). Utilization of glucose as a carbon
source was found to enhance the biofuel producing potential of
Scenedesmus sp. (Pancha et al., 2014). Increased accumulation of
neutral lipid (88.69 ± 2.1% of total lipid) was observed in nitrate
starved hydrogen carbonate supplemented cells of Scenedesmus
sp. (Pancha et al., 2015a,b,c). When cultivated in Porphyridium
medium containing 5 g L 1 glucose, highest DHA content (51.12%
of the total fatty acids and 7.79% of the cell dry weight) was
observed in marine dinoflagellate Crypthecodinium cohnii ATCC
30556 (Jiang et al., 1999).
2.1.4.4. Silica. Several reports convocated diatoms as one the
potential source of PUFAs mainly EPA and/or DHA. Diatom cell
walls are basically composed of silica and therefor presence or
 C. Paliwal et al. / Bioresource Technology 244 (2017) 1216–1226 1221

Table 2
Effect of abiotic stress factors on microalgal pigment.

Microalgae Pigments Abiotic stress factors applied Major observations References

Chlamydomonus reinhardtii
Dunaliella salina
Synechocystis sp.
Chamaesiphon sp.
Dermocarpa sp.
Xenococcus sp.
Phormidium sp.
Pseudanabaena sp.
Fremyella diplosiphon
Spirulina fusiformis
Oscillatoria sp.
Carotenoids High light stress Induced synthesis of violaxanthin Couso et al. (2012)
Carotenoids Salt, light and nutrient depletion Upregulates carotenoid production Ramos et al. (2008)
Carotenoids Salinity Higher production of b-carotene Paliwal et al., 2015a,b
C-PC and C-PE Chromatic adaptation Upregulates production of C-PC and C-PE Tandeau de Marsac (1977)
C-PC and C-PE Chromatic adaptation Upregulates production of C-PC and C-PE Tandeau de Marsac (1977)
C-PC and C-PE Chromatic adaptation Upregulates production of C-PC and C-PE Tandeau de Marsac (1977)
C-PC and C-PE Chromatic adaptation Upregulates production of C-PC and C-PE Tandeau de Marsac (1977)
C-PC and C-PE Chromatic adaptation Higher production of C-PC and C-PE Mishra et al. (2012)
C-PC and C-PE Salt stress and chromatic adaptation Increased production of C-PC and C-PE Singh and Montgomery (2013)
C-PC Salt stress Increased production of C-PC and C-PE Rafiqul et al. (2003)
C-PC and C-PE Chromatic adaptation and temperature Upregulates production of C-PC and C-PE Singh et al. (2012)

absence of silica in the growth medium may affect the biomass and
lipid profile of diatoms. In silicate limited cultures of Nitzschia lae-
vis, increased accumulation of EPA was reported by Wen and Chen
(2000). The reason behind this situation may be attributed to the
change in the metabolism of diatom cells during silica replete con-
ditions, as they may divert the energy allocated for silicate uptake,
towards lipid storage.
2.1.5. The effect of pH
Fluctuations of the medium pH also alter the microalgal lipid
composition. The highest percentage of total PUFAs (38.75%) and
EPA (23.13% of the total fatty acids) were observed in Pinguiococcus
pyrenoidosus 2078 at a pH level of 7 (Sang et al., 2012). In a study
with Chlorella sp. CHLOR1, alkaline pH stress directed accumula-
tion of TAG was observed with simultaneous decrease in the mem-
brane lipid content (Guckert and Cooksey, 1990).
2.1.6. UV- radiation
The effect of UV- irradiance in microalgae is mainly concen-
trated on the influence of UV-A and UV-B radiation on microalgal
growth and biomass nutrient profile (including lipids). Exposure
to UV-A radiation significantly increased PUFA content of N. oculata
(Srinivas and Ochs, 2012). In a study carried out by Skerratt et al.
(1998), increased accumulation of PUFA was observed in Phaeocys-
tis antarctica under low UV-B radiation. In Tetraselmis sp., UV-B
radiation resulted in an overall increase in the SFAs and MUFAs
content with a 50% decrease in the PUFA content (Goes et al.,
1995). Under combined effect of UV-A and UV-B, increase in the
MUFA content was reported in Chaetoceros mualleri (Liang et al.,
2006). In Pavlova lutheri, exposure to UV radiation trigger the accu-
mulation of storage lipids at the expense of EPA and DHA content
(Guihéneuf et al., 2009). Generally, it has been found that abiotic
stress conditions which lead to the formation of ROS and lipid per-
oxidation are directly proportional to the elevated PUFA content.
PUFAs actively participate in the membrane repair mechanism of
cells with free radical scavenging activity. An increase in the EPA
content (up to 19.84%) of Phaeodactylum tricornutum was observed
when exposed to UV light (Liang et al., 2006).
2.2. Pigments
Pigment content of microalgae (including cyanobacteria) can be
altered under the effects of various abiotic stress factors like low
light intensity, chromatic adaptation, temperature, salinity and
nutrient availability (Table 2).
2.2.1. Phycobiliproteins
Phycobilisome –the light harvesting apparatus in cyanobacteria
and red algae, which is composed of water-soluble phycobilipro-
teins covalently bound by linker peptides or proteins in a configu-
ration designed to transfer energy to the photosynthetic reaction
center of the organism. Phycobiliproteins are highly fluorescent
because of their covalently bound, linear tetrapyrrole chro-
mophores known as bilins.
Natural colorants such as phycobiliproteins are gaining impor-
tance over synthetic ones, as they are nontoxic and non-
carcinogenic. They are widely used in cosmetics and as label for
antibodies and receptors. Bio-sensor, Neuroprotective, Antioxi-
dants, anti-inflammatory, anti-nephrolithe, anti-hyperglycemic
and hepatoprotective properties are also manifested by Phyco-
biliproteins (Ghosh et al., 2016; Bhayani et al., 2016; Paliwal
et al., 2015a,b).
2.2.1.1. Complementary chromatic adaptation (CCA). Photosynthetic
organism like cyanobacteria regulates their relative pigment pro-
duction and content in response to changes in light intensity and
light wavelength. The effect of light wavelength on the pigment
content of the cells, known as complementary chromatic adapta-
tion (CCA) in cyanobacteria. The term ‘‘complementary chromatic
adaptation” was used by Engelmann. During CCA, cyanobacterial
cells change from brick red to bright blue green, depending on their
light color. In this type of adaptation, changes in cell pigmentation
in response to specific spectral illuminations result from modera-
tion of the relative amounts of the red colored phycoerythrin
(PE) and the blue-colored phycocyanin (PC), with a predominance
of PE in green-light-grown cells and of PC in red-light grown cells.
These phycobiliproteins (PE and PC) are the major light harvesting
pigments used to drive photosynthesis. Therefore, this mode of
chromatic control allows cells to trap the available light energy
with maximum efficiency (Tandeau de Marsac, 1983). Several
studies have shown that the regulation of CCA is complex and
involves total three pathways. One is regulate by a phytochrome-
class photoreceptor that is responsive to green and red light and
a complex two component signal transduction pathway, whereas
another is based on sensing the redox state. Thus, CCA can only
occur in cyanobacterial species that make both PC and PE, although
not all such species are capable of this process (Tandeau de Marsac,
1977). Pseudanabaena sp. grown under different quality of light.
Here, maximum phycoerythrin production was observed in green
light (39.2 mg L 1), while phycocyanin production was maximum
in red light (10.9 mg L 1) (Mishra et al., 2012). Cyanobacterial spe-
cies that undergo CCA must have stockpile the structural and reg-
ulatory genes needed for this response. Fujita and Hattori (1960)
demonstrated that in the soil cyanobacterium Tolypothrix tenuis
(PCC 7101), CCA was controlled by a photoreversible pigments.
These results suggested that CCA was controlled by a photorecep-
tor rather than via photosynthesis or another cellular process.
Within species that produce both PE and PC, three response groups
were defined. Group I strains, which made up about 27% of the
total, were not capable of CCA because they altered neither PE
nor PC abundance in response to changing light colors. Group II
strains comprised 16% of the total. These had elevated PE levels
1222 C. Paliwal et al. / Bioresource Technology 244 (2017) 1216–1226
in green light but did not vary PC levels in response to green-red
light shifts. The remaining 57% were group III strains, which varied
both PE and PC, increasing PE in green light and PC in red light. It
has become clear that many additional cellular responses are
affected by shifts between green and red light, including changes
in cell and filament assembly (Bogorad et al., 1982; Bennett and
Bogorad, 1973), cell differentiation states (Damerval et al., 1991;
Lazaroff and Schiff, 1962), and the abundance of many RNAs and
proteins that do not encode PBS components (Stowe-Evans et al.,
2004; Gendel et al., 1979).
There is considerable morphological changes and cellular
response during CCA. Under red light, cells are smaller and more
rounded than during growth in green light. Filament length is
approximately 10 times less in red light due to the conversion of
approximately 20% of the cells to necridia (dead cells in filament)
(Bogorad et al., 1982; Bogorad, 1975; Bennett and Bogorad,
1973). The synthesis of hormogonia is CCA regulated in F. diplosi-
phon. Hormogonia are short, differentiated, motile filaments that
develop pili and gas vesicles. They are resistant to adverse environ-
mental conditions and appear to play a role in survival (Tandeau de
Marsac, 1983; Rippka et al., 1979). CCA also affects the growth rate
of F. diplosiphon cells grown with glucose in darkness. A daily five-
minute green-light exposure depresses the dark growth rate by up
to 50%, and this effect can be reversed by brief exposure with red
light (Diakoff and Scheibe, 1975). The above findings suggest that
there are many changes in protein abundance during CCA in addi-
tion to those involved in the restructuring of PBS, several studies
have confirmed this. Protein-labeling experiments demonstrated
that the abundance of several unidentified membrane-associated
polypeptides changes during CCA (Gendel et al., 1979). CCA
responses in cyanobacteria are responsive to the redox state of
photosynthetic electron transport chain and some photoreceptors
that are maximally sensitive to green and red light (Haury and
Bogorad, 1997; Campbell et al., 1993).
The redox state of the plastoquinone pool controls the cellular
differentiation of green-light-acclimated cells. Shifting these cells
to red light partially oxidizes the plastoquinone pool, leading to
inhibition of heterocyst development and the production of hor-
mogonia (Campbell et al., 1993).
2.2.1.2. Salinity. Salinity is one of the most important factors that
limits the growth and productivity of microorganisms (Inabha
et al., 2001). Salinity has a noticeable effect on agriculture, affect-
ing huge part of the world’s irrigated land area. Salt has been
shown to have detrimental effects on growth in cyanobacterial sys-
tems. Salt adaptation in the cyanobacterium initiates two mecha-
nisms; the initial mechanism stimulates photosynthetic activity
and modification of the photosynthetic apparatus with the aggre-
gation of sucrose as an osmoregulator. The secondary mechanism
involves the adaptation of N2 fixation activity and protein biosyn-
thesis (Blumwald and Tel-Or, 1982). 200 mM NaCl in combination
with CCA was found to maximally inhibit cell growth and chloro-
phyll levels, and accumulation of PE and PC under green and red
light, respectively. NaCl also affected cellular morphology resulting
in a larger cell size under both light conditions. Cell length
decreased while width increased under green light in the presence
of salt, and both cell length and width were increased under red
light with salt. The addition of osmoprotectant glycine betaine
(GB) to the growth medium in the presence of salt resulted in a
change in the morphology of cells growing in the absence of salt,
whereas GB treatment in the presence of salt did not have a major
effect on PE and PC biosynthesis or accumulation. Salt impacts
photosynthetic pigment content and morphology in Fremyella
(Singh and Montgomery, 2013). The chlorophyll and cellular pro-
tein contents increased, when 50 mM NaCl incorporated in media.
Further increment in NaCl concentration, shows significant
decrease in both chlorophyll and cellular protein with morpholog-
ical variations of organism by having alteration in their size and
volume, thus increased level of protein was recorded in NaCl
adapted cells of A. cylindrica in response to NaCl stress
(Bhadauriya et al., 2007). Studies have shown that protein content
varied from 37.3% to 56.1% under salt stress (Rafiqul et al., 2003).
2.2.1.3. Temperature. Temperature is the most foundational factor
for organisms as it influences metabolic processes and biochemical
composition of cells. The optimal growth temperature and toler-
ance to the extreme values usually changes from strain to strain.
Sudden temperature changes exert stress on the organisms. Envi-
ronmental stress influences the organisms to inhibit or enhance
the functioning and production of some physiologically important
proteins. One such physiologically important group of proteins is
phycobiliproteins. Different components of PBSs evolved indepen-
dently from each other according to need of cyanobacteria under
different stress conditions. Phycocyanin from Synechococcus lividus
has the same amino acid composition, molecular weight, sedimen-
tation, properties as the Phycocyanin from non-thermal blue green
algae (Edwards and Gantt, 1971). Thermo tolerant Oscillatoria sp.
produced phycoerythrin and phycocyanin (PC) at 30 ± 2 °C and
55 ± 2 °C, respectively whereas at 42 ± 2 °C temperature, it pro-
duced phycoerythrocyanin (Singh et al., 2012). Cyanidioschyzon
merolae from sulfuric hot springs and habitat near volcanic areas
accumulates thermostable phycocyanin growing at 83 °C. These
properties make the C. merolae phycocyanin an interesting alterna-
tive to Arthrospira platensis as a natural blue food colorant (Rahman
et al., 2016).
2.2.1.4. Reactive oxygen species and anti-oxidants. Reactive oxygen
species (ROS) have been implicated both as secondary messengers
as well as harmful by-products of various cellular processes. Their
detrimental activities increase under adverse environmental stress
conditions (Das and Roychaudhury, 2014). Among these environ-
mental conditions, heavy metal stresses and UV-B exposure are
among the chief culprits which give rise to harmful ROS in the
organisms. Exposure to UV-B component of sunlight induces the
generation of ROS in many plant cells. However, a ‘low’ exposure
is not sufficient for damage to the cellular components; on the con-
trary, a ‘low’ UV-B exposure prepares the anti-oxidant defences of
the plant cells and prepares them for a ‘fight’. This phenomenon
has been termed as eustress, which is an important part of the
acclimatization process (Hideg et al., 2013). In order to cause dam-
ages, a ‘high’ exposure is often required. However, the threshold
level of intensity which separates ‘high’ and ‘low’ often varies con-
siderably according to the terrain and environment of growth.
To protect themselves from these ROS and their ill effects,
cyanobacteria have evolved different protection mechanisms, chief
among which are ROS scavenging enzymes such as superoxide dis-
mutase, peroxidase and catalase (Zeeshan and Prasad, 2009). They
all have a collective role in scavenging superoxide and peroxide
radicals, converting them to relatively less harmful substances.
Another way to reduce the effects of ROS on cellular functions is
by non-enzymatic scavenging using various anti-oxidants such as
ascorbic acid, reduced glutathione, carotenoids and osmolytes like
proline (Das and Roychoudhury, 2014).
Phycobiliproteins (PBPs) have a significant role as anti-oxidants
in vitro, which has been extensively demonstrated by various study
groups over many years. Their anti-oxidant activity has been
demonstrated both in the form of crude extracts and in their puri-
fied forms (Paliwal et al., 2015a,b; Thangam et al., 2013; Benedetti
et al., 2004). Our group has studied the antioxidant activities of the
water extracts of different cyanobacterial species, with the result
that their phycobiliprotein content has been directly implicated
 C. Paliwal et al. / Bioresource Technology 244 (2017) 1216–1226
as the reason for their anti-oxidative properties (Ghosh et al., 2016;
Paliwal et al., 2015a,b).
However, their in vivo effects are still largely confined to cell
line and animal model studies. A detailed view of the mechanisms
involved in ROS scavenging and corresponding anti-oxidant mech-
anisms in vivo is a lacuna which should be addressed.
2.2.2. Carotenoids
Carotenoids are natural tetraterpenoids produced by all photo-
synthetic organisms to protect themselves from photodamage and
to aid in photosynthesis (Miller et al., 2002). Keeping in view that
photosynthesis is the primary conversion of sunlight to usable
energy, major producers adopt all possible measures to protect
and enhance this process. They are widely used as a nutraceutical
and natural colorant in the cosmetic industry, but they also find
application in the chemotaxonomy and therapeutics (Paliwal
et al., 2016; Ghosh et al., 2015).
2.2.2.1. Light. Microarray and proteomic approaches have been
used to identify genes and proteins induced by high light in
Chlamydomonas. Exposure of dark-grown cells to high light inten-
sity (800 mEs m 2 s 1) seemed to trigger induction of both phy-
toene synthase (psy) and phytoene desaturase (Pds) as after 1 h
of exposure to very high light their transcript levels were improved
by2- and 4-fold, respectively. LCYE and LCYB, involved in the
cyclization of lycopene to yield ‘‘-carotene and/or -carotene,
showed a slight decrease in their mRNA level. For ZEP, which catal-
yses the synthesis of violaxanthin and is directly involved in the
xanthophyll cycle, where it was observed that there is a very slight
decrease in its transcription levels during high illumination condi-
tion. The most interesting results concerned the expression of the
P450 cytochrome–dependent” - and -ring carotene hydroxylase
genes (Couso et al., 2012).
2.2.2.2. Nutrient. The carotenoid synthesis is highly controlled and
the transcriptional regulation of Lycopene beta-cyclase (Lcy-b)
gene expression in D. salina has been investigated previously with
different environmental stress conditions (Ramos et al., 2008). The
study states that there is an increase in Steady-state DsLcy-b
mRNA levels when D. salina cells were subjected to abiotic stress
like high salinity and light condition. Also, excessive salinity and
high light conditions results into highest level of steady-state
DsLcy-b mRNA when combined with nutrient depletion strategy.
These results show that nutrient depletion is critical for b-
carotene accumulation in this microalga. Coesel et al. (2008) also
suggests that D. salina Psy and Pds are also similarly regulated.
There are certain transcriptional inhibitors that stop carotenoid
biosynthesis in D. salina, which are crucial factors in their regula-
tion in D. salina and other carotenogenic organisms. It is also
observed that in other algae, namely H. pluvialis and C. reinhardtii,
the up-regulation of other carotenogenic genes is caused due to
stressful conditions. Similar type of regulatory control also exists
during the development of fruit and flowers in higher plants at
transcriptional level of carotenoid biosynthesis. Moreover, there
is a need to understand about regulatory factors including post-
transcriptional, translational and metabolic like feedback mecha-
nism through metabolites (e.g. retinol and other b-ring-
containing compounds or end-products) and redox control. Salt-
stress up-regulated the carotenoid ketolase (BKT) in Chlorella
zogingiensis and enhanced the accumulation of canthaxanthin
and astaxanthin. ROS generation is stimulated due to high salinity,
triggering the up-regulation of characteristic carotenogenic genes
and improving the carotenoid yield. Although, there are limited
studies on the effect of nutrients on the carotenoid composition
of cyanobacteria, we have found selective carotenoid accumulation
in Synechocystis sp. in our study (Paliwal et al., 2015a,b).
1223
2.2.2.3. Metabolic engineering. There have been various research
focussing on enhancement of astaxanthin in plants due to its
potential health benefits in some of the life-threatening diseases
like cardiovascular diseases, diabetes, etc. (Fassett and Coombes,
2011). The study by Kim and Portis (2004) suggest that under
the influence of chromoplast-associated PDS promoter from green
alga Haematococcus pluvialis in the nectary, CrtO ketolase gene
overexpresses resulting in the high levels of astaxanthin and other
ketocarotenoids. Similarly, overexpression of bacterial CrtZ and
CrtW enzymes (encoding, respectively, b-carotene hydroxylase
and ketolase) lead to high levels of ketocarotenoids in nectary,
and low levels in leaf tissue in tobacco.
2.3. Polymers
Cyanobacteria have many unexplored potential for natural
products with a huge variability in structure and biological activity.
Their products are species specific and growth condition specific.
Under stress conditions they are reported to produce biopolymers
like EPS and PHA, which can be produced extracellularly and intra-
cellularly, respectively.
2.3.1. Polyhydroxyalkanoates (PHA)
Polyhydroxyalkanoates (PHAs) are linear polyesters produced
by microbial fermentation of sugar or lipids storing carbon and
energy. Halophilic bacteria like Halomonas hydrothermalis accumu-
lates PHA under different stress conditions utilizing various raw
substrates are (PHAs) (Bera et al., 2015) Interest in cyanobacterial
PHAs gained attention due to its minimum requirement for its
growth. PHAs have different applications in packaging films, dis-
posable items, bone replacements, blood vessel replacements and
scaffold material in tissue, etc. The stress condition of salinity in
which organism grows almost reduces the contamination problem
A. subsalsa is reported to produce 129.8 mg/g CDW of PHA when
cultivated in saline ASN-III media (Shrivastav et al., 2010). A study
conducted on A. platensis suggested that in phosphate limited con-
dition poly 3-hydroxybutyrate (PHB) biosynthesis occurs even
after 30 days of growth (Panda et al., 2005).
2.3.2. Exopolysaccharides (EPS)
Cyanobacteria extracellularly secretes heteropolysaccharides,
which are frequently associated with small amounts of non-
carbohydrate substituents, such as peptides, fatty acids (De
Philippis and Vincenzini, 1998). Exopolysaccharides (EPS) from A.
platensis secretes sulfated EPS to the surrounding media which
plays an important role in the survival of the producer organisms
under extreme conditions (Potts, 2001). Trabelsi et al. (2009)
observed the role of temperature (33 °C–35 °C) indicating opti-
mum EPS productivity while decrease in temperature increases
the growth of Arthrospira.
3. Future prospective
In order to achieve cost effective and sustainable production of
microalgal products, a suitable biorefinery approach should be
opted based on the biomass nutrient profile of microalgae. Abiotic
stresses can be used to alter the microalgal metabolite profiles and
thereby utilization of nutrient rich biomass for both high value and
low value products. Establishment of sustainable and economically
viable biorefinery urge for development of less harsh cell disrup-
tion methods that do not degrade high value products such as pro-
teins (including phycobiliproteins) and PUFA. To improve the
productivity of microalgae, genetic engineering efforts need to be
directed to improve the stress tolerance. Stress tolerance genes
from other organisms can be expressed in host organism to
1224 C. Paliwal et al. / Bioresource Technology 244 (2017) 1216–1226
improve the stress tolerance to achieve higher biomass productiv-
ity in extreme conditions and also to improve the yield percentage
of desired end products. Conditional expression of genes is another
area which could help us to metabolically re-program microalgae
to behave differently under stress conditions.
4. Conclusion
Microalgal species respond to the changing environmental con-
dition (abiotic stress factors) by modulating their metabolites.
Nitrogen limitation and salinity stress is found to be the most
widely applied stress condition for the commercial production of
feedstock for biodiesel production whereas, temperature can be
considered as the most tuning factor for PUFA production. Various
abiotic stress conditions affect genes which enhance pigment pro-
duction in cyanobacteria; thus, the field of cyanobacterial genetic
engineering will require attention both at basic and applied levels
for higher pigment production.
Acknowledgements
This manuscript was assigned by CSIR-CSMCRI registration no.
047/2017. Authors deeply acknowledge Dr. Amitava Das, Director,
CSIR-CSMCRI and Dr. Arvind Kumar, DC, Salt and Marine Chemicals
for their encouragement. Authors would like to acknowledge DST
and CSIR for financial support through projects GAP 2006, CSC
0105, CSC 0203 and OLP 0084. MM and VBSV would like to
acknowledge CSIR for SRF. All the authors acknowledge AcSIR for
PhD enrolment. KB is thankful to Bhavnagar University for PhD
enrolment.
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