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Accepted Manuscript

Review

Carbon streaming in microalgae: Extraction and analysis methods for high value
products

G Venkata Subhash, Meghna Rajvanshi, B. Navish Kumar, Sridharan


Govindachary, Venkatesh Prasad, Santanu Dasgupta

PII: S0960-8524(17)31111-2
DOI: http://dx.doi.org/10.1016/j.biortech.2017.07.024
Reference: BITE 18444

To appear in: Bioresource Technology

Received Date: 1 April 2017


Revised Date: 2 July 2017
Accepted Date: 6 July 2017

Please cite this article as: Venkata Subhash, G., Rajvanshi, M., Navish Kumar, B., Govindachary, S., Prasad, V.,
Dasgupta, S., Carbon streaming in microalgae: Extraction and analysis methods for high value products, Bioresource
Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.07.024

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Carbon streaming in microalgae: Extraction and analysis methods for high
value products

G Venkata Subhash*, Meghna Rajvanshi, B. Navish Kumar, Sridharan


Govindachary, Venkatesh Prasad and Santanu Dasgupta

Reliance Research & Development Centre, Reliance Corporate Park, Thane-


Belapur Road, NaviMumbai-400701, India
E-mail: subhash_g_venkat@yahoo.co.in; venkata.goriparti@ril.com; Tel: 0091-
22- 44751362

Abstract
There is a growing recognition that carbon-neutral biofuels and microalgae are
eco-friendly options because of their high CO2 sequestering capability and
ability to grow in wastewater/sea water and non-arable land. Also the intrinsic
properties of microalgal systems can be exploited for high value compounds
such as carbohydrates, lipids, pigments and proteins. This article provides a
comprehensive review of various microalgae cultivation practices utilizing
organic and inorganic carbon sources. The merits and demerits of the various
extraction and analytical procedures have also been discussed in detail.
Key words: Microalgae, CO2, Starch, Lipid, Extraction and Analytical methods
1. Introduction:
Aquatic biomes occupy most part of the earth thereby providing a wide habitat
for a large number of organisms. Algae is considered as a diverse aquatic,
photosynthetic organism (Venkata Subhash et al., 2014) and represents more
than 10% of the flora kingdom (Ioannou & Roussis, 2009). Based on its
biological structure, algae were categorized into two major group’s, namely,
microalgae and macroalgae. Microalgae have recently emerged as a potential
biomass feedstock to address the global challenges of sustainable food, feed and
fuel production. Among the photosynthetic organisms algae has been
recognized as a promising biofuel resource due to their capability to efficiently
converting solar energy into chemical energy. Algal biomass is capable of
producing higher oil yield per unit of cultivation area as compared to oil crops
such as corn and soybean. These organisms are known to grow very rapidly
have a doubling time of approximately 24 h and can fix carbon much more
rapidly (atmospheric CO2) compared to other terrestrial plant species.
These photosynthetic organisms are capable of producing high-content of fibers,
antioxidants, carotenoids, sterols, proteins, phycocolloids, lectins, oils, amino
acids, polyunsaturated fatty acids and vitamins, which are shown to have be
commercialized applications (Venkata Subhash et al., 2014). Apart from high
value compounds, algal biomass can be directly used in food and feed industry
and post processing it can produce biodiesel. Compared to fossil-driven,
microalgae-based biofuels are renewable, biodegradable and eco-friendly
(Vicente et al., 2010). These multiple advantages offered by algae makes it
attractive organism for industry.
Apart from photoautotroph, hetero and mixotrophic mode of cultivation have
been explored in order to improve productivity of biomass and high value
chemicals. In spite of extensive efforts, still commercial production of algae is a
difficult task, not only cultivation but also complex and rigid nature of cell
membrane and cell wall, makes extraction of metabolites of interest extremely
challenging. However, extensive research and advent of sophisticated and
sensitive methods in last two decades have resolved some of the problems
associated with extraction and analysis of metabolites of interest. Present
review provides a comprehensive overview of various cultivation methods
adapted for algae and how carbon is sequestrated and channelized to synthesize
high value products. Review also provides detailed account of metabolite
extraction and analytical techniques.

2. Modes of carbon streaming to various metabolites


2.1. Photoautotrophic carbon assimilation
Organisms capable of utilizing light and inorganic carbon to make their own
food are termed as photoautotrophs. These autotrophic microorganisms have the
potential to produce biomass by fixing carbon dioxide (CO2) and CO2 into
industrial products. CO2 utilization by autotrophs requires an energy in the form
of either light (photoautotrophy) or an external inorganic electron donor
(chemolithoautotrophy). Naturally the photoautotrophic microorganisms will be
considered as either oxygenic or anoxygenic. Oxygenic photoautotrophic
cyanobacterial strains are of Synechocystis sp. and Synechococcus sp. and other
potential oxygenic photoautotrophic cell factories are eukaryotic microalgae are
used for the production of lipids and alkanes. Chlamydomonas reinhardtii and a
few other microalgal species are the examples (Gimpel et al., 2015).
Anoxygenic photolithoautotrophic microorganisms have photosystems that
cannot split water; instead they require inorganic electron donors (such as H2,
hydrogen sulfide or sulfur) to generate reducing power. Anoxygenic species of
bacteria, such as Rhodobacter sphaeroides (Nybo et al., 2015), which can be
used for metabolic engineering (Tikh et al., 2014). In addition, R. sphaeroides
has metabolic flexibility that enables aerobic, anaerobic, heterotrophic and
autotrophic growth.

In oxygenic photosynthesis, light dependent reaction is the first phase, during


this process photon captured by pigment in Photosystem II (680) passed via
cytochrome b6-f, plastoquinone to PSI(700). PSI absorbs another photon
directly and reduces NADP into NADPH. In phototrophic eukaryotic algae,
these complexes are present in chloroplast. Cyanobacterial PSI and PSII are
present equal number (3 to 5) in cytoplasm and maximizing electron usage for
photosynthesis (Blankenship, 2013). Phycobilisomes (PBS) of cyanobacteria
are very efficient in light harvesting (490-650 nm) and assisting Chl aduring
photosynthesis. Further, cyanobacteria has the ability to perform photosynthesis
and respiration concurrently (Pessarakli, 2016). State transition (shuttling of
excess light energy from PSII to PSI or PSI to PSII) in cyanobacteria (with
PBS) and green algae (with light harvesting complex II) are different because
of configuration of light harvesting complexes (Pessarakli, 2016). The next
phase of photosynthesis is carbon fixation. Generation of ATP and NADPH
during photosynthesis are utilized during carbon fixation.

2.1.1. Carbon and energy sources


Flue gas produced while burning coal based or gas based power plants and
composed of carbon dioxide (CO2), sulfur dioxide (SO2),nitrogen oxides (NOx)
oxygen (O2), other trace gases, metals, and ash. High level of CO 2, SOx and
NOx are more toxic to environment and human. One of the most important
industrial activities related to GHG is the cement industry that produces huge
amount of carbon dioxide (CO2). Including related combustion emissions, the
cement industry accounts globally about 8% of global CO2 emissions, leading to
about 33.0 billion tons of CO2 emissions for 2010. As well, global consumption
of coal and natural gas is responsible for about 40% and 20% of total CO2
emissions respectively (Agency, 2011). CO2 mitigation of flue gas using algal
cultivation will be a boon. Several research suggest that very few algae like
Chlorella sp (upto 70% tolerance), Scenedesmus obliquus and Chlorella kessleri
(upto 6%) (De Morais & Costa, 2007) can produce biomass.
Carbon fixation to the metabolic products is driven by energy rich molecules
called adenosine tri phosphate (ATP), Flavin adenine dinucleotide (FADH),
Nicotinamide adenine dinucleotide (NADH) and Nicotinamide adenine
dinucleotide phosphate (NADPH). ATP which is a universal currency of
biological system and this ATP molecule breaks into ADP and liberates
−7.3 kcal of energy per mole of glucose which is used in all cellular activities
like biosynthesis, breakdown of molecules, transport, cell division and
movement etc., NADP molecule gains an electron from hydrogen, and forms
NADPH, which is a reductant plays an important role in fatty acid, steroid
synthesis and conversion RNA to DNA. Among the listed energy molecules the
light plays a major role in the leading of all photosynthetic reaction by
microalgae.
2.1.2. Mechanism of carbon assimilation in autotrophy
Biological CO2 fixation is currently achieved through the photosynthesis of all
terrestrial plants and a tremendous number of photosynthetic microorganisms
(Ho et al., 2011). However, plants are expected to contribute only with a 3-6%
reduction of global CO2 emissions (Skjånes et al., 2007). Therefore, since 50
years ago, researches have focus in the evaluation of microalgae and
cyanobacteria (Fernández et al., 2012) since they can grow much more faster
than terrestrial plants, and their CO2-fixation efficiency compared with higher
plants is about 10-50 times higher (Costa et al., 2000). Microalgae can typically
be used to capture CO2 from three different sources: (1) atmospheric CO2, (2)
CO2 emission from power plants and industrial processes, and (3) CO2 from
soluble carbonate (Brennan & Owende, 2010; Wang et al., 2008). Capture of
atmospheric CO2 is probably the most basic method to sink carbon, and relies
on the mass transfer from the air to the microalgae in their aquatic growth
environments during photosynthesis. However, the potential yield from the
atmosphere is limited by low CO2 concentration in air (around 360 ppm)
(Brennan & Owende, 2010; Stepan et al., 2002; Wang et al., 2008). In contrast,
CO2 capture from flue gas emissions from power plants that burn fossil fuels
achieves better recovery due to the higher CO2 concentration of up to 20%
(Bilanovic et al., 2009). Since microalgae CO2-fixation involves
photoautotrophic growth of cells, CO2 fixation capability of specific species
should positively correlate with their cell growth rate and light utilization
efficiency (Jacob-Lopes et al., 2009a; Jacob-Lopes et al., 2009b). However,
microalgae photosynthesis efficiency declines with increasing temperature,
since CO2 solubility is significantly reduced (Pulz, 2001).
Carbon assimilation in algae can be through gaseous form of CO2 or as
bicarbonate. Gaseous form of CO2 can diffuse through algal cell membrane.
Based on the pH (6.4 to 10.3) dissolved bicarbonates transport inside the cell
through bicarbonate transporters present in plasma membrane and chloroplast
envelope. Eventually bicarbonates converted into gaseous CO2 for carbon
assimilation in stroma of chloroplast (Sayre, 2010). Enzyme Ribulose-1,5-
bisphosphate carboxylase/oxygenase, or RuBisCO (EC number 4.1.1.39) is
slow in action (3 molecules per second) but most abundant protein in our planet
and playing vital role in fixing atmospheric CO2. RuBisCO located more in
pyrenoids of eukaryotic algae and carboxysomes of cyanobacteria. All green
algae and most of the cyanobacteria has form 1B of RuBisCO (Barsanti &
Gualtieri, 2005).
RuBisCO has lower affinity with carbon dioxide and hence plant cells facilitate
CO2 availability through Carbon concentrating mechanism (CCM). In algae,
active inorganic (Ci) carbon transporters present in plasma membrane and
chloroplast which are energised by photosynthetically derived ATP whereas in
cyanobacteria many active Ci transporters present in plasma membrane and
require ATP and NADPH (Pessarakli, 2016). Three phases of Calvin-Benson-
Bassham cycle encompasses (i) Carboxylation of ribulose-1,5-biphopshate
(RuBp) generates 3 phosphoglyceric acid (PGA) (ii) reduction of PGA into
glyceraldehyde 3 phosphate (G3P) using 12ATP and 12 NADPH molecules
(iii) regeneration of RuBp from triose phosphates using 6 ATP molecules with
final product 2 molecules of glucose .

Some green algae are reported to easily grown at very high CO2 concentration.
Chlorella species is very common to be used as carbon sequestration. C.
vulgaris ARC1 strain was studied under ambient CO2 concentration (0.036%)
and elevated CO2 concentration (20%) and showed increase in biomass at the
elevated CO2 concentration (6%) and the temperature was 30 C. ChlorellaKR-1
species showed maximum growth at 10% CO2 and good growth rate upto 50%
CO2 and no CO2 fixation at 70% CO2 concentration.

2.2. Heterotrophic carbon assimilation


In heterotrophic mode of growth algal cells derive carbon and energy for growth
by metabolizing organic carbon sources in the absence of light (Chen & Chen,
2006). Various carbon sources have been used to hetrotrophically cultivate
microalgae. Some of the reported carbon sources are glucose (Li et al., 2007;
Shi & Chen, 2002), sucrose (Gao et al., 2010), acetate (Choix et al., 2012),
glycerol, ethanol, whey, molasses, hydrothermal liquefaction (HTL) aqueous
and municipal wastewater, wastewater from food and carpet industry.
Heterotrophy is not a universal phenomenon in algae. Microalgae are primarily
photosynthetic organisms and only some species are facultative heterotrophs.
Basic reasons of obligate photoautotrophy are not clear but it is proposed that
lack of desired transporters to uptake sugars and other carbon sources can be a
probable reason (Morales-Sánchez et al., 2015; Wood et al., 2004). This
phenomenon has been shown in Cyanobacterial strains like Anabaena
variabilis PCC7118, Synechocystis PCC6308, PCC7008, Synechococcus
PCC7942 and diatom Phaeodactylum tricornutum. P. tricornutum was able to
grow on glucose, following insertion of gene for glucose transporter. Another
hypothesis for inability to grow heterotrophically is incomplete tricarboxylic
acid (TCA) cycle, where enzyme α-ketoglutarate dehydrogenase is missing.
Incomplete TCA cycle generates precursors for biosynthetic pathways but
unable to produce ATP. In such cases organism has to depend on photosynthetic
activity to generate ATP and NADPH to fuel metabolic processes (Chen &
Chen, 2006; Wood et al., 2004). Some of the commonly studied species with
heterotrophic mode of nutrition are, Chlorella kessleri, Chlorella
protothecoides, C. sorokiniana, Chlorella pyrenoidosa, Chlorella vulgaris,
Nannochloropsis sp., Scenedesmus obliquus, Botryococcus braunii, Neochloris
oleoabundans, Nitzschia laevis, Tetraselmis, Dunaliella, Chlorococcum to name
a few. Some cyanobacteria like Anabaena, Spirulina and Synechococcus also
display the ability to grow heterotrophically (Perez-Garcia & Bashan, 2015).
For an extensive list of heterotrophic microalgae, refer to the review written by
Perez-Garcia and Bashan (Perez-Garcia & Bashan, 2015).
2.2.1. Merits and demerits of heterotrophic cultivation
In comparison to autotrophic cultivation, heterotrophic cultivation has several
advantages and constraints. These aspects have been discussed by Perez-Garcia
et al. and Morales-Sanchez et al. (Morales-Sánchez et al., 2014; Perez-Garcia &
Bashan, 2015; Perez-Garcia et al., 2011). We present the key highlights here.
Heterotrophic cultivation eliminates light requirement and therefore, culture can
grow continuously, irrespective of day and night cycle. Heterotrophy, enables
the production of high dense cultures, which cannot be obtained through
autotrophic mode of cultivation because of higher energy content per mole of
the substrates, in comparison to CO2 (Perez-Garcia et al., 2011). High cell
density makes scalability and harvesting easier. Biomass composition can also
be modified by changing nutrient source. Besides, heterotrophic cultivation also
helps in wastewater treatment. Because of these advantages, many algal
companies have adopted heterotrophic mode of cultivation for production of
high value chemicals. For example, TerraVia (USA) makes products for human
consumption, DHA and cosmetics from microalgae through heterotrophic
cultivation. Similarly, Alltech (USA) produces algae protein and DHA using
heterotrophic mode of cultivation. Fermentalg (France) predominantly uses
heterotrophic and mixotrophic environment to cultivate microalgae.
Heterotrophic mode of cultivation is still feasible for production of priced
commodity. However, its use for production of low cost products, biomass for
feed and biofuel has not been proven economically viable yet. This is primarily
due to the fact that maintenance of axenicity and uni-algal condition is
extremely important in heterotrophic cultivation, in order to avoid other
microbial contaminants. These extraneous microorganisms can be fast growers
and can quickly outnumber the targeted algal species (Perez-Garcia & Bashan,
2015). Sterile cultivation in fermenters with oxygen and organic carbon source
supply increase the production and operation cost significantly. Also
heterotrophy is limited to a small number of microalgal species and metabolites.
The metabolites that are only formed in cells exposed to light, are missing in
heterotrophic mode of growth (Morales-Sánchez et al., 2013).

2.2.3. Production of valuable products in heterotrophic algal cultivation


Microalgae are known to produce valuable compounds like pigments
polyunsaturated fatty acids (PUFA) and lipids, which have tremendous
nutritional and commercial value. Many factors affect the production of these
valuable products from microalgae. Carbon and nitrogen source used for
cultivation, C/N ratio, availability of other nutrients, like phosphorous, silica (in
case of diatoms) and environmental conditions significantly affect production of
these valued compounds.

a) Lipid and Poly Unsaturated Fatty Acids (PUFA): Under hetero- and
mixotrophy biochemical composition of cells is widely different compared to
autotrophic cultivation. Microalgae accumulate more lipid and carbohydrate
under heterotrophic and mixotrophic growth regimes. Chlorella zofingiensis
resulted in 900% increase in lipid yield under heterotrophic mode, relative to
autotrophic mode. Heterotrophic cells accumulated more neutral lipids (NL)
that accounted for 79.5% of total lipids, whereas photoautotrophic cells mainly
synthesized membrane lipids such as glycolipids (GL) and phospholipids (PL)
(Liu et al., 2011). Apart from growth regime, type of nutrient source (carbon
and nitrogen source) also affects lipid content and fatty acid profile. It is known
that acetate when used as sole carbon source, improves lipid and
docosahexaenoic acid (DHA, 22:6) production significantly in Crypthecodinium
cohnii, if compared to the cells grown on glucose as sole carbon source (de
Swaaf et al., 2003b; Ratledge et al., 2001). In a batch culture using glucose to
obtain a biomass concentration of 28 g/L, the production of lipids and DHA
(22:6), accounts for 14 and 7%, respectively (de Swaaf et al., 1999). Improved
DHA yield on acetate as sole carbon source, is probably relates to the direct
conversion of acetic acid into acetyl CoA through enzyme acetyl-CoA
synthetase, the key intermediate in lipid biosynthesis. In the contrary, the
conversion of glucose into acetyl CoA is a multistep process (de Swaaf et al.,
2003b). When sodium acetate is used as carbon source, pH of the medium
increases since acetate ions are slowly replaced by hydroxyl and other anions.
Therefore, strategy of cultivating C. cohnii in pH auxostat mode, using acetate
for pH maintenance, is superior strategy for high levels of lipid and DHA
accumulation per se (Ratledge et al., 2001). Fed-batch cultivations with a
controlled feed of pure ethanol leads to high final cell densities and DHA
production in C. cohnii. About 83 g /L dry biomass with 35 g /L total lipid and
11.7 g/L DHA were produced in 220 h, which is quite higher than what is
reported for acetate as carbon source (De Swaaf et al., 2003a). Nitrogen source
is another key component in medium, which affects lipid yield. Wen and Chen
(2001) have tested N-sources like urea, nitrate, ammonium and tryptone or yeast
extract. They found that combination of complex nitrogen sources at a
concentration of 1.6 g/L and 0.8 g/L enhances eicosapentaenoic acid (EPA,
20:5) to 2.74% (w/w) in Nitzschia laevis (Wen & Chen, 2001).

Nitrogen concentration in the medium is a key factor, which affects lipid


content. High C/N ratio, i.e. nitrogen limitation, induces lipid production.
Morales-Sanchez et al. (2013), reported that in balanced C/N ratio of 17 i.e.
nitrogen sufficient condition, major cellular fraction in Neochloris
oleoabundans is the proteins. However, at high C/N ratio of about 278 in a
batch cultivation, i.e. under nitrogen limiting condition, synthesis of nitrogen
containing compounds declines and the accumulation of storage lipids ensues.
This culminates in the lipid content of 52% (w/w). In the case of cells grown
under balanced C/N, the accumulation of storage lipids is limited to 24% (w/w).
Fed-batch cultivation with C/N ratio of 278 and pulsing of nitrogen resulted in
the accumulation of starch (54% w/w). Nitrogen pulsing resulted in stages of
low and high level of nitrogen in the medium. Such short term nutritional stress
resulted in accumulation of starch rather than lipid (Morales-Sánchez et al.,
2013). Lipid accumulation due to high C/N ratio is also reported in other
microalgae (Wen & Chen, 2003).

Apart from nutritional factors, environmental factors also tremendously impact


lipid content and fatty acid profile. Low temperature (150C) results in higher
DHA content in C. cohnii (Jiang & Chen, 2000b), which might be due to the
fact that PUFAs help in maintaining membrane fluidity at low temperatures. pH
is also an important factor which determines growth in heterotrophic
cultivation. Recent study on Chlorella vulgaris, grown heterotrophically at pH
ranging between 5 to 8 showed that pH 7.5 is the optimal pH resulting in
maximum specific growth rate (0.541 d -1) and lipid content (53% w/w)
(Sakarika & Kornaros, 2016). Similarly, initial medium pH of 7.2 was found to
be optimal for growth and DHA production in C. cohnii ATCC 30556 (Jiang &
Chen, 2000a). pH shift strategy was employed in the case of heterotrophically
grown Chlorella protothecoides to over produce lipids. Lower pH favored
cell’s growth but retarded lipid synthesis and vice versa. A constant pH at 5.0
for first 93 h and switching to 6.5 thereafter, results in lipid concentration
reaching to 3.75 g/L. This was 24.2% more compared to constant pH 6.0
operations (Liang et al., 2011). Combined effect of multiple environmental
factors was verified using multiple parameter optimization method. pH, salinity
and carbon concentration exerted significant influence on the lipid synthesis.
High total lipids (55% w/w) was observed in high carbon, minimal salinity, and
low temperature conditions. Whereas, maximum neutral lipids concentration
(13.6% w/w) was observed in high salinity and low carbon conditions at
acidophilic microenvironment (Chiranjeevi & Venkata Mohan, 2016).

b) Carotenoids: Pigments play an important role in photosynthesis. Apart from


capturing light and transferring the excitons to the reaction centers of the two
photosystems, they also exhibit protective role against damage by light and free
oxygen radicals. Presence of carotenoids is mandatory in autotrophy and
therefore, all are naturally produced. There are some pigments, which are also
produced during heterotrophic dark cultivation. Xanthophylls, like lutein and
astaxanthin have been reported to be produced heterotrophically. Various
strategies have been employed to induce stress in algae for astaxanthin
production in dark. In Chlorella zofingiensis chemically generated reactive
oxygen species (ROS) and hydroxyl radicals were used to induce astaxanthin
production. 0.1 mM H2O2 treatment enhanced astaxanthin yield from 9.9 mg/L
to 12.58 mg/L (Ip & Chen, 2005a). Similarly, C. zofingiensis was subjected to
reactive nitrogen species (RNS), the peroxynitrite and reactive nitrogen
intermediate (RNI), the nitryl chloride, to induce carotenogenesis, which results
in enhanced astaxanthin formation in heterotrophic culture. 1 mM peroxynitrite
increased the yield of astaxanthin from 9.9 to 11.78 mg/L (Ip & Chen, 2005b).
In this strain, another strategy is employed to induce astaxanthin production
with the addition of organic acids at late exponential phase of cultivation.
Pyruvate, citrate and malic acids, the intermediates of TCA cycle also promote
the secondary carotenoids and astaxanthin synthesis. As under aerobic
condition, TCA cycle plays important role in formation of carbon skeleton for
biosynthesis of carotenoids and lipids (Bhosale, 2004). Haematococcus
pluvialis has been extensively used for astaxanthin production. However, its
poor growth under autotrophic condition followed by exposure to stress for
astaxanthin induction, led to low yields. Recently, heterotrophic cultivation was
integrated with induction of astaxanthin accumulation under autotrophic
conditions. With this approach, about 10.5 mg/L/d of astaxanthin could be
produced (Zhang et al., 2016). In an another study, heterotrophic cultivation for
biomass production followed by autotrophic mode of cultivation combined with
nitrogen starvation resulted in very high astaxanthin production (7% of dry
weight) in H. pluvialis (Kang et al., 2005).

Lutein is another important pigment produced by microalgae. Chlorella is


extensively studied microalga for lutein production. It is reported that Chlorella
pyrenoidosa can synthesize lutein and other xanthophylls under heterotrophic
cultivation using glucose as the carbon source (Sun et al., 2016). In another
study, seven Chlorella strains belonging to three species viz. pyrenoidosa,
vulgaris and protothecoides were tested in heterotrophic medium for lutein
production. Basal medium supported higher growth (4.6 g/L biomass) and the
production of lutein (≈4.6 mg/g dry cell weight) (Shi et al., 1997). Heterotrophic
fed-batch cultivation enhanced lutein yield even more, which reached to 225.3
mg/L with biomass concentration of 46.9 g/L (Shi et al., 2000). Lutein synthesis
can also be enhanced via inducing non-physiological conditions. For example,
addition of chemicals such as H2O2 and NaClO, the oxidative stress inducers in
the presence of Fe2+ (Fernández-Sevilla et al., 2010), triggers the induction of
lutein accumulation through nitrogen limitation, as reported in C. protothecoides
in fed-batch cultivation (Shi & Chen, 2002).

In summary, it is clear that heterotrophic mode of cultivation can be a viable


option for production of valuable compounds from microalgae. As heterotrophy
supports higher growth, combining this mode of cultivation with the induction
of non-physiological conditions at appropriate time of the growth phase can
help to achieve high yields of lipid, PUFA and pigments.

2.3. Mixotrophic carbon assimilation :


Mixotrophy is a mode of cultivation, where heterotrophic and autotrophic
modes work simultaneously, leading to utilization of inorganic and organic
carbon in the presence of light. Inorganic carbon is fixed through photosynthesis
in the presence of light, whereas organic carbon is assimilated and metabolized
to generate energy and metabolites for biosynthesis through aerobic respiration
i.e. in mixotrophic mode, light is not a stringent requirement for growth (Perez-
Garcia & Bashan, 2015). As listed by Perez-Garcia and Bashan (2015), several
microalgal species have the ability to grow mixotrophically. They are as
follows; Chlorella sp, Chlorococcum, Cyclotella cryptica, Euglena gracilis,
Haematococcus pluvialis, Nannochloropsis, Navicula saprophila, Nitzschia,
Ochromonas minima, Phaeodactylum tricornutum, Rhodomonas reticulate,
Scenedesmus obliquus, Dunaliella, Botryococcus braunii, Pleurochrysis
carterae, cyanobacteria genera Anabaena, Spirulina and Synechococcus (Perez-
Garcia & Bashan, 2015; Wang et al., 2014).
2.3.1. Merits and demerits of mixotrophic cultivation
Presence of two sources of energy (organic carbon and light), gives microalgae
more flexibility when grown in mixotrophy mode. This flexibility in growth,
due to complementarity between photoautotrophy and growth on organic
carbon, can help in achieving high growth rates and biomass productivity. Apart
from this, CO2 released in respiration can be utilized for autotrophic growth,
thus making mixotrophic cultivation more efficient than auto or heterotrophic
mode. Mixotrophy also reduces night time biomass loss, as microalgae is not
solely dependent on stored carbohydrate for catabolism. In mixotrophy, light
not being obligate requirement for growth, makes photo limitation or photo-
inhibition less effective in hampering microalgal growth (Wang et al., 2014).
Apart from reaching high cell densities, both mixotrophic and heterotrophic
cultivation results in dramatic reduction in chlorophyll content. This reduction
helps in improving efficiency of down-stream processing of lipid for biodiesel
production. As chlorophyll interferes with the trans-esterification process for
converting lipid into fatty acid methyl esters (FAME) (Bhatnagar et al., 2011).
Mixotrophy also suffers from constraints associated with heterotrophic
cultivation. For example, cost of organic carbon source, susceptibility to fast
growing contaminants and thus making it restricted to run the process with
axenic monoalgal strains. However, unlike heterotrophic cultivation,
mixotrophy is not restricted by inability to produce light induced metabolites
(Perez-Garcia & Bashan, 2015). Tubular, flat plate and column photobioreactors
are popular for cultivating microalgae in mixotrophic mode but this increases
cost of operation significantly, making the applicability of this cultivation
economically unviable.

2.3.2. Comparative account of biomass productivity in different cultivation


modes
In contrast to auto-, and heterotrophic mode of cultivation, mixotrophy can
support higher biomass and lipid productivity because of the advantage of
having complementary organic substrates for deriving energy for growth, in
addition to light and inorganic carbon source. There are several studies which
shows superiority of mixotrophy over other modes of cultivation.
Scenedesmus sp. AARL tested in mixotrophic mode with different carbon
sources clearly showed growth advantage over autotrophic mode. From this
study, it is evolved that suitable carbon source was 0.05M glucose, which
supports the biomass yield of 2.78 ± 0.86 g/L and 233.68 ± 35.34 mg/L of crude
lipid, respectively. The 16 h:8 h of light-dark cycle is enough to reach the
highest lipid yield (Dittamart et al., 2014). Similarly, biomass and lipid
productivities of Chlorella vulgaris were investigated under different cultivation
modes. Results showed that though lipid content was higher in autotrophic
cultivation, biomass and lipid productivity was much higher in mixotrophy
(Liang et al., 2009). Under mixotrophic condition with glycerol as carbon
source, Chlorella vulgaris, Chlorella minutissima and Chlorella pyrenoidosa,
growth is maximal (Sharma et al., 2016). In the case of green alga,
Haematococcus pluvialis specific growth rate in the mixotrophic condition
(acetate + light) corresponded well with the sum of the specific growth rates of
the heterotrophic (acetate + dark) and autotrophic (no acetate + light)
conditions, indicating an additive effect of two modes of cultivation in
mixotrophy (Kobayashi et al., 1992).
3. Mechanism of biosynthesis of valuable metabolic products
Once carbon is assimilated by the cells through photoautotrophic, heterotrophic
or mixotrophic cultivation, cells further utilize the metabolites in energy
generating pathways (glycolysis, TCA) and biosynthesis of various
macromolecule (starch, lipid, pigments, proteins etc), required for cell structure
and function. Brief account of biosynthesis of starch, lipid, and carotenoids have
been discussed below to understand the channelization of assimilated carbon
into building blocks of the cell.
a) Starch: Glycogen and starch are the main storage carbohydrates in algae
and cyanobacteria. Green algae mainly store carbohydrate in the form of starch,
whereas red algae store amylose-free starch granules called “floridean” (Viola
et al., 2001). In cyanobacteria, glycogen is the main storage polysaccharide.
However, in Chroococcales (a small group in cyanobacteria), starch is the main
storage polysaccharide. The three main enzymes involved in synthesizing both
starch /glycogen are ADP-glucose pyrophosphorylase (Agp), glycogen/starch
synthase and the branching enzyme (Ball et al., 2011). CO2 assimilated through
the Calvin cycle forms glyceraldehyde-3-phosphate, which through a series of
chemical reactions (gluconeogenesis) is converted into glucose-6-P. In case of
heterotrophic condition, organic carbon is used to synthesize glyceraldehyde-3-
phosphate through glycolysis/ gluconeogenesis. Which is then transported into
chloroplast. The exact form of carbon compound, which enters chloroplast is
unclear yet (Scott et al., 2010). As mentioned above, glyceraldehyde-3-
phosphate is then converted into glucose-6-phosphate through gluconeogenesis.
Further, glucose-6-phosphate to glucose-1-phosphate conversion is catalyzed by
an enzyme called phosphogluco mutase. Glucose-1-phosphate is an important
intermediate, as it is used to synthesize substrate (ADP-glucose) for starch/
glycogen synthase. Agp catalyzes this first committed irreversible reaction in
glucan biosynthesis, forming ADP-glucose and ATP from glucose-1-P. ADP-
glucose is the only nucleotide sugar, which is used as substrate by starch/-
glycogen synthase. Glucan synthase catalyzes transfer of glucosyl units from
ADP-glucose to the elongating chain of linear α-1,4-glucan. Finally, branching
enzyme mediates the formation of α-1,6-linkages to produce branched polymer
(starch/glycogen). In green algae, starch biosynthesis takes place in chloroplast.
In contrast to the green lineage, assimilated carbon is exported to the cytosol as
dihydroxyacetone phosphate (DHAP) in red algae by the triose-phosphate
translocator (TPT), leading to cytosolic storage of polysaccharide. In red algae
instead of ADP-glucose, UDP- glucose acts as substrate for starch synthesis
(Viola et al., 2001). Apart from having difference in storage location, green and
red algae also have very striking differences in terms of level of complexity in
starch metabolism. Green algae shows very high level of redundancy (involving
30-40 genes), whereas, red algae has simple starch metabolism, involving less
than 12 genes in the metabolism (Ball et al., 2015).

Apart from storage carbohydrate, some algal species (Porphyridium cruentum,


Botryococcus braunii and many cyanobacteria (Geitlerinema sp., Oscillatoria
sp., Spirulina platensis, Spirulina maxima, Synechocystis sp., Synechococcus
sp., Cyanothece sp. etc) also synthesize exopolysaccarides (EPS) (Donot et al.,
2012). EPS can be associated with cell surface (sheath, capsule, slime) or
released into the medium (RPS). EPS are mainly synthesized to protect the cells
from un-favorable environmental conditions. It protects the organism from
dehydration, phagocytosis and lysis by viruses. Cyanobacterial EPS being
negatively charged, gives it the ability to chelate metal ions, thus making them
available to cells. Cyanobacterial EPS are complex heteropolysaccharides,
which have more than 6 types of monosaccharides. Like glycogen biosynthesis
in cyanobacteria, EPS biosynthesis starts with conversion of monosaccharide
into sugar nucleotide in cytoplasm. Lipid linked oligosaccharide are assembled
at plasma membrane through a reaction, catalyzed by glycosyltransferases. This
is followed by polymerization in periplasmic space and export to the cell
surface (Pereira et al., 2009).

b) Lipid: Algal lipids are of two types namely, polar and non-polar lipids. Polar
lipids, such as phosphoglycerides and glycosylglycerides are structural elements
of algal membranes and play an important role in transport and cell signaling.
On the other hand, non- polar lipids are storage lipids and help to cope with the
stress conditions. Many microalgal species can produce large amount of
triacylglycerols (TAG- storage lipid) under stress conditions, accounting for 20-
50% of the dry cell weight. Increase in TAG content in stressed or stationary
phase cells is primarily due to metabolic shift from membrane lipid biosynthesis
to storage lipids synthesis. As in microalgae, cyanobacteria do not have TAGs.
Detailed review on lipid biosynthesis is reported elsewhere (Hu et al., 2008;
Zienkiewicz et al., 2016). However, key biosynthetic steps are listed here.

Fatty acids (FA) are the building blocks for lipid synthesis and are primarily
synthesized in the chloroplast. First committed step in fatty acid synthesis is
conversion of acetyl CoA into malonyl CoA through an enzyme acetyl CoA
carboxylase (ACCase). Glyceraldehyde-3-P formed in Calvin cycle is converted
into Acetyl CoA through a series of enzymatic reactions in chloroplast.
Cytosolic glycolysis is also a probable source for Acetyl CoA. Phosphoenol
pyruvate is transported to plastid, where it is converted to Acetyl CoA. After
first committed step, malonyl group is transferred to protein co-factor on acyl
protein carrier (ACP). Malonyl ACP is the carbon source for subsequent
elongation reactions, forming saturated 16:0 and 18:0-ACP. Unsaturated fatty
acids are produced by the activity of enzyme sterol ACP desaturase (Hu et al.,
2008). Monounsaturated fatty acid (oleic acid, 18:1, ω-9) converts into linoleic
acid (18:2, ω-6) and linolenic acid (18:3, ω-3). Linolenic acid through
desaturation and elongation steps, forms ω-3PUFAs; EPA and DHA (Guschina
& Harwood, 2006). For triacylglycerol biosynthesis in algae, fatty acids
produced in the chloroplast are transported into cytosol. Where FA are
sequentially transferred to glycerol backbone, forming TAGs. Though TAG
content and FA profile is to some extent governed by genetic makeup of the
organism. However, stress conditions like nutrient limitation, temperature, light
intensity and growth stage significantly affect and alter TAG content and FA
profile. For instance, under photo oxidative stress, TAG synthesis increases, as
it serves as an electron sink. 24 NADPH are consumed in TAG synthesis,
releasing NADP+, which can accept excess electrons in electron transport chain
and thus relaxing the cell under stress condition (Hu et al., 2008).

c) Carotenoid: Microalgal pigments mainly comprise of chlorophyll,


phycobilins and carotenoids. Chlorophyll is ubiquitous in algae, cyanobacteria
and plants. However, carotenoid types and phycobilins are specific to particular
classes of algae. For details on carotenoid distribution in different classes of
algae, refer to (Takaichi, 2011). Phycocyanin and phycoerythrin are water
soluble accessory phycobiliproteins found in cyanobacteria and red algae,
respectively. Carotenoids are widely distributed among various classes of algae.
They are 40-carbon polyene chains, classified as carotenes and xanthophylls.
Carotenes are purely hydrocarbons in nature, whereas, xanthophylls contain
oxygen in the form of epoxide or hydroxyl group. Carotenoids are also
classified as primary and secondary carotenoids. Primary ones are essential for
survival and have structural and functional role in photosynthetic apparatus
(example: lutein, β-carotene). Secondary carotenoids are accumulated by
microalgae only after exposure to stress (example: astaxanthin) (Takaichi,
2011). Carotenoids not only assist cells in light harvesting and protecting
against excessive light but owing to their antioxidant properties, they also help
in relieving oxidative stress. Also they are essential in assembly of chlorophyll-
protein complex (PSII). In most microalgae, carotenoids are synthesized and
accumulate in chloroplasts. However, in some microalgae (for example,
Haematococcus sp.), xanthophyll accumulate in cytoplasm (Takaichi, 2011).

Isopentenyl pyrophosphate (IPP- a 5 carbon compound) is the precursor for


synthesis of carotenoids and phytols in chlorophylls. It is synthesized through
two pathways. Mevalonate (MVA) and 1-deoxy-D-xylulose-5-phosphate
(DOXP) pathway. MVA pathway is found in cytoplasm of plants, animals and
bacteria. In this pathway, acetyl CoA is converted to IPP and dimethylallyl
pyrophosphate (DMAPP) through a series of reactions, forming mevalonate as
an intermediate compound. DOXP pathway is present in cyanobacteria, plastids
of algae, plants and certain bacteria (Takaichi, 2011). DOXP pathway is
initiated by reaction of glyceraldehyde 3-P (GA3P), pyruvate and thiamine
pyrophosphate (TPP) forming DOXP. DOXP is further converted into IPP
through C-skeleton rearrangement. Process involves multiple reactions,
catalyzed by reductases, dehydratases and kinase (Lichtenthaler, 1999). IPP
further forms geranylgeranyl diphosphate (GGPP- a C20 compound). Carotenoid
biosynthesis begins with condensation reaction of two GGPP molecules,
forming C40 phytoene. Phytoene is sequentially modified to lycopene with
addition of carbon-carbon double bonds at each step (Mikami & Hosokawa,
2013). β-carotene is formed from lycopene by action of β-cyclase, resulting in
cyclization of both the ends. Identical hydroxylation of both the β rings form
zeaxanthin. Which through epoxidation reactions form violaxanthin and
neoxanthin. α-carotene is derived from lycopene by reaction catalyzed by β-
cyclase and ε-cyclase. Hydroxylation of β and ε rings in α-carotene results in
lutein formation. Astaxanthin is synthesized from β-carotene by action of β-
carotene ketolase and carotenoid hydroxylase.

Starch, EPS, lipids, fatty acids and carotenoids have great industrial relevance.
For instance, lipid is important for commercial production of biodiesel. Their
roles in diverse physiological functions in humans make EPA and DHA
attractive candidates for nutraceutical and pharmaceutical market penetration or
expanding the existing formulations. Owing to their antioxidant properties and
unique pigmentation, carotenoids are invaluable for food, beverages, cosmetics,
aqua culture and nutraceutical markets. EPS on the other hand has wide range of
applications in industries related to textile, cosmetics, adhesives, oil recovery
and brewing. It is also used extensively as food additives and thickening agent.
Due to the wide applications and industrial significance of these metabolic
products, efficient extraction, recovery and quantification is essential. The next
section deals with various methods of extracting and quantifying these valuable
products.

4. Biochemical characterization and analysis of high value compounds


4.1. Lipids extraction from dry algal biomass
Lipids which constitute major portion of biological macro molecules are highly
soluble in organic solvents. They consists of neutral and polar lipids that are either
saturated or unsaturated. Lipid content differ in various algal species because of
their respective cultivation processes. Factors like growth medium composition,
temperature, pH, quality and quantity of light, ratio of light/dark cycle, aeration
rate, etc. will influence lipid yield, its composition and fatty acid profile (Halim et
al., 2012). Determination of individual lipid composition by different analytical
techniques involves three main steps - extraction, separation by analytical methods,
identification and quantification.
Lipids which are usually embedded inside the matrix do not appear in free form
and hence an extraction step will be needed for downstream analysis. Depending
upon the experimental goal, one can perform the following three different
techniques for the lipid analysis: (i) Lipids separation from the matrix, (ii)
Removal of any non-lipid compounds; (iii) Fractionation and isolation of lipids
from the extract.
Lipid extraction mainly depends on the method of extraction and the type of
solvents used for the process (Table.1). Methods used are mainly categorized as
either mechanical or chemical (Halim et al., 2012).Mechanical methods mainly
include oil expeller, microwave assisted extraction and ultrasonic based extraction
whereas chemical methods consist of soxhlet extraction, supercritical fluid
extraction and accelerated solvent extraction (Halim et al., 2011).
Table.1
a) Mechanical methods
(i) Expeller press
Mechanical or expeller press will help to break the cells and compress out the oil
(75%) from the large quantity of dry biomass (Harun et al., 2010). For this kind of
technique algae needs to dry before pressed. Though 75% of the oil is extracted,
this technique is being used in combination with other chemical based techniques
for making it commercially viable.
(ii) Ultrasound assisted extraction of oil
Intracellular lipids present in microalgae are blocked by thick extra cellular
membrane for the extraction. Use of solvent extraction and mechanical press
technique in such cases usually yields less lipid (Neto et al., 2013). In such
scenario, the ultrasound assisted extraction method can be used to break the cell
wall and lipid extraction. Propagating sound waves (higher than 20 kHz) into the
liquid media results in alternate high and low-pressure cycles. During this process
small vacuum bubbles are formed collapsing rapidly, leading to a phenomenon
called cavitation. The high pressure and high speed liquid jets generate shearing
forces around algae cells and break the cell structure. This mechanical disruption
of the cells improves material transfer and extraction of lipids as well (Lee et al.,
1998). By employing ultrasonication method, about 50–500% increase in oil yields
is achieved in Nannochloropsis oculata and Chlorella vulgaris (Adam et al.,
2012).
iii) Microwave assisted extraction (MAE) of oil
In this method, microwave generated electromagnetic radiations, penetrate through
biomaterial there by interacting with polar molecules like water in the biomass.
During this process entire sample is heated uniformly, helping in the mass transfer
of targeted compounds like lipid from the matrix. Higher oil yield with superior
quality and reduced extraction time are the main advantages of microwave-assisted
extraction (MAE). Iqbal and Theegala (Iqbal & Theegala, 2013) have studied this
phenomenon extensively for microalgal lipid extraction. Chen et al., measured
neutral lipids from 13 different green algae strains using microwave with
optimized timing of 50 and 60 seconds by incorporating Nile Red fluorescence
detection. This two-step staining protocol was found to be much more effective for
quantifying the lipid content of algae (Chen et al., 2011).
b) Chemical methods
Organic solvents like hexane, petroleum ether and supercritical carbon dioxide are
generally used in chemical methods for the extraction of simple esters of fatty
acids and acylglycerols. Selection of these solvents depend on the polarity of the
target compound as it dictates reciprocal solubility. Polar compounds are better-
extracted using polar solvents and vice versa. Phospholipids, lipoproteins,
glycolipids and free fatty acids (polar lipids and complex) require more polar
solvents (e.g., methanol or acetonitrile). The process of lipid or oil extraction from
microalgae is energy intensive and requires extra amount of energy for recovering
the solvents from lipids after extraction (Balasubramanian et al., 2011). Here we
discuss the various methods used for lipid extraction based on chemical methods.
(i) Extraction by solvents
An ideal solvent requires high levels of specificity towards lipids especially
acylglycerols and must be volatile enough to get separated from extracted lipid as
well. Polar and non-polar solvents can be used for the lipid extraction from algae
biomass. Non-polar solvents disrupt hydrophobic interactions between non-polar
and neutral lipids present in the algae biomass. Solvents used for extracting lipid
from microalgae biomass are n-hexane, ethanol, 1-butanol, 1,8- diazabicyclo-
[5.4.0]-undec-7-ene (DBU), dimethyl ether, and mixtures of chloroform/methanol,
n-hexane/ethanol, n-hexane isopropanol, n-hexane/2-propanol, methanol/1-ethyl-3-
methyl imidazoliummethyl sulfate, DBU/ethanol, DBU/octanol, methylene
chloride/methanol, dichloroethane/ methanol, dichloroethane/ethanol, and
acetone/dichloromethane (Neto et al., 2013). Amongst, chloroform/methanol (1/2
v/v) is the most widely used organic solvent system for extracting lipid from
biomass (Wang & Wang, 2012). Total lipid extraction and purification in algae
using Bligh & Dyer and the modified forms of this method were published. In this
method, methanol and chloroform are solvents and water is a co-solvent.
Performance of five different solvent systems chloroform/methanol (2:1, v/v),
hexane/ isopropanol (3:2, v/v), dichloromethane/methanol (1:1, v/v),
dichloromethane/ ethanol (1:1, v/v) and acetone/dichloromethane (1:1, v/v)
through bead beating was evaluated in Botrycoccus braunii. Of all these solvent
systems higher lipid yield of 28.6% was reported with chloroform/methanol (2:1
v/v) solvent system.
In study of Balasubramanian et al. (Balasubramanian et al., 2013) lipids were
classified into three categories based on solid-phase extraction column, namely
neutral lipid, free fatty acid (FFA) and polar lipid. Factors like biomass drying,
moisture content and solvent systems such as chloroform:methanol (2:1) and
hexane:methanol (3:2) were investigated for the intracellular lipid extraction. It
was found that FFA content of the lipid extracted from solar dried algae biomass
was three times higher than freeze-dried algae biomass and extraction of oil using
chloroform– methanol solvent system showed maximum lipid extraction efficiency
(Balasubramanian et al., 2013).
Soxhlet extraction process was developed with continuous refilling of biomass
with fresh solvent and minimize the solvent consumption (Halim et al., 2012).
Guckert et al. used Chlorella strain for lipids and they recovered larger amounts of
neutral lipid similar to modified Bligh and Dyer's method (methylene chloride and
methanol solvent systems. Though the solvent extraction techniques are cheaper
and easy to implement, they are more toxic to experimenter. They are also energy
intensive, during the distillation process for separating lipid from the solvents
(hexane and chloroform) (Halim et al., 2012).
(b) Supercritical CO2 extraction
Supercritical CO2 (SCCO2) is used as a primary solvent and commonly used in
supercritical fluid extractions. This is because of its moderate critical pressure (7.4
MPa) and low critical temperature (31.1°C) properties.
Optimum conditions for pressure, modifier addition, temperature, and fluid flow
rate are important to reduce energy requirement and for maximum lipid yield by
SCCO2 extraction. Supercritical fluids are found to be much more suitable as an
extraction solvent because of the variations in fluid power and density by varying
extraction pressure and temperature. This helps in producing much more solvent
free crude lipids (Santana et al., 2012). In a study by Crampon et al. using N.
oculata, SCCO2 method was employed for extraction with air flow drying and
freeze drying as pretreatment methods (Crampon et al., 2013). It was concluded
that airflow drying was the most adequate pretreatment method for extracting 90%
by weight of triglycerides without any phospholipids. SCCO 2 extraction
performance and hexane based extraction on the yield and fatty acid composition
was evaluated in Chlorococcum sp. for biodiesel production as reported by Halim
et al (Halim et al., 2011). Yield of lipid declined with increase in temperature and
pressure using SCCO2 extraction
Optimized conditions (35 MPa, 40°C) for SCCO 2 extraction of lipids from
microalgae Shizochytrium limacinum with 95% ethanol as co-solvent was reported
by Tang et al. (Tang et al., 2011). Highest lipid yield in C. pyrenoidosa extracts
was obtained at 32°C, 40 MPa, 20 L/h with dosage of modifier as 1 ml of ethanol
per gram sample for 3 h (Hu et al., 2007). Optimal conditions obtained for
extracting lipids from B. braunii was (22–25 MPa and 50°C) was reported by
Santana et al. (Santana et al., 2012). It was found that the amount of unsaturated
compounds in the extracted lipid also enhanced as the pressure and density of CO 2
increased. This further helped in increase of unsaturated fatty acids in the form of
triglycerides.
In Nannochloropsis sp, about 33% of lipid was extracted at the best extraction
conditions of 40°C and 30 MPa with a CO2 flow rate of 0.62 g/min (Nobre et al.,
2013). SFE method was also used to study the effect of modifiers (oil mixed with
microalgae and ethanol with SC \CO2) for extracting lipid and pigments from C.
vulgaris at 30 MPa and 40°C (Gouveia et al., 2007).
4.2.Oil extraction from wet algae
During the lipid extraction process, drying algae is both energy and cost intensive.
In order to circumvent this bottleneck, the concentrated wet algae biomass can be
used for extracting lipid, wherein drying process can be bypassed (Lardon et al.,
2009). Kanda et al. had reported the use of liquid dimethyl ether (DME) as a
solvent for refining hydrocarbons and lipids from wet B. braunii (Kanda et al.,
2012). Using osmotic treatment process, a two-fold increase in the lipid extraction
from wet biomass of Chlamydomonas reinhardtii is observed (Yoo et al., 2012).
Patil et al. reported a single step supercritical process for simultaneous extraction
and transesterification of wet algae (Nannochloropsis sp.) (Patil et al., 2011). Lipid
extraction procedure developed by Sathish and Sims seem to extract about 79% of
transesterifiable lipids aided by acid and base hydrolyses. For this, wet biomass
from mixed cultures of Chlorella and Scenedesmus sp were used. A water miscible
solvent process was used (1,2-dimethoxyethane) for the extraction of algal oil from
wet cells of B. braunii and found that the water content of algae biomass
significantly affected the lipid extraction efficiency (Liu et al., 2013). Additional
information on lipid-extraction techniques can be found in specialized reviews
(Halim et al., 2011).
4.3. Analysis of lipids
Analysis of lipids by analytical methods is influenced by the chemical and physical
properties of the lipids. Different analytical techniques may be used to characterize
lipids, as illustrated in Table 2. Most of the extracted material are contaminated
with impurities and thus needs to be excluded for further lipid analysis. Hence
during analytical process, more than one component needs to be analyzed to
overcome this constraint.
Table. 2

4.3.1. Chemical, chromatographic and electrophoretic methods


Lipid quality and compound characterization of the samples is usually performed
by employing chemical and chromatography techniques. Compared to chemical
techniques, chromatography based ones are very useful in identifying different
components of lipid samples. Saponification value (SV), Iodine value (IV), acidity,
and peroxide value (PV) are most commonly used parameters in characterizing the
quality of fats. These parameters are widely used to determine purity (SV), number
of unsaturated compounds (IV) and free fatty-acid content (acidity). Though these
classical methods are widely used, they are increasingly replaced by new faster
methods.
From long time, gas chromatographic (GC) separation with flame-ionization
detection (FID) is used for lipids analysis. Limitation of this analytical method is
the thermal stability of the compound being analyzed, so that enough vapor
pressure is generated for injection. This issue can be resolved by derivatization of
the analytes. For complex biomolecules, a multiple reaction steps are essential.
Sample preparation for this technique includes pre-separation of lipid classes,
hydrolysis, derivatization, or pyrolysis. The fatty-acid composition of fats and oils
is normally resolved by GC from the corresponding methyl ester fatty acid
(FAME) derivatives. In fat and oil, fatty-acid esters of glycerol (acylglycerols) is
the predominant constituent, while fatty-acid esters of sterols and long chain
aliphatic alcohols found in negligible quantities. Transesterification is another
method to generate FAMEs from fatty-acid esters. An alkali- or acid can be used as
chemical catalyst in transesterification reaction to generate FAMEs in a methanolic
medium, which is very important for the lipid analysis.
GC can also be used for direct separation of triacylglycerols based on the carbon
number (CN). Analysis is normally performed at high temperatures on non-polar
or medium-polar low-bleeding columns. In the case of medium-polar, separation is
done at the level of family group of triacylglycerols. High resolution of the CN
family group peaks could be achieved when the samples are at polar stationary
phase.
Another advantage of GC is that it can be coupled with Fourier transform IR (GC-
FTIR). As of today, three types of GC-FTIR interfaces are commercially available:
matrix isolation, direct deposition and most commonly used light Pipe. GC-FTIR
can be used to identify various kinds of FAMEs. It has an added advantage of
isomer discrimination and structure elucidation of compounds having very closely
related structures.
a. Thin-Layer Chromatography (TLC)
Thin-layer chromatography (TLC) is the earliest chromatographic method used to
assess lipids, which is in use till date. It is a well-established technique that allows
for rapid screening of samples, delivering highly useful information with little
effort. It permits the separation of lipid mixtures at different polarities in a single
run. In recent years, High-Performance Thin-Layer Chromatography (HPTLC),
commonly coupled with densitometric quantification, has been developed to
improve separation efficiencies.
b. High Performance Liquid Chromatography (HPLC)
Liquid chromatography (LC) or high-performance LC (HPLC) has a much broader
application than gas chromatography for lipid analysis. This is mainly due to the
availability of various HPLC protocols for carrying out lipid analysis. Based on the
relative polarity, two different modes are defined in partition chromatography:
(i) Normal Phase LC (NP-LC)
Which typically is used for separation of lipids based on the class of compounds
(Dołowy & Pyka, 2015). Schaefer et al. developed an NP-LC method for
identification and quantitative estimation of 12 lipid classes.
(ii) Reverse Phase LC (RP-LC)
Which is working on retention time differences caused due to fatty-acyl
composition (Dołowy & Pyka, 2015). Separation is based on combined effect of
chain lengths triacylgycerol and their degree of unsaturation. Each double bond
reduces the retention by the equivalent of about two carbon atoms. The equivalent
CN (ECN) has been proposed for describing the elution chromatographic order
which can be calculates as ECN = CN 2n, where n is the sum of double bonds per
triacylglycerol (Balasubramanian et al., 2011). Its separation pattern are different
from those obtained from usual GC which is based on molecular mass (CN
separation). RP-LC is generally used for fast analysis of free fatty acids (FAMEs)
and for determining positional isomers of triacylgylcerols. Also it is used for
identification of conjugated linoleic acids (CLAs) and their geometrical isomers
from larger biological samples.
(c) Mass spectrometry
GC based electron ionization MS (GC-EI-MS) emerged as famous and powerful
tool for identification of lipid structures. This facilitated in building large number
of searchable databases containing structural information about many fatty acids
and lipids. Till date, several forms of soft ionization were developed such as Fast
Atom Bombardment (FAB), Matrix Assisted Laser Desorption/ Ionization
(MALDI), Chemical Ionization (CI), Electrospray Ionization (ESI), Field
Desorption (FD), Atmospheric Pressure Chemical Ionization (APCI), Atmospheric
Pressure Photo Ionization (APPI), Lipid Secondary Ionization MS (LSIMS) and
Thermospray (TS), providing excellent interfaces to HPLC and MS.

(d) Spectroscopy techniques


In recent years, spectroscopic techniques are regarded as attractive and promising
analytical tools for lipid analysis. This section presents characteristics, advantages,
limitations and potential of some spectroscopy based techniques for lipid analysis.
(i) Nuclear magnetic resonance (NMR)
Nuclear magnetic resonance (NMR) spectroscopy has become a universal method
in lipid analysis. It allows structure elucidation, qualitative and quantitative
analysis of defined molecules and even complex mixtures. NMR is a powerful tool
for the elucidation of molecular structures of purified lipids (1H-NMR and 13C-
NMR), structure and the dynamics of lipid membranes (1H-NMR, 2H-NMR and
31P-high-resolution and solid-state NMR). For the analysis of phospholipid
mixtures, 31P-NMR is by far the most advisable approach. The linear response and
relatively high speed of 31P-NMR allows accurate, selective measurement with
high sample throughput.
Compared to common chromatographic analysis (e.g., GC and HPLC), NMR
spectroscopy has some advantages. NMR is a non-destructive method and for
quantification purposes, no specific standards are necessary. However, compared
to MS, NMR techniques have only moderate sensitivity. Moreover, NMR-based
lipid-analysis methods are typically limited by overlapping signals in the 1H-NMR
spectrum and the low natural abundance of 13C (Mahrous et al., 2008).
(ii) LC-NMR
LC-NMR is another powerful methods for separation and structural elucidation of
unknown compounds in mixtures. Recent progress in pulse-field gradients, solvent
suppression, probe technology improvement and construction of high-field
magnets have given new stimulus to this technique. LC-NMR thus represents a
potentially interesting complementary technique to LC-MS in the analysis of
complex biological matrices and also in studying on-line structural analysis of
products. But till date not much has been reported about its application in lipid
analysis.
(iii) Mid-IR (MIR)
Mid-IR (MIR) spectroscopy has an illustrious history in lipid chemistry, together
with the two other forms of vibrational spectroscopy (i.e. near-IR (NIR) and
Raman spectroscopy) traditionally having less prominent roles.
Quantitative IR lipid analysis began with the development of FTIR spectroscopy.
With the advent of FTIR spectrometers, which use interferometry to resolve IR
energy into its component wavelengths, has resolved much of the limitations
associated with conventional dispersive IR instruments. This is achieved by higher
energy throughput, scan speed, multiplexing and improved wavelength accuracy.
4.4. Pigments Extraction and Analysis
(a) Supercritical fluid extraction (SFE):
Mendes et al. reported the supercritical extraction method to extract value-added
compounds with pharmaceutical significance (Henry & Yonker, 2006; Mendes et
al., 2003). This method was used for microalgae like Botryococcus braunni,
Chlorella vulgaris, Dunaliella salina and Spirulina maxima. Using supercritical
CO2 as an extraction media resulted in uncontaminated and undamaged extracted
components. Assessment of the extraction of astaxanthin and its esters besides
other carotenoids using supercritical CO2 was done using Haematococcus pluvialis
by. They optimized the parameters for the process of astaxanthin and its esters
extraction.
Mendes et al. described the extraction of antioxidants and antimicrobial
components from Spirulina platensis, applying supercritical fluid extraction (SFE)
using CO2 and the mixture of CO2, ethanol (Mendes et al., 2003). Extraction using
CO2 with co-solvent (ethanol) gave higher yields. Adding co-solvent to the
extraction agent also resulted in higher antioxidant activity. Therefore adding a co-
solvent overcomes this drawback and the extraction efficiency increases (Mendes
et al., 2003).
4.4.1. Pigments analysis (carotenoids)
Mendes et al. (Mendes et al., 2003) used HPLC to analyse astaxanthin,
canthaxanthin and β-carotene as well. For Liquid-Liquid pigment-containing
extracts, HPLC coupled with diode array detector was utilized as an analytical
method. (Ramus, 1972) demonstrated the sample pre-treatment before being
analysed; methanol was used for dissolving the residue, while fat-soluble
impurities were extracted with hexane. The use of Diode Array Detection (DAD)
in the HPLC system has proved its usefulness in compounds identification. A
challenge is presented in the simultaneous extraction and analysis of dissimilar
compounds (Ramus, 1972).
5. Conclusion
Microalgae have been widely recognized as feed stocks for renewable energy
generation by capturing atmospheric CO2 and organic carbon source. Products
from these microorganisms can are used for biofuel, cosmetics and in health care.
Successful commercialization of microalgae largely relies on growth optimization,
pretreatment methods, effective solvent extraction, recovery and conversion of the
same to biofuel. In this review, current day high throughput analytical technologies
like HPLC, GC, TLC, MS NMR are widely discussed to critically evaluate the
pros and cons of their adoption in technology development. Also, various
cultivation modes and analytical techniques for resolving bottlenecks were
discussed.
Supplementary information: For detailed account of organic carbon assimilation,
exo-polysaccharide extraction and analysis, schematic of production of metabolites
of interest and extraction and analysis methods and comparative analysis of
biomass productivity under different cultivation modes, refer Electronic annex.

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Table 1: Different extraction techniques for algae extracts
Temp. (°C)
Method of Chemicals used Chemicals
and
extraction for extraction Extracted
Pressure
Dichlorome hane
75-85°C
Soxhlet extraction and acetone for
during hot Ulvan
followed by hot Soxhlet extraction
water polysaccharide
water extractions and water for hot
extraction
water extraction
Dichloromethane
Soxhlet extraction and acetone for 120-180°C Lipids
Acetone
Ethanol solution
Room
containing BHT.
Liquid-Liquid temperature
KOH was added in Carotenoids
Extraction and dark
the procedure,
conditions
refer to the text.
Sonication under
Ethanol, ascorbic
ultrasound and the
acid , hexane, and
output was re- ----- Tocopherols
distilled water
extracted applying
L-L extraction.
Esterification
Acetylchloride,
followed by water Esterificatio Fatty acid methyl
methanol, n-
and n-heptane L-L n at 80°C esters
heptane and water
extraction.
Three
different
Pressurized liquid Hexane Ethanol
temp.: 50, Various for the aim
extraction (PLE) Water
100, 150, of characterization
and 200°C

Acetone Β-carotene
Supercritical CO 2 Carotenoids
Astaxanthin and
Supercritical CO 2
Canthaxanthin
Supercritical fluid
extraction (SFE) Supercritical CO 2 Lipids
39.95 and
59.95 and
Hexane and pressures up Hydrocarbons
supercritical CO2 to 35.0 MPa
(40 and 60),
pressure
Ethanol (200 and 300 Β-carotene
bar),

275 bar and Antioxidant


57°C for activities
antioxidant
activity and
Ethanol and CO 2 220 bar and
27 for Antimicrobial
optimum activity
antibacterial
activity
360 bar and
74°C for
antioxidant
activity. At
Lipid composition
Pure CO2 361 bar,
analysis
55°C for
highest
antimicrobia
l activity
Sulphate content
125°C
Pre-treatment by Total sugar content
120°C
Autoclaving in
water Successive Uronic acid
Water 115°C
filtration,
concentration and Neutral sugar
precipitation of the 115°C analysis
polysaccharides.
Molecular weight
120°C
Table 2: Different analytical methods and conditions for algae products
Analytical Proffered Mobile phase
Compound
technique Columns solvents
β-
carotenoids
Reversed-phase A mixture of
HPLC, UV-Vis Astaxanthin
column, 250 mm acetonitrile and
detector and
long methanol (90/10 v/v)
canthaxanthi
n
C30 analytical Mixture of acetone
HPLC-DAD Carotenoids
column (5 μm, and water (84%
47
250 × 4.6 mm acetone and 16%
i.d.) water) for the first 21
min, followed by a 4
min linear gradient to
97% acetone and 3%
water for the
remainder of the 50
min run.
Lichrosorb Si 60-
HPLC
5 (250 × 3 mm
equipped with
i.d.) Chrompack Mixture of n-hexane
an automatic
column protected and isopropanol Tocopherols
injector and a
by a silica pre- (99.3:0.7; (v:v))
fluorescent
column S2-SS
detector
(10 × 2 mm i.d).
Solvent A: 0.4%
triethylamine in 20
mM ammonium
acetate buffer
solution (pH 6.30 by
acetic acid)–
acetonitrile
(9:1). solvent B, 0.4%
YMC-Pack ODS-
triethylamine in 20 Neutral
HPLC AQ (250 mm ×
mM ammonium sugar
4.6 mm, 5 m)
acetate buffer
solution (pH 6.30
with acetic acid)–
acetonitrile (4:6);
gradient, 10–14% in
9 min, 14–64% from
9 to 30 min, 64%.
The following 5 min
at 1 mL/min.
YMC carotenoid
HPLC with
column (3 μm Acetonitrile:methanol
diode array
particle size, 250 (75:25) Carotenoids
detector
mm × 4.6 mm)

SunFire C18 Acetonitrile:


HPLC-ESI-MS column (150 mm methanol (0.1 M
Carotenoids
× 4.6 mm × 3.5 ammonium
μm formate):dichloromet
48
hane (71:22:
7, (v/v)
The column
used was a 30 m
× 0.25 mm i.d.
fused silica
GC-MS capillary column Helium as the carrier Volatile
coated gas (7 psi) compounds
with a 0.25 μm
layer of SE- 54
(HP-5MS,
Agilent)
Gas
chromatograph Capillary column
(Varian Star 30 m in length,
3400 Cx 0.25 mm in
Equipped with diameter, and
an auto- with 0.25-μm Polyethylene
Fatty acids
sampler and film thickness glycol
fitted with a (DB-WAX,
flame J&W Scientific,
ionization Folsom, CA,
detector at USA)
250°C.
BTR Carbowax
Gas Ethyl esters
column 30 m,
Chromatograph ____ of the
(0.25 mm inner
y various fatty
diameter)
acids
DBWax
Chromatograph Lipid yield
polyethylene
y and
glycol capillary
unit equipped Helium composition
column (30 m _
with a mass of the post-
0.25 mm id
spectrometry methylated
and 0.25 lm film
detector lipid
thickness)
Verify the
Petroleum
chemical
(20 cm X 20 cm) ether:acetone:diethyla
composition
TLC plates covered mine mixture in the
of the
with silica gel proportion 10:4:1
extracts and
(v/v)
were
qualitative
49
or only
quantitative
Investigate
the types of
(10 cm × 20 cm) Petroleum compounds
TLC
plates covered ether:acetone (75:25) responsible
with silica gel for the
antioxidant
activity
------ ------ Elemental
Combustion analysis
Kjedhal ------ ------
Protein
Analysis
Chemical
IR and 1H structure of
------
NMR ------ the extracted
polysacchari
de
Spectrophotom
etry:
(Neutralization
----- Methanol Antioxidants
of free radicals
activity
of DPPH by
the extract
antioxidants)
Fused silica To provide a
Micellar
capillary with 75 preliminary
electrokinetic 50 mM sodium
_m i.d., 37 cm analysis on
chromatograph tetraborate, 100 mM
total length and the
y with diode SDS at pH 8.8
30 cm length to composition
array detection
the detector of the
(MEKC–DAD)
extracts
Barium
chloride– ------- -------- Sulphate
gelatin content
Method
Phenol–
sulfuric acid ----------- ----------- Total sugar
method

50
51
Highlights
 Review describes about metabolic pathways of microalgae
 Algal metabolic pathways towards high value compounds production
were described
 Review gives a clear view on extraction and analysis methods of algal
compounds
 Merits and demerits during extraction and analysis was discussed

52
Carbon streaming in microalgae: Extraction and analysis methods for high
value products
G Venkata Subhash*, Meghna Rajvanshi, B. Navish Kumar, Sridharan
Govindachary Venkatesh Prasad and Santanu Dasgupta
Reliance Research & Development Centre, Reliance Corporate Park, Thane-
Belapur Road, NaviMumbai-400701, India
E-mail: subhash_g_venkat@yahoo.co.in; Venkata.goriparti@ril.com; Tel:
0091-22- 44751362
Graphical Abstract

53