1 Carbon sequestration by microalgae: A green pathway

2
3 Shailesh Kumar Patidarab, Sandhya Mishraac
a
4 Salt and Marine Inorganic Chemicals Discipline, Central Salt and Marine Chemicals Research

5 Institute (CSIR-CSMCRI), Bhavnagar, India.

b
6 Department of Life Sciences, College of Natural Science, Hanyang University, Seoul, South
7 Korea
c
8 Academy of Scientific and Innovative Research, Central Salt and Marine Chemicals Research

9 Institute, Bhavnagar.

10 Graphical abstract

Nutrients
C, N, P, K, Ca
Mg ,S and
Trace metals
Carbohydrate ----- Ethanol

Flue gas
and Biodiesel
waste- Microalgae Lipids PUFA
water PHA

Proteins, Pigments ,
Cosmetics, EPS
Calcites/
Calcium Changes in growth mode and
Carbonates/
carbonates carbon chemistry
Bicarbonates/
CO2

Organic
metabolites

11

12
13 Global climate change and GHGs
14
15 Global climate change is one of the great adversities which earth is experiencing,resulting in
16 to array of catastrophic/ undesirable changes which affects human lives, global economy,
17 biodiversity and almost all the natural resources. It is evident from global warming records of
18 last few decades that anthropogenic activities disturbed natural ecosystems in larger
19 magnitude that elevated GHGs level mitigation is becoming a first priority for sustainable
20 development (IPCC, 2015).
21 Out of all the GHGs; CO2 is considered as major contributor for climate change because of
22 its consistent increment trend in troposphere and larger proportion in magnitude than other
23 GHGs. The CO2 level of preindustrial era was 280 ppm which is 410 ppm up to 2015.
24 Preindustrial span of 8000 years also had shown the increment of CO2 level but it was only
25 20 ppm. The CO2 concentration has shown enormous increment of almost 100 ppm
26 since1750 (House et al., 2002; Giles et al., 2007; Kutzbach et al., 2010), which is computed
27 as 1.5 ppmv/ Yr of increasing rate (3 Pg C/year (1 Pg = petagram = billion ton) up to 2001
28 (IPCC, 2001). The dissolution of CO2 in the aquatic system is very poor on atmospheric
29 pressure. Though, some of the anthropogenic source originated CO2 captured by both
30 terrestrial and aquatic ecosystems and fraction of the CO2 remains as dissolved in
31 hydrosphere; majority of the CO2emitted (by various anthropogenic sources) persists in the
32 troposphere, which contributes the global warming. The CO2 level of the earth is expected to
33 reach up to 535-983 ppm in the year 2100 (IPCC, 2007; Mondor& Tremblay, 2010). The
34 characteristic absorption of IR emitted from the earthon exposure of solar radiations by green
35 house gases makes our atmosphere warmer which we call Green house effect. The green
36 house effect and libido of the earth have good impact on thriving of life, heat budget,
37 biogeochemical processes and links with it to physiological and biochemical processes of
38 almost all livings systems, that is why when elevated CO2levelmakes earth more warmer it
39 gives rise to undesired changes, which was not an usual phenomenon before preindustrial era
40 (Nature, NRC).The major CO2 contributor to troposphere through the anthropogenic means
41 are combustion of coal, liquid and gaseous fuels, majorly contributed by cement and thermal
42 power plants, followed by automobile vehicular emission (Boden et al., 2013). However,
43 some natural sources such as volcanic eruptions (e.g. Emission by Mount Pinnatabo in the
44 year 1991) also contribute to higher carbon influx in troposphere and, in addition disturbed
45 stressed ecosystems releases more CO2 under the influence of adverse conditions.
46 Mismanaged land use and accelerated deforestation and, unusual biogeochemical events
47 make it more vulnerable (Quret al., 2013; Holgerson & Raymond et al., 2016; Lal, 2004).
48 The radiative enforcing of GHGs according to report of IPCC 2001, is 1.46 w/m2 for CO2,
49 0.5 w/m2 for CH4 and 0.15 w/m2 for N2O. These gases has led to increment in cumulative
50 radiative enforcing and resulted in to overall increase in the average global surface
51 temperature of 0.6 C since the late 19th century, with the warming rate of 0.17 C per
52 decade which is the subject of worrisome (Lal et al., 2004). Out of all the GHGs; CO2 is
53 being considered as a major contributor for climate change because of its consistent
54 increment trend in troposphere with higher impact on biological world and larger proportion
55 in magnitude than other GHGs (Patidar Thesis, 2015). All the artificial and natural
56 processes which are absorbing/capturing/ storing/converting CO2 in to green
57 chemicals/minerals/biological macromolecules by any physiochemical or biological process
58 is called as carbon sequestration.
59 There is an emergent need of efficient biological carbon sequestration, which could
60 complement the existing physical and chemical processes.
61 Carbon Budget
62 Regulation of carbon in carbon cycle fundamentally understood through various modern
63 sensitive analytical techniques with the aid of modelling software systems. The removal of
64 carbon from the atmosphere and storing of carbon on land and oceans as sink is quite
65 complex process (IPCC, 2007); yet not understood very well due to differences in dynamic
66 changes in varied ecosystems (Jung et al., 2007). The cumulative carbon emissions of the
67 earth are the sum of the total CO2 emitted in to atmosphere by all physical, chemical and
68 biological processes occurring on the earth during any given unit period of time. Total
69 cumulative emissions from larger point sources (LPS) during 1870 to 2014 were 40020 GtC
70 due to combustion of fossil fuels in thermal power plants and cements, and 14550GtC
71 contributed from land use change. This total of 54555 GtC was partitioned among the
72 atmosphere (2305 GtC), ocean (15520GtC), and the land (16060
73 GtC).(http://www.globalcarbonproject.org/carbonbudget/15/hl-full.htm; Qur et al., 2015).
74 Globally, forest vegetation and soils collectively contain about 1146 GtC as a sequestered
75 stock (Dixon et al., 1994)
 76
77 Carbon exchange between land and atmosphere
78 Carbon flux between terrestrial plants and the atmosphere are substantial to understand the
79 dynamic carbon pool and, often calculated at annual scales. The major activities which
80 contribute to carbon exchanges through biological means are respiration and photosynthesis.
81 Global land flora fixes about 120 GtC/yr by photosynthesis (Gross Primary Production).
82 Moreover, 50% of this carbon is lost through respiration (photorespiration and dark
83 respiration) in the form of CO2 by the vegetation. The Net Primary Production of global flora
84 is about 60 GtC/yr (Archer& Barber, 2004). Heterotrophs (soil flora and organotrophic
85 microbes) in the soil contribute a similar magnitude of carbon to the atmosphere
86 (Heterotrophic or soil Respiration). The resultant difference between NPP and HR is called
87 Net Ecosystem Exchange or NEE). Carbon from land is lost through non-respiratory
88 processes, for example, forest fires, litter and dead animal degradation; carbon loss also
89 occurs as CO2 and CH4 efflux from paddy fields and animal manure in addition to harvests,
90 insect damage, river run-off, etc. under the control of land surface-atmosphere interactions
91 also play significant role (IPCC, 2000). The resulting balance between carbon gains and
92 losses is called Net Biome Production, NBP. Currently, it is estimated that NBP is about 1-3
93 GtC/yr. The terrestrial carbon sink have been continuously increased as 1.1 GtC/yr during
94 1960s to 4.1GtC/yr in the year 2014 and trend also reflected increment in atmosphere
95 growth rate as 1.7 in the 1960s to 3.9 GtC/yr in the year 2014 (Qur et al., 2015).
96 Carbon exchange between atmosphere and oceans
97 There are also significant exchanges of CO2 between the atmosphere and the oceans, of the
98 order of about 90 GtC/yr, with a net sink of about 2 GtC/yr. Carbon in the surface ocean
99 layers is used by the marine biota, and more is exchanged with the intermediate to deep
100 layers in the ocean. The carbon reserves in the ocean are significantly larger than on land
101 (IPCC, 2000; Qur et al., 2015).
102 CO2 sequestration through bioremediation of flue gas by employing the scrubbers containing
103 carbon absorbent solution/s e.g. green chemicals such as by amine compounds is one of the
104 popular chemical methods (Rochelle, 2009;Pires et al., 2012). Major chemical/ physical
105 processes involve absorption, adsorption, gas separation membranes and cryogenic
106 distillation for CO2 capture (Verma et al., 2015). In addition, some carbon composite
107 monoliths prepared from phenolic resin are being used with carbon nanotubes with their
108 desired transformation and physical activation (Jin et al., 2013).The carbon capture by
109 geological means is also demonstrated wherein the technologies implied for the injection of
110 CO2 in to carbonate and calcite rich rocks directly from the point source like thermal power
111 plants. These technologies need large investments and costly, further there is a risk of the
112 leakage and in turn there is no economic incentives so make it lesser economical sustainable
113 (Figueroa et al., 2008).The carbon fixation through biological means is an alluring strategy
114 because there is no net emission of CO2 (efflux to atmosphere). Further, natural
115 photosynthetic processes employing microalgae fix carbon efficiently and utilize in to
116 biomass based fuel with valuable products and co-products (Patidar et al., 2015; Patidar et
117 al., 2014; Mitra et al., 2016).
118 Why microalgae are promising for CO2 sequestration and climate change abatement?
119 Algae contribute around50% of total carbon sequestered in the form of biomass of the total
120 earth (Beardall and Raven, 2004). Out of the total carbon emitted from industrial processes,
121 only3-4% carbon is sequestered by terrestrial plants (Chinnasami et al., 2009).The global
122 algal net primary productivity is 111-117 PgC per year, which includes both fresh water and
123 ocean ecosystems(Giordano et al., 2005). Microalgae are microscopic photosynthetic
124 organisms which may be unicellular or multicellular and essentially contain
125 Chlorophyll-a; they may be both prokaryotic and eukaryotic cells. They synthesize
126 bioorganic macromolecules and reserve storage material in the form of chrysolaminarian
127 (polymer of carbohydrate, especially found in diatoms), starch and TAGs (Khan et al., 2009).
128 Carbon sequestrations through algae have many attractive features (Patidar Thesis, 2015):
129 Higher carbon fixation rate through efficient photosynthesis with higher lipid
130 productivity (Yoo et al., 2010; Brennan et al., 2012; Mata et al., 2012). Out of wide range of
131 microalgae, green algae have comparative higher lipid productivity (Walker et al., 2009),
132 although the feasibility depends upon solution towards lot of challenges being faced realizing
133 the facts and not only its fantasy.
134 Many microalgae may opt for heterotrophic, autotrophic and mixotrophic growth mode
135 under optimum suitable organic carbon concentration in addition to other environmental
136 conditions (Yangand Shimizu, 2000; Liang et al., 2009, Patidar et al., 2014, Patidar et al.,
137 2016, Ghosh et al., 2014, Mishra et al., 2012). However, microalgal strains have a
138 physiological variability for choosing favourable growth mode among available options.
139 They can use variety of organic carbon sources for the growth and have adaptability of
140 utilization of different chemical species of DIC (Patidar et al., 2014; Patidar et al., 2016).
141 Symbiosis or mutualism with the certain growth promoting beneficial bacteria and
142 their nature to cope up with co-culturing conditions with value added product generation
143 lures technocrats to utilize microalgae for multi-purpose environmental, food and energy
144 goals.
145 Aerial biomass productivity o f m i c r o a l g a e is higher than any conventional
146 plant crop (Benemann et al., 1987; Savage, 2011).Under open conditions for many promising
147 microalgae it is reported up to 15-45 g/m2/day under optimum scaled up conditions. Though
148 accumulated carbon % varies as far as the C% composition in the dried biomass is concerned,
149 majority of microalgae contains 20-65 % C in dried/ lyophilized conditions(Chaeetal., 2006,
150 Bhattacharya et al., 2015), although the dissolution rate of CO2 at normal atmospheric
151 pressure and temperature (NTP) is weak (VanDenHendeet al., 2012).
152
153 Microalgae have an excellent potential to grow in polluted environment (Patidar et al.,
154 2015;Subhashchandrabose et al.,2013; Dwivedi et al., 2008). Microalgae remediate wide
155 range of organic and inorganic nutrients with higher efficiency. Many microalgae are reported
156 as accumulator for some specific metals while on the other hand they sequester carbon
157 through photosynthesis (Dwivedi et al., 2008; Patidar et al., 2014; Patidar et al., 2015,
158 Bhattacharya et al., 2016).
159
160 Microalgae do not need fertile land (Demirbas et al., 2011) hence; these can be
161 cultivated in wasteland, coastal waterways (Savage, 2011) and saltfarm (Mishra et al., 2014).
162 Generally the uncultivable lands are ideal conditions for microalgae if water supply in the
163 form of wastewater or seawater is available (Bhattacharya et al., 2016).
164
165 Susceptibility of contamination is lower than any bacteria, fungi and yeast population
166 hence; intensive culture can be obtained (Borowitzka, 1999). Many microalgae have
167 resistance for the contamination by the bacteria and other undesired cyanobacterial
168 contaminants through the allelochemicals. However, some bacteria are proved to be growth
169 promoting, hence the consortium also play vital role through mutualisitic interactions to
170 contribute in indirect higher carbon fixation rates (de-Bashan et al., 2002).
171
172 These can be grown in both closed (photobioreactors) and open(open salt pans/ponds/
173 open type photobioreactors) environment (Borowitzka, 1999; Mishra et al.,2014).
174

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175 Microalgae is source of single cell protein along with variety of bioactive compounds
176 like phycocyanin, phycoerythrin, carotenoids, astaxanthin, -Tocopherols,EPA,DHA,
177 exopolysaccharides, PUFA etc. (Pulz et al.,2004; Mishra et al., 2012).
178
179 Microalgae may produce biofuels like biodiesel,biomethane (Mishra et al., 2014),
180 bioethanol (Harun et al., 2010) and biohydrogen (Benemann et al., 1999) etc.
181
182 Microalgae yield series of products and co-prodcuts to offset the cost expended
183 during cultivation and can generate carbon credits (Michiki, 1995). Microalgae have a better
184 avenues to establish an integrate processes for the biodiesel production along with suitable
185 yields for EPA, DHA (Mitra et al., 2016, Mitra et al., 2015) and glycerol (Patidar et al., 2015,
186 Ghosh et al., 2014).
187 Emerging cheaper harvesting methods like autosettling of biomass, skimming of
188 naturally floating microlagal mats for biodiesel production promises its low cost desired
189 products (Mishra et al., 2014; Patidar et al., 2015).
190
191 Microalgae and carbon sequestration process: Mechanisms, efficiency and limitations
192 Microalgae have efficiency to utilize inorganic carbon (as CO2 or HCO3) and transform in to
193 reserve storage such as starch/ chrysolaminarian through photosynthetic process. Once
194 carbon is fixed, it is partitioned in to the other macromolecules after starch synthesis for e.g.
195 proteins and TAGs. However, photosynthetic carbon fixation rate varies with the light
196 quality and quantity (Mishra et al., 2012), pH, chemical species of inorganic carbon,
197 temperature, physiological adaptation potential and genetic variability of the strain. Since, all
198 these factors may affect the photosynthesis and other allied metabolic processes to fix and
199 convert the inorganic carbon in to sugar/ starch etc; hence carbon sequestration process of
200 the natural microalgal communities are climate dependant too. In light of the current and
201 projected elevated CO2 levels, it would be quite imperative that RUBISCO and allied CCM
202 machinery would be less competitive and adaptable to fix higher CO2 concentration through
203 its incorporation and stabilization in to macromolecular synthesis for potential carbon
204 capture after certain extent (Giordano et al., 2005;Keeley & Rundel, 2003; Raven et al.,
205 2008). CO2 tolerance of microalgae varies; there are different reports on microalgal CO2

23
206 tolerance as well as lipid and biomass productivities even in single genus, and again it is the
207 subject of ecophysiological variability and long term adaptation (Tuba et al., 1999; Beardall,
208 2001; Solovchenko et al., 2013). Though some microalgae are reported for 20-100 % CO2
209 tolerance but there are so many microalgae which may tolerate only 1-2 % or even equal to
210 the atmospheric concentration only (Solovchenko et al., 2013; Patidar Thesis, 2015). To
211 understand the varied growth on different pH and in different medium with different
212 tolerance we may take a glimpse of growth characteristics of some microalgae isolated from
213 coastal Gujarat sites, India (Patidar Thesis, 2015).
214 Table 1. Mean biomass productivity (mg/L/day) of some of the selected microalgal

215 strains isolated from Gujarat coast, India ( at 50M photons /m2/sec. light and 30C
216 temp.).
BG11 ZM ACB BBM Growth
inhibition
Name ofthe strain/genus by minimum
CO2
Initial 7.1 6 3 10 8 6 3 8 6 3 6.8 4
(%)
pH

Mean 10.80 6.72 5.27 6.06 7.24 8.56 3.82 9.35 13.04 NG 20.01 9.88 2
Chlorococcum sp.
SD 0.01 0.26 0.13 0.79 0.01 0.53 0.09 1.32 0.66 - 0.13 0.66

Mean 12.51 11.06 5.66 8.95 22.52 5.53 5.40 8.56 5.79 NG 5.27 NG 1
Synechococcus sp.
SD 0.40 0.40 0.04 0.79 0.40 0.13 0.13 0.04 0.03 - 0.03 -

Mean 11.59 10.40 8.95 5.93 3.95 6.58 0.01 6.32 7.51 NG 3.95 NG 1
Synechocystis
sp. SD 0.40 0.26 0.01 0.13 0.05 0.13 NG 0.40 0.13 - 0.01 -

Mean 5.79 3.95 NG 23.57 24.75 8.30 NG 12.11 15.93 NG 14.48 NG 2

Chlorella variabilis
SD 0.26 0.01 0.01 0.66 1.05 0.13 0.01 0.40 0.13 - 0.20 -

Monoraphidi-um Mean 12.11 5.66 NG 23.83 24.89 9.22 NG 22.91 16.06 NG 9.48 NG 2
minutum
SD 0.13 0.53 0.00 0.26 0.66 0.00 NG 0.13 0.40 - 0.00 -

Mean 5.53 5.79 NG 14.22 18.04 12.51 4.08 16.06 5.53 NG 5.93 NG 0.5
Chroococccus sp.
SD 0.13 0.53 0.00 0.26 0.66 0.00 0.26 0.13 0.04 - 0.10 -

Mean 8.43 3.82 9.37 9.16 7.03 NG 11.31 NG 9.66 NG 0.5

Scenedesmus 13.04 12.64
sp. SD 0.13 0.00 0.00 0.26 0.13 0.13 0.00 0.13 0.26 - 0.40 -

217
218
219 Italic value in the table shows optimum biomass productivity. NG
220 indicatesthatno growth was observed.
221

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222 Microalgae experience inorganic carbon limitation in seawater and other natural water bodies
223 wherein they inhabited at atmospheric pressure since the solubility of the CO2 in the water
224 (104 moles/liter/atm in seawater) is poor (Li et al., 1971). During the evolution processes
225 they adapted through CCMs to cope up with carbon limitation (Price et al., 2011). The pH
226 range of 6-10.3 is predominantly having bicarbonate concentration as dissolved inorganic
227 carbon; hence, most of the microalgae are adapted to uptake bicarbonate by catalysing the
228 dehydration reaction via carbonic anhydrase enzyme and RUBISCO complex capture CO2 to
229 provide it to calvin cycle for synthesis of stable organic compounds. However, this enzyme
230 complex can concentrate around 10000 times more CO2 than it remains in the surrounding
231 medium in nature. But, oxygenase and carboxylase enzymes have competition for CO2 and
232 O2 (influx and efflux). The oxygenase enzymes catalyse the exchange of oxygen through the
233 plasmalemma. Although the CCMs efficiency varies within genus but in general it may be
234 stated that the diatoms have higher efficient CCMs along with the haptophytes and,
235 coccolithophores have comparatively less efficiency (Mlanon et al., 2016). The
236 coccolithophores contribute higher % assemblage within microalgal groups in seawater and
237 contribute significantly to calcification and carbon sequestration (Iglesisas-Rodriguez et al.,
238 2008).
239 Eventhough, there are possibilities of different routes for operational CCMs but commonly
240 bicarbonate is taken up by microalgae through the carbonic anhydrase. Due to some
241 physiological variability some microalgae are able to uptake CO2 and carbonate and, often
242 on elevated CO2 diffusive entry in the cell take place. Further, photosynthetic carbon
243 fixation also varies because of different metabolic pathway of C3, C4 and CAM. Some
244 microalgae are also evident to have C4 pathways (Badger et al., 1994; Reinfelder et al.,
245 2004; Giordino et al., 2005; Raven et al., 2005).Some microalgae have shown that CO2
246 uptake and carbonic anhydrase activity is not completely dependent. Under high CO2
247 concentration free diffusion of CO2 takes place through the plasmalemma. The role of
248 carbonic anhydrase in some microalgae is not clear. It is reported in some microalgae most
249 of the DIC uptake was accomplished in the absence of carbonic anhydrase,and transport
250 process of CO2 was observed distinct from bicarbonate in some cyanobacteria which was
251 observed to be dependent on metabolic energy. It was suggested that the bicarbonate,

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252 carbonate and CO2 were distinctly recognized by the transport system (Millere et al.,
253 1988;Tsuzuki & Miyachi 1989; Miller et al., 1990).
254 Nannochloropsis oculata showed light driven alkalization, and on high pH up to 10.5,
255 there was a dominant uptake of bicarbonate and further it was confirmed that external
256 carbonic anhydrase was not involved in transport at high pH, efficiency can be improved by
257 providing higher illumination with only sufficient air flow without adding sodium
258 bicarbonate in it (Merrett et al., 1990; Shene et al., 2016). Both CCMs and consequent
259 photosynthesis are light dependent and, numerous abiotic factors such as limitation of
260 nutrients and sufficient nutrient regime along with variation of physical factors such as pH,
261 temperature, light quality and quantity etc. may affect it. Due to global climate change and
262 global warming the surface water temperature increased along with the decrement in pH,
263 hence, consequently the carbonate is decreasing in compare to CO2 and bicarbonate under
264 ocean acidification (Raven et al., 2011), while the coastal eutrophication increased due to
265 changing carbonate chemistry with addition of anthropogenic enrichment of nutrients
266 (Borgesa&Gypensb et al., 2010; Patidar et al., 2015; Anderson et al., 2002).The overall
267 photosynthetic activity of phytoplankton which is fixing carbon will be enhanced, if other
268 factors such as iron, nitrogen etc. will not be in limited conditions, but many of the
269 phytoplankton species are pH sensitive. So the changing climate change scenario will affect
270 microalgal community composition, carbon fixation and other such relevant processes. This
271 may certainly affect the global carbon budget as well as individual microalgae to respond
272 with inorganic carbon species as well as photosynthetic carbon fixation and sequestration.
273 The residence time for the sequestered carbon is also going to be shifted. To keep in the
274 mind of the role of microalgae, the global carbon sequestration can be improved through the
275 artificial cultivation of microalgae or to support the natural microalgal growth/ carbon
276 fixation by providing artificial optimum conditions. The CO2 concentration may substantial
277 affect the quantity and quality of total chlorophyll (Chl), carotenoids, carbohydrate, and
278 protein in in cells. The Chl a/Chl b value shown to be increased profoundly with CO2
279 enrichment in Chlorella pyrenoidosa , but not in Chlamydomonas reinhardtii (Xia and Gao,
280 2005). Elevated pCO2 level and light interactively affect the growth rates as well as
281 photophysiological parameters of coastal diatoms. Elevated pCO2 do not significantly affect
282 RbcL (RUBISCO) and PsaC (PSI) protein subunits but these proteins significantly affected

26
283 by alteration in magnitude of light (Li and Campbell, 2013).Marine phytoplankton faces
284 dissolved inorganic carbon limitation. Carbon chemistry and low affinity of RUBISCO for
285 inorganic carbon substrate are important causes for limited production (photosynthesis) in
286 seawater. During evolution microalgae evolved to fix the carbon in seawater which is
287 comparatively low (10-30 M; Reinfelder, 2011).
288 The efficient sequestration generally maybe attained through the suitable strain which
289 could attain high cell density in less growth period, but two major factors such as light
290 and CO2 supply may affect the cell growth (Beardall et al., 2013). For this purpose
291 Chlorella sp. reported with high efficiency to tolerate high CO2 and with attainment of
292 higher growth and carbon fixation at optimum 10 % CO2 and, in addition Chlorella is also
293 reported for wastewater remediation which is considered as a good resource for
294 sustainable utilization (Maeda et al., 1995; Chinnasamy et al., 2009). However, strain
295 specific response varies with different tolerance and carbon sequestration efficiency.
296 Carbon supply mode dominantly affects the carbon sequestration efficiency as previously
297 demonstrated in photobioreactors studies in Chlorella and Monoraphidium minutum
298 (Patidar et al., 2015; Patidar et al., 2014). The CO2from flue gas released by point sources
299 such as thermal power plants and cement plants can be sequestered by microalgae;
300 wherein pH is one of the major problems, since dissolution of CO2 in water leads to acidic
301 pH. Some of the microalgae may grow in acidic conditions while in some cases pH can be
302 controlled through CO2 gas flow and by using base/ acids in the optimum range for
303 maintaining the growth and efficient sequestration in artificial structures (Hirata et al.,
304 1996; Hanagata et al., 1992; Patidar et al., 2014, Patidar et al., 2016). To maintain
305 efficient photosynthetic growth, the optimum pH is required or the cells should be
306 adapted for particular pH, since the cells which are adapted for higher pH suffers if
307 medium becomes acidic, lead the impeded growth with low carbon sequestration yield in
308 terms of biomass. The lower pH often inhibits the growth of microalgae, since most of
309 the microalgae found in nature have neutralised or alkaline range of adaptation for pH
310 except acidophilic microalgae. The higher electron flow in PS1 is essential for high
311 CO2adaptationfor the growth of the cells (Satoh et al., 2002). However, the high CO2
312 adapted cells are reported to have higher efficiency even in low CO 2 environment but
313 their uptake for specific carbon species varies (Patidar et al., 2016). In low pH,

27
314 microalgae favour CO2 uptake while in higher pH, it favours bicarbonate uptake; upon
315 the consideration of their adaptive/ acquisitive strategy and ecophysiological variability.
316 Efficient operational carbon sequestration through microalgae relies on the system design
317 apart from the other factor such as strains own characteristics, so different types of PBR
318 (photobioreactor) are being designed to attain the maximum efficiency. Factors such as
319 light intensity, duration of light hours, color, temperature, pH, gasmixing,
320 CO2concentration, residence time, CO2 supply mode, pH control strategies and some
321 other operational conditions are needed to be optimized and modelled for successful
322 microalgal cultivation in photobioreactor to attain higher fixation of carbon (Patidar et al.,
323 2016; Patidar et al., 2014, Fulke et al., 2010, Pires et al., 2012).For example, the outdoor
324 open thin-layer photobioreactor for flue gas phycoremediation was evaluated by mass
325 balance and mathematical model approach, exhibited50% flue gas sequestration
326 efficiency (decarbonization) and could remediate 4.4kg of CO2 for1Kg of algal
327 biomass(dryweight) production in optimum growth conditions (Douchaetal.,2009). While,
328 in the parallel type PBR integrated with flue gas sparging could attain up to 58 % of CO2
329 removal efficiency (Chiu et al., 2008). The reports on Chlorella are even higher in
330 mutated thermotolerant strain studied in bubble column and open type outdoor PBR up to
331 60% CO2 removal efficiency along with 70% NOx and 50 % NO2from flue gas (Ong et
332 al., 2010; Chiu et al., 2011, Jacob-Lopesetal.,2008).Many other microalgae such as
333 Duanliella tertiolecta SAD,Chlorella vulagaris,Spirulina platensis, Chlorella variabilis,
334 Monoraphidium minutum, Scenedesmus spp, Chlaymydomonas, Synechococcus and
335 Botryococcus braunii are also reported for the higher carbon fixation rates (Patidar et al.,
336 2014; Patidar et al., 2016; Sydney et al., 2010; Fulke et al., 2010). The microalgal
337 cultivation in wastewater and at wastelands elevates transportation cost to transfer CO2
338 from the point source like Thermal power plants and Cement industries to cultivation site.
339 Conversion of CO2 in to bicarbonate as nutrient for cultivation of microalgae is lucrative
340 because it will reduce the cost of transportation. The trapping of CO2emitted from point
341 source in water requires alkaline pH so CO2 can be stored as bicarbonate. Study suggested
342 to use alkaliphilic microalgae which can generate carbonate which can be again utilized
343 for the capture of CO2 as adsorbent (Chietal.,2011). One very good example is the
344 production of calcite during the Spirulina platensis while remediating the CO2; 0.14 g/L

28
345 of calcite could be deposited as reported in the study (Ramanan et al., 2010). Some other
346 strains like Chlorella, Chalmydomonas and Synechococcus sp are also reported for calcite
347 production and CO2 remediation (Fulke et al., 2010). The algal cells are also reported to
348 produce a great quantity of calcite, aragonite and vaterite crystals by fixing dissolved
349 inorganic carbon along with sequestration of other metal ions like calcium, which then
350 sink to the bottom of the ocean (Mishra et al., 2004; Mishra et al., 2010).
351 Beside some disadvantages such as the cost of photobioreactors etc, open ponds attracts
352 economically viable algal cultivation despite the risk of contamination and productivities
353 variation due to climatic variability is yet to be explored for the further research. Some
354 scientists also emphasized the use of natural sites for carbon sequestration. United States
355 Environmental Protection Agency stated that USA alone has more than 7000 facultative
356 lagoon systems (U.S.EPA, 2002; Christenson et al., 2012). From the perspective of algal
357 cultivation, and lagoon treatment of wastewater, lagoon suffices the requirement of land,
358 water, and nutrients for project establishment and infrastructure development for effective
359 carbon sequestration project. Without losing the major ecosystem services, lagoons stand
360 out as promising potential algae production facilities for carbon sequestration and biomass
361 generation. The Logan Regional Wastewater Treatment Plant (Utah, USA) consists of
362 vast 460 acres of lagoon area, and algae-based approach to treat wastewater with
363 additional production of valuable products and byproductsis ongoing (Grifths, 2009;
364 Christenson et al., 2012). Some of the coastal bays of the Gujarat, India are highly
365 affected by anthropogenic input of nutrients and natural microalgal mat is proliferating,
366 the study done by CSIR-CSMCRIs team concluded that naturally floating microalgal mat
367 can be utilized for bioremediation, carbon sequestration as well as for biofuel production
368 (Patidar et al., 2015). The transportation cost of flue gas from larger point sources such as
369 thermal power plant (having less wastewater), cement plant and steel plant to the
370 wastewater pond/ lagoon would be one of the troubles to overcome (Christenson et al.,
371 2012; Christenson and Sins, 2011); so overall process may become economical. Study is
372 also needed on effect of toxic compounds of flue gas on the microalgae, although many
373 microalgae can remediate these but effects are not explored. Flue gas contains CO2, N2,
374 H2O, O2, NOX, SOX, CXHY, CO, particulate matter, aerosols, halogen acids and heavy
375 metals etc., and collectively about 142compounds. However, some of these compounds

29
376 maybe important nutrient up to certain concentration after dissolution in cultivation
377 medium (Patidar et al., 2014; Van Den Hende et al., 2011). Nitrogen,phosphorous and
378 potassium are the essential nutrients after carbon which is needed for microalgal growth
379 and metabolic functioning to build macromolecule and organelles. The cost of these
380 macronutrients can be a cost on the overall process for cultivation of microalgae; hence
381 utilization of wastewater containing these nutrients with other trace metals can facilitate
382 economically attractive and prudent management of wastewater. Bioremediation of
383 heavy metals, phosphate and nitrate is commonly known for most of the microalgae.To
384 integrate multiple solutions like bioremediation of wastewater, carbon sequestration and
385 biofuel production with value added products can be achievable and affordable with low
386 cost of the cultivation and biofuel (Kumaretal.,2010;Wangetal.,2008).
387 To utilize marine microalgae such as cocolithophores for highly efficient carbon
388 sequestration candidate are yet to be developed. Coccolithophores are important
389 regulators of carbonate chemistry due to their calcifying nature in seawater and sensitive
390 to the change in pH. For example, Emiliania huxleyi is sensitive to low CO2and low
391 bicarbonate as well as low pH. Calcification and photosynthesis do not function in same
392 efficient way hence, process of calcification and photosynthesis should be understood
393 thoroughly with changing pH and carbonate chemistry prior to utilise it for effective carbon
394 sequestration. The overall process of carbon sequestration by oceans in the changing global
395 climate scenariosis still needs more explanatory study (Bach et al., 2013).
396 The intermittent CO2 can be used to achieve maximum bicarbonate uptake in
397 alkaline pH (Aishwaryaet al., 2012) and can increase the lipid content in microalgae
398 such as Monoraphidium and Chlorella as well as maintain the intracellular pH of the cell,
399 such processes also improve lipid content by enhancing nitrogen limitation in the
400 cell(Patidar et al., 2015). The CO2 sequestration from the flue gas requires high lipid
401 productivity (with higher saturated fatty acids) containing strain with high CO2 and
402 temperature tolerance to integrate it with biofuel production (Chinnasami et al., 2009;
403 Patidar et al., 2014). The Cyanidium caldarium (Tilden) Geitler can grow under 100%
404 CO2 and in acidic pH even<1(Seckbachetal., 1970).Unfortunately, there is no follow up
405 on these specific characteristics containing microalgae (KuranoandMiyachi,2004). The
406 microalgae Cyandium caldarium reported to produce up to 2folds fresh weight biomass

30
407 when it grown inpure CO2(100%) than air (Grosetal.,1989). It is thermophilic and also
408 reported to grow heterotrophically in dark with glucose, showing pigment bleaching
409 (Doemeletal.,1971).It can be a promising strain for carbon sequestration from flue gas and
410 wastewater. Dunaliella acidophila also reported to grow in acidic pH (1.0) hence, can
411 remediate CO2 in acidic environment, wherein no need for the pH maintenance through
412 additional alkali (Ben-Amotzet al., 2003).
413 Many efforts have been concentrated on generating biofuel like biodiesel from microalgae
414 while using the wastewater for organic carbon/nitrogen-phosphate/ heavy metals
415 bioremediation in addition to the CO2 sequestration. The U.S. Department of Energys
416 Office of Fuels Development during 1978-1996 seeded an aquatic species program to
417 develop microalgal biofuel by extensive screening of potential strains which can utilize
418 waste CO2 from coal fired plants. The programme recommended metabolic engineering of
419 microalgae for consistent lipid production. Approximately 3000 species of microalgae
420 were isolated and, biochemically and technically characterized for their lipid productivity
421 and CO2 capture potential in this programme and, out of this 300 species were selected for
422 further development of microalgal biodiesel production program (Sheehan et al., 1998).
423 Tropical countries with higher wasteland area are environmentally more suitable to attain
424 higher biomass productivity. According to the technical report of NREL on resource
425 potential for microalgae cultivation for carbon sequestration and biodiesel production, it is
426 projected that India has about 55.27 Mha of wasteland (approximately 18% of the total
427 land area).If India dedicates only10%(5.5Mha) of its wasteland for employing algal mass
428 cultivation, it could yield 2255 Mt of algal lipid which would substitute 45%100% of
429 current diesel consumption. The generation of this huge pile of algal biomass would
430 sequester about 169423 Mt of CO2 which would offset 26%-67% of the present
431 emissions (Milbrandtetal.,2010) and in addition many co-products may be created which
432 can offset the price of the overall cost of the cultivation (Mitra et al., 2016; Mitra et al.,
433 2015; Mitra et al., 2015). Microalgae proliferate in mixotrophic mode with potential
434 biomass productivity in wastewater while utilize CO2, hence, significant biomass and oil
435 yield can be achieved by modulating and optimising the factors, hence the complete bio-
436 refinery for high value and low value products is not so far (Pittman et al., 2011; Devi
437 Prathima et al.,2012; Mitra et al., 2015; Mitra et al., 2016; Patidar et al., 2015; Mishra et

31
438 al., 2014; Patidar et al., 2014; Dibenedetto et al., 2016; Mohan et al., 2016).
439

440

32
441 Acknowledgement
442 CSIR-CSMCRI communication no. 116/2016. Dr. Arvind Kumar, DC, SMC is greatly
443 acknowledged for motivational support. All the labmates are also acknowledged for their
444 timely help during the work.

33
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