Accepted Manuscript

Bicarbonate-based Cultivation of Dunaliella salina for Enhancing Carbon Uti-

lization Efficiency

Ga-Yeong Kim, Jina Heo, Hee-Sik Kim, Jong-In Han

PII: S0960-8524(17)30490-X
DOI: http://dx.doi.org/10.1016/j.biortech.2017.04.009
Reference: BITE 17904

To appear in: Bioresource Technology

Received Date: 29 December 2016

Revised Date: 31 March 2017
Accepted Date: 3 April 2017

Please cite this article as: Kim, G-Y., Heo, J., Kim, H-S., Han, J-I., Bicarbonate-based Cultivation of Dunaliella
salina for Enhancing Carbon Utilization Efficiency, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/
j.biortech.2017.04.009

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 1 Bicarbonate-based Cultivation of Dunaliella salina for Enhancing Carbon Utilization

2 Efficiency

4 Ga-Yeong Kima, Jina Heob,c, Hee-Sik Kimb,c, Jong-In Hana,*

a
5 Department of Civil and Environmental Engineering, KAIST, 373-1 Guseong-dong,

6 Yuseong-gu, Daejeon 305-701, Republic of Korea

b
7 Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology

8 (KRIBB), Daejeon 305-806, Republic of Korea

c
9 Biotechnology, University of Science and Technology (UST), Daejeon 305-350, Republic

10 of Korea

*
11 Corresponding author: Jong-In Han (E-mail address: hanj2@kaist.ac.kr; Tel: +82-42-350-

12 3629; Fax: +82-42-350-3610)

13

14 Abstract

15 In this study, bicarbonate was proposed as an alternative carbon source to overcome

16 exceedingly low CO2 fixation efficiency of conventional microalgae cultivation system. 5 g

17 L-1 of sodium bicarbonate was found to well support the growth of Dunaliella salina,

18 showing 2.84-fold higher specific growth rate than a bicarbonate-free control. This

19 bicarbonate-fed cultivation also could yield biomass productivity similar to that of CO2-based

20 system as long as pH was controlled. While the supplied CO2, because of its being a gas, was

21 mostly lost and only 3.59% of it was used for biomass synthesis, bicarbonate was effectively

22 incorporated into the biomass with 91.40% of carbon utilization efficiency. This study
23 showed that the bicarbonate-based microalgae cultivation is indeed possible, and can even

24 become a truly environment-friendly and workable approach, provided that a CO2

25 mineralization technology is concomitantly established.

26

27 Keywords

28 CO2 capture and utilization (CCU); microalgae; Dunaliella salina; sodium bicarbonate

29 (NaHCO3); bicarbonate cultivation.

30

31 1. Introduction

32 Ever-increasing concerns about the climate change due to CO2 have strongly

33 demanded ways to reduce the greenhouse gas emission and even use it in a beneficial way.

34 These approaches are collectively referred to as carbon capture and utilization (CCU)

35 technology, and microalgae is one of those promising options.

36 Microalgae can assimilate CO2 through photosynthesis, and its biomass can then be

37 processed to produce profitable products, such as biodiesel, pigments, proteins, or functional

38 chemicals (González-Delgado and Kafarov, 2011). There have been a good many studies that

39 microalgae were cultivated in such a way that consumes CO2 contained in flue gases,

40 focusing on the aspect of CCU. Despite the theoretical feasibility of this technology, however,

41 there are three critical obstacles: low quality of flue gas, high cost for CO2 transportation, and

42 limited CO2 utilization efficiency.

43 The flue gas contains toxic components, such as SOX and NOX; thus, its direct

44 application to algal culture may inhibit the algal growth (Negoro et al., 1991). The need for

45 transportation adds another burden. Land available for algae cultivation around power plants
46 is generally limited and hence CO2 has to be transported in a highly pressurized form with a

47 typical pressure of 150 atm (Chi et al., 2011). Besides, the gaseous CO2, when supplied to

48 algae pond, is utilized by microalgae only to a very limited degree on account of its low water

49 solubility. All these happen due to gaseous being of CO2; therefore, it is our hypothesis that

50 by employing aqueous bicarbonate and bicarbonate-utilizing microalgae species, such

51 problems can be solved.

52 Sodium bicarbonate (NaHCO3), which is obtained from CO2 mineralization process,

53 can be a good candidate for the alternative carbon source. In fact, our research group has been

54 developing a method of electrochemically producing bicarbonate from CO2

55 (PCT/KR2015/008405). Solid or liquid forms of NaHCO3 have no need of energy intensive

56 compression and can be transported through a simple water pipe line. The high water

57 solubility of NaHCO3 (9.21% (w/w) at 25˚C) also leads to high carbon utilization efficiency,

58 and all these make the overall process simple and easy to control.

59 Up until now, however, only a few studies have demonstrated that bicarbonate ion

60 indeed has a positive effect on microalgal cell growth, and they have mainly focused on the

61 basic mechanism at the genetic level (Chi et al., 2011; Moroney and Ynalvez, 2007; Price et

62 al., 2008; Wang et al., 2011). Among microbes known to be able to grow on bicarbonate ion,

63 prokaryotic cyanobacteria have been mainly studied because of their superior genetic

64 manipulability to eukaryotic algae, and works on eukaryotic microalgae were scarce (Ma et

65 al., 2014).

66 The objective of this study was, therefore, to explore the ability of eukaryotic green

67 algae to utilize NaHCO3 as its major carbon source from an engineering point of view. To this

68 end, Dunaliella salina, which is a halophilic green microalgae often found in marine habitats,

69 was selected as a model eukaryotic algae species. Dunaliella salina is known to be the richest

70 natural source of carotenoid, beta carotene, and this property makes it possible to capture CO2
71 in an economically beneficial way (Borowitzka, 1990).

72 The bicarbonate-based microalgae cultivation can be more advantageous especially

73 when combined with these seawater algal species. Seawater, which has a salinity value of 35

74 g salt per kg seawater, has the following carbonate species concentrations at pH 8.1 and 25˚C:

75 [CO2] = 10.4 µmol kg-1, [HCO3-] = 1818 µmol kg-1, and [CO32-] = 272 µmol kg-1 with pK1

76 and pK2 values of 5.86 and 8.92, respectively (Zeebe and Wolf-Gladrow, 2001). This

77 condition that is low in CO2 but high in bicarbonate is expected to be beneficial to the

78 bicarbonate-based cultivation rather than the CO2-based one.

79 The effect of bicarbonate on the growth of Dunaliella sp. was investigated, and

80 optimal culture conditions, such as NaHCO3 concentration, culture temperature, and light

81 intensity, were also determined. In addition, the potential of this bicarbonate-based

82 microalgae cultivation was assessed in terms of biomass and carotenoid productivity

83 compared to the conventional CO2-based cultivation. Finally, carbon utilization efficiency,

84 which is the most important criterion as a CCU technology, was estimated.

85

86 2. Materials and methods

87 2.1. Bicarbonate uptake of Dunaliella salina

88 Dunaliella salina JDS 001 (KT778301.1) was isolated from the salt pond in the

89 western sea, Korea, and it was maintained in Modified Johnsons (MJ) medium with 2 M of

90 NaCl (Borowitzka, 1988). To check whether this Dunaliella species is able to grow with

91 bicarbonate as a carbon source, it was cultivated with various concentrations of NaHCO3: 0

92 to 30 g L-1 of NaHCO3 were supplemented into the MJ medium. For this experiment, a new

93 equipment named PhotoBiobox was employed (Heo et al., 2015). This high-throughput

94 device made it possible to cultivate microalgae in a 96-well plate with a wide range of
 95 temperature and light intensity. In each of 96 wells, 200 μL of the Dunaliella culture, which

96 had 0.1 of initial optical density at 680 nm (OD680), was added, and the plate was sealed with

97 an air permeable membrane (Breathe-Easy, Diversified Biotech). Dunaliella sp. was then

98 cultivated for 2-3 days with each chosen condition in triplicate until it reached stationary

99 phase. OD680 was measured with a microplate reader (Sunrise, Tecan) three times a day, and

100 the specific growth rate (SGR) of Dunaliella sp. was calculated only at the exponential

101 growth phase by the equation (1) where X1 and X2 represent OD680 on day t1 and t2,

102 respectively.

103 (1)

104 Temperature and light intensity were maintained as 25˚C and 340 μE m-2 s-1 (based on

105 the surface of membrane), respectively, and the same experiment was repeated by using the

106 Dunaliella cells, which had been cultured in the first batch, as an inoculum. New MJ media,

107 which contained each concentration of NaHCO3, were supplemented and each well served as

108 an inoculum to make initial OD680 of 0.1 with all other conditions remained the same.

109

110 2.2. Optimization of temperature and light intensity

111 Optimum temperature and light intensity for bicarbonate-based Dunaliella cultivation

112 were obtained with 5 g L-1 of NaHCO3 and compared to those obtained from CO2-based

113 cultivation. 5% (v/v) of CO2 was supplied at a steady flow, and 5 g L-1 of NaHCO3 was

114 included in the MJ medium at the outset only. In the PhotoBiobox, 96 different combinations

115 were simultaneously tested: 12 different levels of temperature, from 19 to 31˚C, and 8 levels

116 for light intensity, from 85 to 500 μE m-2 s-1. Dunaliella sp., which had been acclimated to

117 each of carbon sources and stored at 25˚C with 30 μE m-2 s-1 of light intensity under the LED

118 fluorescent light, was used as an inoculum. It was then cultivated for two days, and optimal
119 conditions were determined on the basis of its SGR.

120

121 2.3. Lab scale cultivation of Dunaliella salina

122 The effectiveness of aqueous bicarbonate as a substitute carbon source was confirmed

123 in a larger flask scale. For that, bicarbonate-based cultivation with 5 g L-1 of NaHCO3 and

124 CO2-based cultivation with 2% (v/v) of CO2 were comparatively analyzed. In the

125 PhotoBiobox experiment, CO2 was provided through an air-permeable membrane after being

126 filled in a closed compartment. In the flask cultivation, however, CO2 was directly bubbled

127 into the culture medium, so it was supplied with a reduced amount of 2% (v/v) rather than 5%

128 (v/v).

129 Dunaliella cells were inoculated into 250 mL baffled culture flasks containing 220 mL

130 of either MJ medium with 5 g L-1 of NaHCO3 (for bicarbonate-based cultivation) or MJ

131 medium (for CO2-based cultivation) in such a way that initial OD680 values fell into 0.2.

132 Cultivations were proceeded with the operational conditions of shaking at 160 rpm,

133 temperature at 30ºC, 280 μE m-2 s-1 of light intensity at the surface of culture medium, and 0.2

134 vvm of air or 2% (v/v) CO2 supply. All cultivations were done in triplicate, and growth was

135 monitored in terms of OD680.

136 This experiment was repeated with pH controlled and with all the other conditions

137 remained unchanged. The pH was adjusted as 8 by adding hydrochloric acid on a daily basis.

138

139 2.4. Analytical methods

140 The cell number of Dunaliella sp. was counted by using an automated cell counter

141 (Cellometer, Nexcelom). Ash free dry weight (AFDW) was measured by Zhu’s method with

142 ammonium formate as a washing solution, and concentration of carotenoids by

143 Lichtenthaler’s method (Lichtenthaler and Wellburn, 1983; Zhu and Lee, 1997). Carbon
144 utilization efficiency was defined as the percentage of fixed carbon in total input carbon.

145

146 (2)

147

148 In the bicarbonate-based cultivation, total input carbon was defined as the sum of (1)

149 a reduced amount of total inorganic carbon (TIC), which was obtained by total organic

150 carbon analyzer (Multi N/C 3100, Analytikjena), and (2) an amount of carbon introduced

151 through air bubbling. For the CO2-based cultivation, the total input carbon was calculated

152 based on an amount of carbon supplied in the form of CO2 throughout the cultivation.

153 In order to estimate the mass of fixed carbon, amounts of carbon contained in final

154 microalgae biomass were obtained by an element analyzer (FLASH 2000 series, Thermo

155 Scientific), and the carbon content was multiplied by AFDW. The detailed calculation process

156 was described in the supplementary materials.

157

158 3. Results and discussions

159 3.1. Bicarbonate uptake of Dunaliella salina

160 Dunaliella sp. was able to well utilize NaHCO3 as a carbon source, and their response

161 was concentration-dependent. As the concentration of NaHCO3 increased, the SGR of

162 Dunaliella sp. also increased especially at a low concentration range. Specifically, 0.34 day-1

163 of initial SGR was raised to 0.70 day-1 (2.06 times) in the presence of 5 g L-1 of NaHCO3 (Fig.

164 1). In addition, in the second round of batch cultivation, when the Dunaliella cells had

165 already exposed and adapted to bicarbonate, SGRs were further increased by 2.84 times

166 (from 0.37 day-1 of initial SGR to 1.05 day-1).

167 In general, when microalgae are exposed to a high level of sodium, sodium ions are
168 accumulated to an excessive degree, leading to the inactivation of some enzymes and

169 eventually to the growth inhibition (Sudhir and Murthy, 2004). It is this reason that a majority

170 of microalgae species, even some marine species, typically have a peak SGR at a certain

171 concentration of NaHCO3, and above it, their growth is inhibited (White et al., 2013). On the

172 contrary, Dunaliella sp. behaved differently. It reached a stationary state at 5 g L-1, and SGRs

173 were maintained even above it. This was attributable to the fact that Dunaliella sp., which

174 had been isolated from saltworks where sea salts are produced through evaporating the

175 seawater, potentially had developed resistance to high sodium conditions. While a

176 conventional culture medium for Dunaliella sp. contains only 0.043 g L-1 of NaHCO3

177 (Borowitzka, 1988), the NaHCO3 concentration for the optimal growth was found to be 5 g L-
1
178 ; this concentration was used in all subsequent experiments.

179 SGR with respect to NaHCO3 concentration could be also nicely depicted by the

180 nonlinear fitting of Monod equation (Fig. 1), and its kinetic parameters were determined as

181 maximum SGR (µmax) of 1.11 day-1 and half saturation constant (KS) of 0.42 g L-1.

182

183 3.2. The optimization of culture condition

184 Optimum temperature and light intensity were also investigated for both bicarbonate-

185 based and CO2-based Dunaliella cultivations, and the optimum SGRs were compared to see

186 how the carbon source affected growth behaviors.

187 The highest SGRs were found to be not much different regardless of the carbon

188 source: 0.97 day-1 with bicarbonate and 0.95 day-1 with CO2. Conditions to support the best

189 growth, however, were somewhat different: 30.2ºC and 375 μE m-2 s-1 of light intensity with

190 bicarbonate and 27.9ºC and 425 μE m-2 s-1 with CO2 (Fig. 2).

191 Contrary to a widely accepted notion that microalgae prefer CO2 to bicarbonate

192 (Moroney and Somanchi, 1999), Dunaliella sp. responded similarly to both of the two.
193 Dunaliella sp., which had long been exposed to its natural habitat of the seawater, where CO2

194 is scarce but bicarbonate is rich ([CO2] : [HCO3-] : [CO32-] ≈ 0.5% : 86.5%: 13%) (Zeebe and

195 Wolf-Gladrow, 2001), is likely to be adapted to the bicarbonate, similarly to CO2. This ability

196 of Dunaliella sp. to utilize bicarbonate as a carbon source, even with comparable SGR to the

197 conventional CO2-based cultivation, has high applicability. Gas-free microalgae cultivation

198 yet with equivalent biomass productivity has a great many operational advantages.

199 With CO2, however, Dunaliella sp. could have high SGRs (> 0.7 day-1) in wider

200 range of temperature and light intensity than bicarbonate: in about half of conditions tested

201 for the CO2-based cultivation, but in only about a quarter for the bicarbonate. This implied

202 that it is critical to maintain a certain controlled condition so as to obtain the high biomass

203 productivity with the NaHCO3-based approach.

204

205 3.3. NaHCO3-based cultivation vs. CO2-based cultivation

206 To confirm the experimental results obtained from the PhotoBiobox, Dunaliella sp.

207 was cultivated with a larger-scaled reactor. In the 200 μL-scaled test, the instantaneous SGRs

208 were similar in both bicarbonate-based and CO2-based cultivations, but it was not the case in

209 the flask test, particularly in a prolonged cultivation.

210 The bicarbonate-based cultivation was found to be inferior to the CO2-based one; the

211 final OD680 value under the CO2 condition was almost twice as high as that under the

212 bicarbonate condition (Fig. 3). This retarded growth in the bicarbonate condition appeared to

213 be related with a sudden rise in pH. While pH in the CO2 condition was maintained around 7-

214 8, it was escalated rapidly to 10 in three days in the bicarbonate condition. It was from this

215 third day that the OD values started to show significant difference. This pH rise was because

216 OH– ions were accumulated in the process of metabolizing bicarbonate (Chi et al., 2014).

217 Bicarbonate must be first converted into CO2 to be fixed by Rubisco, but in so doing H+ is
218 consumed and OH– is left in the algal cell according to this equilibrium equation, H+ +

219 HCO3– ⇌ CO2 + H2O. This OH– is then neutralized by H+ uptake from the extracellular

220 environment; thus, the reduction of H+ in the culture medium unavoidably increases the pH.

221 To prove this hypothesis, the growth of Dunaliella sp. with bicarbonate was re-

222 examined, but in this time with pH controlled. The pH was adjusted to 8 instead of 7-7.5,

223 which was the pH range in the CO2 condition. Considering the aim of this study to cultivate

224 microalgae with bicarbonate, pH 8, at which bicarbonate is abundant according to the pKa

225 values of carbonate species, was the most desirable condition. In addition, to lower the pH by

226 using chemical leads to an increase in overall production cost. Taking these reasons together,

227 the pH 8 was determined to be the best value in terms of both efficiency and economic

228 feasibility.

229 As expected, almost the same amount of OD680 and cell number were achievable at

230 the end of cultivation in both conditions (Fig. 4). The AFDW also supported this with 3.17 ±

231 0.15 g L-1 in the bicarbonate condition and 3.47 ± 0.25 g L-1 in the CO2 condition. Although it

232 has been a rather unshakable belief that CO2 is superior and irreplaceable for the effective

233 microalgae cultivation, our results strongly suggested that bicarbonate also can produce

234 similar performance to CO2 as long as pH is maintained.

235 This bicarbonate ion had positive effects not only on the biomass productivity of

236 Dunaliella sp. but also on the productivity of target value chemicals, carotenoids, showing

237 the highest carotenoids concentration in the bicarbonate condition, 20.43 ± 2.84 µg mL-1 (Fig.

238 5). This value was significantly higher than that of a similar previous study, 7.10 ± 0.08 µg

239 mL-1 with 12.6 g L-1 of NaHCO3 (Srinivasan et al., 2015). Usually, Dunaliella sp. has a

240 separated induction stage, particularly for the accumulation of carotenoids, and this induction

241 process is carried out by several stress conditions, such as nitrogen starvation, high salinity,

242 or high temperature (Pisal and Lele, 2005). Further in-depth research may prove that
243 NaHCO3-based stress can indeed be an easy and effective induction method, and even more

244 so when it is combined with the NaHCO3-based cultivation in a sequential manner.

245

246 3.4. Carbon utilization efficiency

247 The most important factor of microalgae cultivation as a CO2 utilization technology is

248 how much carbon is converted into algal biomass. To assess it, carbon utilization efficiency

249 was estimated on the basis of the 9th day when Dunaliella cells were still actively dividing.

250 In the bicarbonate condition, TIC decreased from 533.5 mg L-1 to 152.6 mg L-1 with

251 the growth of Dunaliella sp. on the 9th day, and Dunaliella sp. grown with bicarbonate

252 contained 49.59 ± 0.42% of carbon and with CO2 51.06 ± 0.67% (Table 1), suggesting that

253 the form of utilized carbon was not a determining factor of carbon contents. The amounts of

254 fixed carbon were obtained by multiplying these carbon contents by AFDW of Dunaliella sp.:

255 1.85 g L-1 in the bicarbonate condition and 2.22 g L-1 in the CO2 condition.

256 The overall carbon utilization efficiency in the bicarbonate-based cultivation was

257 found to be 91.40%, and in the CO2 cultivation 3.59%. It is typical that open-culture systems

258 are not effective in terms of CO2 utilization. CO2 supplied in a gaseous form easily escapes

259 from the culture medium, rather than being captured and utilized by the cell, due to its poor

260 mass transfer efficiency. It is also because the medium is generally neutral and the reactors

261 are too shallow to keep the gas in the medium long enough. All these consequently result in a

262 high CO2 outgassing rate and low CO2 fixation efficiency of only 10-30% (E. W. Becker,

263 1994; Weissman and Goebel, 1985). In this study, this inherent inefficiency was further

264 exaggerated as 3.59% on account of smallness and shallowness of the bioreactor. Even taking

265 it into consideration, however, this huge discrepancy (3.59% and 91.40%) indicated that most

266 of CO2, which was continuously supplied throughout the cultivation just as in the

267 conventional system, was lost to the atmosphere. Besides, the solubility of CO2 decreases
268 with increasing salinity (Hangx, 2005). The exceedingly high salinity (2 M NaCl) of MJ

269 medium might also play a role in the high outgassing rate. With bicarbonate, however, such

270 an uncontrollable loss did not take place because of its being ions in water; thus, it appeared

271 to be clear that carbon utilization efficiency is much higher when bicarbonate is used as a

272 carbon source. This also implies that substantially more is required with the gaseous form of

273 CO2 than with an ionic form of bicarbonate to yield the same biomass productivity.

274 It is true that the bicarbonate-based culture system also has several weaknesses; for

275 instance, high cost for bicarbonate supply and the necessity of pH control. This bicarbonate-

276 based algae cultivation, however, can serve as a truly eco-friendly way of producing algae

277 biomass when an innovative technology for CO2 mineralization, such as electrochemical

278 conversion of CO2 to bicarbonate, is industrialized (PCT/KR2015/008405; Xie et al., 2014).

279

280 4. Conclusions

281 This study demonstrated that NaHCO3 can be a carbon source for microalgae

282 cultivation, and it can be equally effective to the gaseous CO2. The proposed bicarbonate-

283 based approach is particularly advantageous from an operational standpoint because

284 continuous supply of CO2 gas, even with its cost ignored, is rather a hassle. The gas-free

285 microalgae cultivation makes the overall bio-process easier to control. Furthermore, it has a

286 significant merit from a CCU perspective as well. To realize the potential of this ideal way of

287 supplying carbon, however, economic methods of producing bicarbonate and controlling pH

288 must be concomitantly developed.

289

290 Supplementary materials are available online.

291
292 Acknowledgements

293 This research was supported by the Advanced Biomass R&D Center (ABC) through

294 the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT &

295 Future Planning (NRF-2011-0031348) and by Korea Electric Power Corporation (KEPCO)

296 with Contraction number of CX72-16-0009.

297

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355
356 Fig. 1. The specific growth rates of Dunaliella salina with various concentrations of NaHCO3.

357 Fig. 2. The optimization of temperature and light intensity (a) with 5% CO2 (v/v) supply, and
358 (b) with NaHCO3 5 g L-1: red color represents high growth rate (day-1), and purple for low
359 growth rate.

360 Fig. 3. Cultivation of Dunaliella salina.

361 Fig. 4. Cultivation of Dunaliella salina with pH controlled: (a) OD680 and cell number; and (b)
362 pH.

363 Fig. 5. Carotenoids concentration of Dunaliella salina.

364 Fig. 1. The specific growth rates of Dunaliella salina with various concentrations of NaHCO3.
365

366
367

368

17
369 Fig. 2. The optimization of temperature and light intensity (a) with 5% (v/v) CO2 supply, and
370 (b) with NaHCO3 5 g L-1: red color represents high growth rate (day-1), and purple for low
371 growth rate.
372

373

374
375

18
376
377 Fig. 3. Cultivation of Dunaliella salina.
378

379

380

381

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382 Fig. 4. Cultivation of Dunaliella salina with pH controlled: (a) OD680 and cell number; and (b)
383 pH.

384

20
385
386
387

388 Fig. 5. Carotenoids concentration of Dunaliella salina.

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389

390

391

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392 Table 1 Element composition of cultured Dunaliella salina

Contents (w/w %) Carbon Nitrogen Hydrogen

NaHCO3 5 g L-1 49.59 ± 0.42 6.90 ± 0.31 7.44 ± 0.04

2% (v/v) CO2 51.06 ± 0.67 6.83 ± 0.07 7.65 ± 0.03

393

394

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395 Highlights

396

397  To utilize bicarbonate as carbon source for microalgae cultivation was investigated.

398  The specific growth rate of Dunaliella sp. was significantly increased with NaHCO3.

399  pH control was important to obtain high biomass productivity with bicarbonate.

400  NaHCO3 positively affected not only for growth but also for carotenoid production.

401  With bicarbonate, carbon utilization efficiency was apparently higher than with CO2.

402

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