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Optimizing the growth of Haematococcus pluvialis based on a novel microbubble-

driven photobioreactor
--Manuscript Draft--

Manuscript Number: ISCIENCE-D-21-01398

Full Title: Optimizing the growth of Haematococcus pluvialis based on a novel microbubble-
driven photobioreactor

Article Type: Research Article

Order of Authors: Kebi Wu

Kezhen Ying

Jin Zhou

Dai Liu

Lu Liu

Yi Tao

James Hanotu

Xiaoshan Zhu

Zhonghua Cai, Ph.D.

Abstract: Haematococcus pluvialis ( H. pluvialis ), the richest bioresource for natural

astaxanthin, encounters a challenge of achieving high growth rate when it comes to
mass biomass production.  Base on substrate consumption model and Redfield ratio,
rapid algae growth benefits from a proper carbon supply. However, the conventional
cultivation schemes with limited CO 2 supply and inefficient carbon mass transfer may
restrict the carbon capture and growing ability of H. pluvialis . We hypothesize that
optimal H. pluvialis growth improvement may be achieved by efficient CO 2 supply.
In this study, we first identified the carbon consumption of H. pluvialis during
exponential growth. Then, a novel microbubble-driven photobioreactor (MDPBR) was
designed to satisfy the carbon demand. The novel microbubble photobioreactor
improve the CO 2 supply by reducing bubble size, then the CO 2 mass transfer
significantly elevated. With only 20% of gas flow rate, higher cell growth rate (0.49 d -1
) has been achieved in MDPBR.

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1 Optimizing the growth of Haematococcus pluvialis

2 based on a novel microbubble-driven photobioreactor

3 Kebi Wua,b1, Kezhen Yingb1, Jin Zhoub, Dai Liu b, Lu Liu a,b, Yi Taoc, James Hanotud, Xiaoshan

4 Zhua, Zhonghua Caia,b*

5 a
School of Life Sciences, Tsinghua University, Beijing, 100086, China

6 b
Shenzhen Public Platform for Screening and Application of Marine Microbial Resources, Shenzhen

7 International Graduate School, Tsinghua University, Shenzhen 518055, China

8 c
Guangdong Provincial Engineering Research Centre for Urban Water Recycling and Environmental

9 Safety, Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China

10 d
Department of Chemical and Biological Engineering, The University of Sheffield, S13JD, United

11 Kingdom

12 1
These authors contributed equally to this work and should be considered co-first authors

13 *Corresponding author: Zhonghua Cai

14 Shenzhen International Graduate School, Tsinghua University

15 E-mail: caizh@sz.tsinghua.edu.cn

16

17

18

1
19 SUMMARY

20 Haematococcus pluvialis (H. pluvialis), the richest bioresource for natural astaxanthin,

21 encounters a challenge of achieving high growth rate when it comes to mass biomass

22 production. Base on substrate consumption model and Redfield ratio, rapid algae growth

23 benefits from a proper carbon supply. However, the conventional cultivation schemes

24 with limited CO2 supply and inefficient carbon mass transfer may restrict the carbon

25 capture and growing ability of H. pluvialis. We hypothesize that optimal H. pluvialis

26 growth improvement may be achieved by efficient CO2 supply. In this study, we first

27 identified the carbon consumption of H. pluvialis during exponential growth. Then, a

28 novel microbubble-driven photobioreactor (MDPBR) was designed to satisfy the carbon

29 demand. The novel microbubble photobioreactor improve the CO2 supply by reducing

30 bubble size, then the CO2 mass transfer significantly elevated. With only 20% of gas flow

31 rate, higher cell growth rate (0.49 d-1) has been achieved in MDPBR.

32

2
33 INTRODUCTION

34 Microalgae play a promising role in CO2 fixation and provide high-value products for

35 healthy diet and medical care. Among those algal bioproducts, astaxanthin (C40H52O4,

36 3,3′-dihydroxy-β,β′-carotene-4,4′-dione) gains its name as a potent antioxidant, widely

37 applied for anti-inflammation, anti-aging, immune system enhancement, cancer

38 prevention etc (Cui et al., 2019; Fakhri et al., 2020; Kim et al., 2019; Medhi and Kalita,

39 2021; Olaizola, 2000). Chan et al.(2012) reported that involving astaxanthin in diet can

40 significantly reduce MCP-1, TNF-α, IL-6, and ROS in diabetic rats. Recently, Talukdar

41 et al.(2020) suggested that algal-derived astaxanthin has considerable possibility as a

42 supplementary medicine for clinical help against COVID-19 for its anti-oxidative benefit.

43 As the market demand has surged over the years, the industrial production of astaxanthin

44 becomes a critical issue. The commercial astaxanthin used to be dominated by synthetic

45 astaxanthin due to its low production cost and steady yield, only less than 1% of the

46 market was taken by natural astaxanthin (Li et al., 2011). However, due to the difference

47 in isomerism and chemical structure between natural astaxanthin and synthetic

48 astaxanthin, there are concerns about the biological functions and food safety for synthetic

49 astaxanthin (Li et al., 2011; Milledge, 2011). To date, the chemical astaxanthin has not

50 been approved for direct human consumption, instead, it can only be applied as an

51 additive to fish and livestock industry (Li et al., 2011; Panis and Carreon, 2016).

52 Significantly, natural astaxanthin has been confirmed safe for direct human consumption

53 in food and supplements(Guerin et al., 2003), and becomes competitive in the global

54 market (Pérez-López et al., 2014).

3
55 Among various sources for natural astaxanthin, microalgae Haematococcus pluvialis (H.

56 pluvialis) is considered to be the richest bioresource for its considerable astaxanthin

57 accumulation under stress condition. (Ambati et al., 2014; Kaewpintong et al., 2007).

58 Consequently, there is a rising interest on developing the bioprocess for H. pluvialis

59 cultivation.

60 During cultivation process, since H. pluvialis has two distinctive morphologies--green

61 vegetative cell and red rest cell (Xi et al., 2016). To attain an effective astaxanthin

62 production and to make the scale-up feasible, achieving a high vegetative cell density

63 before subjected to the stress conditions for astaxanthin accumulation is one of the

64 promising ways (Kang et al., 2009; Wang et al., 2013; Yoo et al., 2012). Thus, research

65 communities and industries have mainly resorted to two-stage cultural mode, intending

66 for high biomass and astaxanthin yield(Fábregas, Jaime et al., 2001; Hong et al., 2015).

67 Although progress has been made in vegetative H. pluvialis cultivation, rapidly achieving

68 high biomass during massive cultivation in vegetative stage remains a challenge and most

69 of the Photo-bioreactor (PBR) cultures have been facing the obstacle of low yield/cost

70 ratio. (Kang et al., 2009; Vega-Estrada et al., 2005; Yoo et al., 2012).

71 The key solution to this difficulty could lie in efficient nutrient supply. Base on substrate

72 consumption model(Zhang et al., 1999), sufficient substrate supply is closely related to

73 algal productivity. The chemical formations of algae partly determine the nutrient

74 demand(Hillebrand and Sommer, 1999). Previous studies have focused more on N, P

75 requirements in the cultivation of H. pluvialis (Imamoglu et al., 2007; Nahidian et al.,

76 2018; Tocquin et al., 2012). Fábregas, J. et al.(2000) claimed that higher cell density was

77 obtained after tripled the nutrient concentrations in Bold Basal Medium (BBM medium).

78 While Tocquin et al.(2012) declared that a lower N/P ratio (below 1) favours vegetative

4
 79 growth of H. pluvialis. The inconsistent results indicated that N, P or N/P ratio supplement

80 in medium may not the sole key parameter in H. pluvialis mass production.

81 Generally, it is believed that the ideal chemical elements present in average phytoplankton

82 biomass demonstrate a Redfield ratio (C: N: P = 106: 16: 1). The idealized chemical

83 reaction of phytoplankton can be illustrated as followed equation:

84 106CO2+16HNO3+H3PO4+78H2O⇌(C106H175O42N16P)+150O2

85 which indicates the potential CO2 requirement and capture ability of phytoplankton.

86 Therefore, CO2 concentration should be a crucial part to consider for algal cultural

87 industry. (Arun et al., 2021; Cleveland and Liptzin, 2007; Judd et al., 2017; Singh, S. P.

88 and Singh, 2014). According to Zimmerman et al.(2011), the optimal CO2 uptake by

89 Dunaliella salina photosynthesis could reach up to 0.3×10-4 mol L-1 min-1, whereas,

90 according to an optimistic estimation on a previous research Ying, K. et al.(2013a), the

91 CO2 mass transfer rate of conventional technologies was in a range of 0.4×10-6 - 0.7×

92 10-5 mol L-1 min-1, far away from meeting the CO2 demand for Dunaliella salina culture.

93 Singh, S. K. et al.(2016) indicated that the maximum biomass productivity of Spirulina

94 (1.03 g L-1 d-1) was achieved when CO2 of cultivation elevated to 6% which is

95 significantly higher than traditional cultivation devices like race pond supplying merely

96 less than 20% of CO2 of the maximum demand.

97 For H. pluvialis, the element stoichiometry was reported as 45.6% C, 8.2% H, 6% N,

98 0.58%(García-Malea et al., 2006; Razon, 2011) , revealing sufficient carbon supply is

99 pivotal for algae biomass enhancement. In order to provide more CO2 for H. pluvialis

100 cultivation, airlifting, liquid pumping, or impeller mixing etc. have been applied

5
101 (Borowitzka, 1999; Carvalho et al., 2006; Tsygankov, 2001), but the growth rate of

102 vegetative H. pluvialis still less than satisfactory. The conventional cultivation schemes

103 with inefficient carbon mass transfer may restrict the carbon capture and growing ability

104 of H. pluvialis. Thus, we hypothesize that optimal H. pluvialis growth improvement can

105 be achieved by increasing CO2 mass transfer for efficient CO2 supply.

106 To tackle the mass transfer issue, microbubble technology was developed which has been

107 widely applied in wastewater treatment industries (Rehman et al., 2015; Yamashita et al.,

108 2010; Yao et al., 2016). With microbubble device, threefold improvement of mass

109 transfer rate was achieved, the sufficient mixing and gas flow benefits wastewater

110 treatment process(Rehman et al., 2015). According to Chisti (Erickson, 1990), the CO2

111 mass transfer rate is mainly determined by the volumetric mass transfer coefficient (KLa)

112 and KLa manifests cubic growth by reducing the bubble size. Studies have shown that

113 the CO2 mass transfer can be significantly elevated by using microbubble technology

114 (Ying, K. et al., 2013a; Ying, K. et al., 2013b; Zimmerman et al., 2011). Based on the

115 feature of high mass transfer, it is possible to improve the H. pluvialis growth by replacing

116 the conventional CO2 feeding technologies with microbubbles.

117 In this study, we identified the carbon consumption of H. pluvialis during exponential

118 growth via studying its growth and carbon consumption kinetics. To validate the

119 hypothesis, a novel microbubble-driven photobioreactor (MDPBR) was applied. The

120 technology could serve as an alternative to enhance H. pluvialis mass production, and

121 could be further applied for CO2 mitigation and bioproduct production.

122

6
123 RESULTS AND DISCUSSION

124 H. pluvialis growth and carbon consumption

125 Effect of carbon concentration on MDPBR growth

126 The growth curves of H. pluvialis cultured with different carbon concentrations were

127 shown in Fig. 2a. The H. pluvialis culture with 2 mM of carbon addition showed the

128 lowest biomass productivity, with the dry weight rising from 0.048 to 0.081 g L-1 within

129 1.5 days. The cultures with 4 mM and 8mM of carbon addition started with an initial

130 biomass concentration of 0.050 and 0.045 g L-1, respectively. However, they achieved a

131 similar final biomass concentration of approximately 0.109 g L-1. Therefore, 8mM of

132 carbon addition presented a slightly higher biomass productivity than 4mM of carbon

133 addition. In general, higher carbon concentration in the culture was found having positive

134 effect on the H. pluvialis biomass growth. Similar results have also been reported on other

135 microalgal species. For instance, Schultze et al.(2015) found that increasing the CO2

136 concentration from the atmospheric concentration to 2% resulted in a significant

137 improvement in biomass productivity for the cultivation of Halochlorella rubescens. Ryu

138 et al.(2009) compared the growth of Chlorella sp. with different CO2 concentrations

139 ranging from 0.5% to 5.0%, and found that the highest cell density was achieved with 5%

140 CO2 injection. However, Ying, K et al.(2014) studied the effect of CO2 concentration on

141 Dunaliella salina, and the results were found opposite. The photosynthetic rate decreased

142 slightly by rising the CO2 concentration from 2 mM to 8 mM. Further enhancing the

143 concentration to 20 mM resulted in the cell death. The discrepancy in those results could

144 mainly attribute to the various levels of tolerance on carbon concentration for various

145 algal species. Besides, extremely high CO2 concentration could lead to a lower

146 intracellular pH level which may inhibit the activities of photosynthetic enzymes (Ying,
7
147 K et al., 2014). In our case, the pH for each culture was in the range of 7- 8 during the

148 entire experimental period, which was suitable for H. pluvialis growth (Choi et al., 2017;

149 Wan et al., 2014). Therefore, inhibition was not observed even with 8 mM of carbon

150 concentration. However, an attenuation of the improvement on the productivity was

151 observed when rising the carbon concentration from 4 mM to 8 mM, indicating that the

152 upper limit of carbon concentration is close to 8 mM. It is therefore possible that the

153 growth might be inhibited by keeping increasing the carbon concentration above the

154 threshold value of 8 mM.

155 Regarding to the overall specific growth rate (μ), the value was calculated for every 12

156 hours, plotted in Fig. 2b. 8 mM of carbon addition resulted in the highest μ at each stage

157 of time, followed by 4 mM and 2 mM. Nonetheless, the μ for the culture with either

158 carbon concentration exhibited a descending trend across the whole culture period. The

159 value of μ declined from around 0.41, 0.63, 0.78 d-1 to 0.30, 0.47, 0.56 d-1 for 2, 4 and 8

160 mM of carbon supply, separately. The phenomenon can be well explained by the Monod

161 first order model (Eq. 4). The instantaneous specific growth rate is positively correlated

162 to the instantaneous concentration of the limiting substrate (carbon concentration in this

163 case). Therefore, the culture with higher concentration of carbon supplement lead to a

164 greater μ at any stage. However, as the microalgae grew, the residual concentration of

165 carbon decreases due to the photosynthetic consumption, consequently the specific

166 growth rate slows down with time.

167 Effect of carbon concentration on carbon uptake

168 Fig. 2c presented the residual carbon concentration at each day. From Fig. 2c, the carbon

169 concentration declined with time for each culture, while it remained almost the same in

170 each control set. It can be therefore assumed that the reduction of carbon concentration
8
171 was mainly caused by algal consumption. For 2, 4 and 8 mM of carbon supplement, their

172 corresponding cultures started with the initial carbon concentrations of 2.2, 4.3 and 8.3

173 mM, and ended with the final carbon concentrations of about 2.0, 3.9 and 7.7 mM,

174 separately. The total carbon consumption achieved 0.2, 0.4 and 0.6 mM within 1.5 days,

175 resulting in an average carbon consumption rate of about 0.13, 0.26 and 0.40 mM d-1,

176 separately. In total, higher concentration of carbon addition lead to more carbon

177 consumption. As discussed in 3.1.1, higher carbon concentration had positive impact on

178 biomass growth. As a result, more carbon was consumed to contribute the growth.

179 Similarly, Ying, K. et al.(2013b) also found that the carbon uptake was proportional to

180 the biomass increase in Dunaliella salina culture. Therefore, considering both Fig. 2a and

181 Fig. 2c, the carbon consumption can be strongly related to the biomass increase, which

182 was shown in Fig. 2d. Fig. 2d demonstrated that the residual carbon concentration

183 decreased with the increase in biomass concentration (dry weight) for each culture. The

184 amount of carbon being consumed for every unit of biomass increase, represented by the

185 slope of each linear regression, was nearly the same for each culture, which was about

186 7×10-3 mol g-1. The reciprocal of the slope in Fig. 2d (142.9 gCell molC-1) stands for the

187 biomass produced per unit of carbon consumption, known as yield coefficient (Yw/C).

188 Even though higher concentration of carbon caused more carbon consumption by

189 microalgae, the yield coefficient was however not affected. It is highly supported by the

190 theoretical assumption that the activities of the enzymes for nutrient assimilation should

191 be constant under a fixed environment including temperature, pH, salinity etc.(Najafpour,

192 2015).

9
193 Growth and carbon consumption kinetics

194 The overall specific growth rate for each culture was calculated based on Eq. 1, while the

195 corresponding carbon concentration was estimated as an average value for 1.5 days. The

196 reciprocal of overall specific growth rates (1/μ) versus the reciprocal of carbon

197 concentrations (1/CC) were plotted (data not shown), where the slope and intercept of the

198 linear regression represented the KC/μmax and 1/μmax, respectively, according to the

199 linearized form of Eq. 4. The saturation constant on carbon (KC) and the maximum

200 specific growth rate (μmax) were therefore found to be 2.0 mM and 0.68 d-1, separately.

201 Based on Eq. 4, to attain the maximum specific growth rate, the instantaneous carbon

202 concentration in the medium should be maintained at 0.4 M, approximately 200 times

203 higher than KC (Erickson, 1990). Nonetheless, carbon is commonly provided via CO2

204 bubbling for microalgae culture, with the equilibrium carbon concentration of 0.04 M

205 achievable under 100% CO2 dosing. Therefore, theoretical μmax is hardly achieved in the

206 real practice. On the other hand, high percentage of CO2 could lead to a relatively low pH

207 level which could cause growth inhibition. Ying, K et al.(2014)reported a strong

208 inhibition on Dunaliella salina growth with 20% CO2 dosing, and 50% CO2 was found

209 fatal to the growth. As a trade-off, 1%-5% CO2 mixture gas dosing along with the addition

210 of certain concentration of carbonate in the medium, providing approximately 5 - 12mM

211 of equilibrium carbon concentration, could be employed to attain nearly 70-85% of the

212 maximum growth potential and meanwhile to maintain the pH at a suitable range (7-7.5)

213 for H. pluvialis growth.

214 The first order kinetic model describing the H. pluvialis growth and carbon consumption,

215 including equations and values of the coefficients, was summarized in Table 1. By using

216 this model, the instantaneous concentration of carbon and biomass for the above

10
217 experiments were simulated. The computed results were thereafter compared to the

218 experimental results, an identical match was found between them (data not shown),

219 indicating a strong validity of this model. The scenario of H. pluvialis ideal growth (at

220 vegetative stage) along with its corresponding daily carbon consumption were then

221 simulated based on this model, illustrated in Fig. 3. Assuming a) nitrogen and phosphorus

222 are replete and the light limitation does not occur, b) the daily carbon uptake by H.

223 pluvialis growth can be traded off by sufficient carbon supplement achieved through 1%

224 CO2 dosing, and c) the instantaneous concentration of total carbon in the medium is

225 maintained at around 5 mM for the whole culture period, therefore H. pluvialis would

226 grow at a constant specific growth rate of 0.49 d-1 (about 70% of maximum potential). As

227 can be seen from Fig. 3, under the ideal conditions the H. pluvialis is expected to grow

228 exponentially from initial 0.01 g L-1 to 0.30 g L-1 within 7 days, with the corresponding

229 daily carbon consumption rising from about 4.4×10-5 mol L-1 d-1 to 1.3×10-3 mol L-1 d-

1
230 . Therefore, to maintain the optimal growth for 7 days, the CO2 mass transfer rate of

231 1.3×10-3 mol L-1d-1 is required according to a conservative consideration.

232 The simulation of H. pluvialis culture with conventional photobioreactor was also shown

233 in Fig. 3. In most of photobioreactor cultures, CO2 was dosed into the medium via

234 conventional bubbles (approximately 3 mm in diameter) at a volumetric flow rate of

235 nearly 5% V/V. For the case of 5L culture applying 1% CO2 conventional dosing, the

236 CO2 mass transfer coefficient was estimated to be 5.6×10-5 min-1 in a preliminary

237 experiment (data not shown), which could provide a maximal mass transfer rate of

238 roughly 3.2×10-5 mol L-1 d-1 (Eq. 9). Such mass transfer rate could only maintain H.

239 pluvialis optimal growth for about 1day. Thereafter, the H. pluvialis would grow linearly

240 rather than exponentially. Therefore, the growth at the first day was estimated according

11
241 to the questions listed in Table 1. The growth between day 2 and day 7 for the

242 conventional PBR culture was calculated as Eq. 11, where Wt1 and Wt2 mean the dry

243 weight of biomass at time t1 and t2, separately. △Ct1,t2 represents the overall carbon

244 consumption rate between t1 and t2, which is limited to the mass transfer rate of

245 conventional bubbles.

Ct1,t 2 (t2  t1 )
246 Wt 2  Wt1 + Eq. 11
Yw/ c

247 Mass transfer of microbubbles

248 Impacts of microbubble size, flowrate, H/D ratio and liquid volume on CO2 mass
249 transfer

250 The CO2 mass transfer rate is mainly determined by the volumetric mass transfer

251 coefficient (KLa) and the driving force (C*-CT, the concentration gradient between

252 equilibrium concentration and instantaneous concentration of dissolved CO2). Since the

253 CO2 equilibrium concentration in the liquid depends on the partial pressure of CO2 in the

254 mixture gas (i.e. CO2 volume percentage) according to Henry’s law (Ying, K et al., 2014),

255 KLa becomes the only variable determining the mass transfer rate when the CO2 volume

256 percentage is fixed. Regarding to KLa, KL can be considered as a constant under the fixed

257 conditions regarding to gas-liquid properties, including temperature, density, viscosity

258 and diffusivity etc., while the interfacial area (a) is a function of gas holdup and bubble

259 diameter (Erickson, 1990; Ying, K. et al., 2013a). The gas holdup directly depends on the

260 liquid volume, bubble rising velocity, liquid height and gas flowrate. The bubble rising

261 velocity and liquid height are determined by the bubble size and H/D (reactor height to

262 diameter ratio), respectively. In total, the bubble size, flowrate, H/D ratio and liquid

263 volume turn to be the key limiting parameters to mass transfer coefficient. The impacts
12
264 of these parameters on KLa were therefore studied, with the results shown in Fig. 4. For

265 either average bubble size (554 μm, 464 μm, or 333 μm), the KLa varied with flowrate,

266 H/D ratio and liquid volume, ranging from 0.0035 to 0.02 min-1, 0.0045 to 0.0375 min-1,

267 and 0.007 to 0.0854 min-1, respectively, but in general, smaller bubble size always

268 resulted in higher KLa. According to Chisti (Erickson, 1990), the bubble size is inversely

269 proportional to the KLa, smaller the bubble size, higher the surface-volume ratio, and

270 consequently the greater KLa.

271 Under the same liquid volume, H/D ratio, and bubble size, KLa was found to be enhanced

272 by increasing the volumetric flowrate. For instance, by increasing flowrate from 0.15 to

273 0.5 L min-1, the KLa rose from 0.007 to 0.017 min-1, 0.0045 to 0.009 min-1, 0.0035 to

274 0.0072 min-1 in 5L deionized water with the H/D ratio of 1:1, for the average microbubble

275 size of 300 μm, 400 μm, 500 μm separately. The same trend was also reported by Ying,

276 K. et al.(2013a). It can be easily understood that increasing the flowrate can lead to a

277 higher gas hold-up which consequently raised the KLa.

278 For the same liquid volume, flowrate and microbubble size, the KLa was also found to be

279 improved with higher H/D ratio. As an example, for the microbubbles of 333 μm at 0.5 L

280 min-1 of bubbling flowrate in 5 L deionized water, the KLa increased from 0.017 to 00854

281 min-1 by expanding the H/D ratio from 1:1 to 5:1. This phenomenon can be supported by

282 theoretical deduction. Higher H/D ratio also means higher liquid height, the bubbles

283 therefore have a longer residence time staying in the liquid which in turn resulted in a

284 larger gas-hold up. As the KLa is in direct proportion to gas hold-up theoretically

285 (Erickson, 1990), increasing the H/D ratio can also enhance the K La. On the other hand,

286 the KLa values decreased when doubling the liquid volume to 10 L, with other parameters

13
287 remaining the same. In consistent with theory, increasing the liquid volume would cause

288 a reduction of gas holdup, and KLa would be decreased consequently.

289 In summary, the KLa can be improved by either increasing the flowrate and H/D ratio or

290 by reducing the bubble sizes. But when increasing the liquid volume, the K La would be

291 reduced if other key parameters remain the same. Therefore, for scale-up cultures,

292 flowrate and H/D should be correspondingly increased or the size of bubbles should be

293 reduced to maintain the similar KLa desired for lab scale studies. Thus, it is important to

294 understand the relationship between the KLa and those key parameters, preferably in a

295 mathematical way, so that the determination of key parameters could be specifically

296 guided for the scale-up.

297 The mathematical relationship between KLa and its relevant parameters

298 Fig. 5a is the schematic diagram interpreting the relationship between KLa and its relevant

299 parameters (bubble size, flowrate, H/D ratio and liquid volume). The interfacial area for

300 mass transfer (a) is well known to be analytically related to the mean bubble size (dB) and

301 to the gas holdup (ε) (Erickson, 1990). The gas holdup is defined as the volume fraction

302 of gas in the gas-liquid dispersion (Najafpour, 2015), which is a function of bubble rising

303 velocity (υB), liquid height (or H/D), and liquid volume (VL). Regarding to the bubble

304 rising velocity, the mean value can be simplified as a function of liquid-gas density (ρL-

305 ρG), viscosity (μ), bubble size (dB) and gas flowrate (Q), according to Stokes law (Bond,

306 1927). Finally, based on certain simplifications shown in Fig. 5a, the KLa is correlated to

307 υB, H/D, VL, and dB via Eq. 12.

432 K L
3
Q Q( H / D)
308 KLa  2
 2
 2
Eq. 12
4 V
 (  L  G ) g( )
3 3
3
( L ) d B3
3 (VL ) d B
 H/D
14
309 According to Eq. 12, KLa should be in direct proportion to the item Q(H/D)2/3/(VL2/3dB3).

310 Among those key parameters, bubble size seems to have the most remarkable influence

311 on mass transfer coefficient, followed by the gas flowrate. By individually increasing the

312 Q and H/D by 10 times, the KLa could be improved by about 10 times and 102/3 times,

313 respectively, while the KLa could be 1000 times greater by reducing the bubble size by

314 only 10 times. Such relationship described by Eq. 12 was also strongly supported by our

315 results (Fig. 4).

316 The diagram of KLa versus the item Q(H/D)2/3/(VL2/3dB3) was plotted to find out the

317 proportionality constant (α) in Eq. 12, shown in Fig. 5a. The linear regression with

318 R2=0.89 was obtained, indicating a strong proportional function between KLa and the item

319 Q(H/D)2/3/(VL2/3dB3). The value of the proportionality constant α was determined to be

320 6×106. The accuracy of Eq. 12 was examined by comparing its computational data with

321 the experimental data, which were plotted in Fig. 5b. For most of computational values,

322 the deviations to their corresponding practical values were less than 30%. Approximately

323 half percentage of the data estimated had less than 20% deviation. After all, Eq. 12 was

324 theoretically derived based on certain assumptions and simplifications, the possibilities

325 of bubble coalescence, bubble entrainment, and internal turbulence etc. were not

326 considered, which are the main reasons causing certain deviation to the real values.

327 However, our model (Eq. 12) at least provided a rough idea on the mathematical

328 relationship between KLa and its relevant parameters. Besides, the model was build

329 following the classic theory (such as two-film theory and stocks law), with the

330 proportionality constant attained from the practical trials, which could be more reliable to

331 predict KLa, comparing to many other empirical equations. Furthermore, the model

332 contains almost all the key parameters affecting KLa, and therefore is more

333 comprehensive than many empirical models which might be limited to specific scenarios.
15
334 Thus, the mass transfer model built in this study could be used as a guidance for the

335 engineering design in the scale-up process.

336 Optimization of mass transfer parameters for H. pluvialis culture

337 As discussed in 3.1.3, for a 5L lab-scale culture, CO2 mass transfer rate of 1.3×10-3 mol

338 L-1d-1 is required to maintain H. pluvialis optimal growth for 7 days, and to achieve a

339 relatively high biomass density (around 0.3g L-1 or 3×105 cells mL-1). To design the

340 microbubble driven bioreactor and meet the mass transfer requirement, the K La values

341 were firstly estimated on three mean bubble sizes (350, 450, 550μm), four flowrates (0.05,

342 0.1, 0.15, 0.3 L min-1), and three H/D ratios (1, 3, 5) by using Eq. 12. The estimated KLa

343 ranged from 0.61×10-3 min-1 to 0.042 min-1, capable of achieving 0.35×10-3 – 2.4×10-2

344 mol L-1d-1 of mass transfer rate, under continuous 1%CO2 dosing. Among them, five sets

345 of combinations achieving the minimal requirement along with relatively low energy

346 input (either low flowrate or low injection pressure) were selected, shown in Table 2. The

347 mass transfer rate of a 5L-conventional PBR with a high H/D ratio was also computed

348 (Set. 6), which was found almost two orders of magnitude smaller, comparing to the one

349 achievable by MDPBR. Among those five sets, Set. 5 requires both the smallest flowrate

350 (0.05 L min-1) and injection pressure, and therefore was selected as the optimal

351 combination of mass transfer parameters for 5L-H. pluvialis culture.

352 H. pluvialis growth in a MDPBR

353 Based on the results from 3.2.3, a 5L MDPBR engaged with a ceramic diffuser capable

354 of creating nearly 550 μm microbubbles was customized for the lab-scale H. pluvialis

355 culture. Meanwhile, another culture in a photobioreactor with the same geometry but a

356 different gas sparger which could generate around 3 mm bubbles was also conducted as

16
357 the control set (i.e. conventional PBR). The gas flow rates were set to be 0.05 L min-1 and

358 0.25 L min-1 for the former and later, respectively. The results were shown in Fig. 6.

359 Generally, the dry weight of H. pluvialis cultured in the MDPBR increased by nearly 30

360 times at the end of a week, with an overall specific growth rate was relatively close to the

361 optimal value 0.49 d-1 as expected. Almost all the cells were confirmed to be vegetative

362 at the final day of cultivation via microscope observation. It was therefore confirmed that

363 our MDPBR can maintain the optimal growth of H. pluvialis under proper light intensity.

364 Besides, the experimental data were found almost in consistence with the simulated data,

365 which confirmed the reliability of the experimental data as well as the validity of the

366 kinetic model for H. pluvialis growth in Table 1. In comparison, the H. pluvialis in the

367 conventional PBR grew linearly instead of exponentially and achieved 0.21 d-1 of specific

368 growth rate with only about 0.06 g L-1 of final biomass concentration, while the MDPBR

369 achieved approximately 5 times higher biomass concentration, but with only 1/5 of gas

370 flow rate, which means the MDPBR could be 25 times more efficient than the

371 conventional PBR, in terms of output-input ratio.

372 In fact, continuous efforts have been made to improve the growth of H. pluvialis. Lee et

373 al.(2015) cultivated H. pluvialis in 4 types of tubular photobioreactors to enhance the

374 carbon conversion, and the specific growth rate was approximately range from 0.07- 0.12

375 d-1. Vega-Estrada et al.(2005) designed a in a split-cylinder internal-loop airlift bioreactor

376 for H. pluvialis, with the maximum specific growth rate at 0.23 d-1.The bioreactor

377 performed by Tjahjono et al.(1994). with sodium acetate as a carbon source showed

378 specific growth rate at 0.25 d-1 Kaewpintong et al.(2007) demonstrated a 1% CO2

379 supplement airlift bioreactor design, achieving specific growth rate achieved 0.31 d-1.

380 Regarding specific growth rate and potential productivity, our MDPBR outperformed

381 their bioreactors, with about 2-5 times of improvement obtained on growth rate.
17
382 Conclusion

383 In this study, the CO2 demand of maintaining H. pluvialis exponential growth was profiled,

384 implying insufficient carbon supply through traditional PBR. To meet the demand, a

385 novel MDPBR was proposed to facilitate mass transfer through minimizing bubble size.

386 Our hypothesis of CO2 supply and H. pluvialis growth has been validated via cultivation

387 trial in MDPBR, in which a significant improvement (5 times higher) of the H. pluvialis

388 biomass productivity has been achieved with energy saving (80% off). Furthermore, our

389 previous work (Wu et al., 2020) which applied higher light intensity suggested that our

390 MDPBR has a potential of reaching higher biomass that achieved so far. This technology

391 offers an efficient alternative for H. pluvialis mass production and could further serve as

392 a useful tool for CO2 mitigation and bioproduct production.

393 Limitations of the study

394 This study designed MDPBR for H. pluvialis massive growth, and the CO2 capture ability

395 of H. pluvialis has been proved. However, the light intensity in this study may not be

396 optimal for maximum photosynthesis, in terms of CO2 supply. The stabilities and

397 efficiency of MDPBR should be especially followed with interests and improved for the

398 practical applications. MDPBR can be adapted to exploit higher algal productivity in

399 industrialized production.

400 METHODS

401 All methods can be found in the accompanying transparent methods supplemental file.

18
402 ACKNOWLEDGMENT

403 This work was financially supported by the S&T Projects of the Economic, Trade and

404 Information Commission of Shenzhen (No. 20180124085935704), Guangdong Basic and

405 Applied Basic Research Foundation (2020B1515120012), as well as the S&T Projects of

406 Shenzhen Science and Technology Innovation Committee (JCYJ20200109142822787,

407 JCYJ20200109142818589, and RCJC20200714114433069).

408 AUTHOR CONTRIBUTIONS

409 Kebi Wu, Kezhen Ying, Jin Zhou, Yi Tao and Zhonghua Cai designed the research.

410 Kezhen Ying, Kebi Wu and Dai Liu, Lu Liu conducted the experiments. Kebi Wu and

411 Kezhen Ying drafted the manuscript and analysed the data. Jin Zhou, Yi Tao, Xiaoshan

412 Zhu and Zhonghua Cai contributed to the critical revision of the article. James Hanotu

413 provided an editorial service.

414 DECLARATION OF INTERESTS

415 There is no conflict of interest, informed consent, human or animal rights applicable. All

416 authors agreed to the authorship and the submission of the manuscript for peer review.

417 REFERENCES

418 Ambati, R. R., Phang, S.-M., Ravi, S., and Aswathanarayana, R. G. (2014). Astaxanthin:

419 sources, extraction, stability, biological activities and its commercial

420 applications—a review. Mar. Drugs, 12.

421 Arun, J., Gopinath, K. P., Sivaramakrishnan, R., SundarRajan, P., Malolan, R., and

422 Pugazhendhi, A. (2021). Technical insights into the production of green fuel from

19
423 CO2 sequestered algal biomass: A conceptual review on green energy. Sci. Total

424 Environ., 755, 142636.

425 Bond, W. N. (1927). LXXXII. Bubbles and drops and stokes' law (Taylor & Francis).

426 Borowitzka, M. A. (1999). Commercial production of microalgae: ponds, tanks, and

427 fermenters Prog. Ind. Microbiol., Osinga, R., Tramper, J., Burgess, J. G., and

428 Wijffels, R. H., ed.(Elsevier), pp. 313-321.

429 Carvalho, A. P., Meireles, L. A., and Malcata, F. X. (2006). Microalgal reactors: a review

430 of enclosed system designs and performances. Biotechnol. Prog., 22, 1490-1506.

431 Chan, K.-c., Pen, P.-J., and Yin, M.-c. (2012). Anticoagulatory and Antiinflammatory

432 Effects of Astaxanthin in Diabetic Rats. J. Food Sci., 77, H76-H80.

433 Choi, Y. Y., Joun, J. M., Lee, J., Hong, M. E., Pham, H.-M., Chang, W. S., and Sim, S. J.

434 (2017). Development of large-scale and economic pH control system for outdoor

435 cultivation of microalgae Haematococcus pluvialis using industrial flue gas.

436 Bioresour. Technol., 244, 1235-1244.

437 Cleveland, C. C., and Liptzin, D. (2007). C:N:P stoichiometry in soil: is there a “Redfield

438 ratio” for the microbial biomass? Biogeochem., 85, 235-252.

439 Cui, L., Xu, F., Wang, M., Li, L., Qiao, T., Cui, H., Li, Z., and Sun, C. (2019). Dietary

440 natural astaxanthin at an early stage inhibits N-nitrosomethylbenzylamine-

441 induced esophageal cancer oxidative stress and inflammation via downregulation

442 of NFκB and COX2 in F344 rats. Onco Targets Ther, 12, 5087-5096.

443 Erickson, L. E. (1990). Airlift bioreactors ( Elsevier applied science).

444 Fábregas, J., Domínguez, A., Regueiro, M., Maseda, A., and Otero, A. (2000).

445 Optimization of culture medium for the continuous cultivation of the microalga

446 Haematococcus pluvialis. Appl. Microbiol. Biotechnol., 53, 530-535.

20
447 Fábregas, J., Otero, A., Maseda, A., and Domínguez, A. (2001). Two-stage cultures for

448 the production of Astaxanthin from Haematococcus pluvialis. J. Biotechnol., 89,

449 65-71.

450 Fakhri, S., Nouri, Z., Moradi, S. Z., and Farzaei, M. H. (2020). Astaxanthin, COVID-19

451 and immune response: Focus on oxidative stress, apoptosis and autophagy.

452 Phytother Res, 34, 2790-2792.

453 García-Malea, M. C., Acién, F. G., Fernández, J. M., Cerón, M. C., and Molina, E. (2006).

454 Continuous production of green cells of Haematococcus pluvialis: Modeling of

455 the irradiance effect. Enzyme Microb. Technol., 38, 981-989.

456 Guerin, M., Huntley, M. E., and Olaizola, M. (2003). Haematococcus astaxanthin:

457 applications for human health and nutrition. Trends Biotechnol., 21, 210-216.

458 Hillebrand, H., and Sommer, U. (1999). The nutrient stoichiometry of benthic microalgal

459 growth: Redfield proportions are optimal. Limnology and Oceanography, 44, 440-

460 446.

461 Hong, M.-E., Hwang, S. K., Chang, W. S., Kim, B. W., Lee, J., and Sim, S. J. (2015).

462 Enhanced autotrophic astaxanthin production from Haematococcus pluvialis

463 under high temperature via heat stress-driven Haber-Weiss reaction. Appl.

464 Microbiol. Biotechnol., 99, 5203-5215.

465 Imamoglu, E., Sukan, F. V., and Dalay, M. C. (2007). Effect of different culture media

466 and light intensities on growth of Haematococcus pluvialis. International Journal

467 of Natural & Engineering Sciences, 1(3), 05-09.

468 Judd, S. J., Al Momani, F. A. O., Znad, H., and Al Ketife, A. M. D. (2017). The cost

469 benefit of algal technology for combined CO2 mitigation and nutrient abatement.

470 Renewable Sustainable Energy Rev., 71, 379-387.

21
471 Kaewpintong, K., Shotipruk, A., Powtongsook, S., and Pavasant, P. (2007).

472 Photoautotrophic high-density cultivation of vegetative cells of Haematococcus

473 pluvialis in airlift bioreactor. Bioresour. Technol., 98, 288-295.

474 Kang, C. D., Han, S. J., Choi, S. P., and Sim, S. J. (2009). Fed-batch culture of

475 astaxanthin-rich Haematococcus pluvialis by exponential nutrient feeding and

476 stepwise light supplementation. Bioprocess Biosyst Eng, 33, 133.

477 Kim, H.-Y., Kim, Y.-M., and Hong, S. (2019). Astaxanthin suppresses the metastasis of

478 colon cancer by inhibiting the MYC-mediated downregulation of microRNA-29a-

479 3p and microRNA-200a. Sci. Rep., 9, 9457.

480 Lee, J. Y., Hong, M.-E., Chang, W. S., and Sim, S. J. (2015). Enhanced carbon dioxide

481 fixation of Haematococcus pluvialis using sequential operating system in tubular

482 photobioreactors. Process Biochem., 50, 1091-1096.

483 Li, J., Zhu, D., Niu, J., Shen, S., and Wang, G. (2011). An economic assessment of

484 astaxanthin production by large scale cultivation of Haematococcus pluvialis.

485 Biotechnol. Adv., 29, 568-574.

486 Medhi, J., and Kalita, M. C. (2021). Astaxanthin: An algae-based natural compound with

487 a potential role in human health-promoting effect: An updated comprehensive

488 review. J. Appl. Biol. Biotechnol., 9(1), 114-123.

489 Milledge, J. J. (2011). Commercial application of microalgae other than as biofuels: a

490 brief review. Rev. Environ. Sci. Bio/Technol., 10, 31-41.

491 Nahidian, B., Ghanati, F., Shahbazi, M., and Soltani, N. (2018). Effect of nutrients on the

492 growth and physiological features of newly isolated Haematococcus pluvialis

493 TMU1. Bioresour. Technol., 255, 229-237.

494 Najafpour, G. (2015). Biochemical Engineering and Biotechnology 2nd Edition (Elsevier

495 Science).

22
496 Olaizola, M. (2000). Commercial production of astaxanthin from Haematococcus

497 pluvialis using 25,000-liter outdoor photobioreactors. J. Appl. Phycol., 12, 499-

498 506.

499 Panis, G., and Carreon, J. R. (2016). Commercial astaxanthin production derived by green

500 alga Haematococcus pluvialis: A microalgae process model and a techno-

501 economic assessment all through production line. Algal Res., 18, 175-190.

502 Pérez-López, P., González-García, S., Jeffryes, C., Agathos, S. N., McHugh, E., Walsh,

503 D., Murray, P., Moane, S., Feijoo, G., and Moreira, M. T. (2014). Life cycle

504 assessment of the production of the red antioxidant carotenoid astaxanthin by

505 microalgae: from lab to pilot scale. J. Cleaner Prod., 64, 332-344.

506 Razon, L. F. (2011). Net Energy Calculations for Production of Biodiesel and Biogas

507 from Haematococcus pluvialis and Nannochloropsis sp Zero-Carbon Energy

508 Kyoto 2010, Yao, T., ed.(Springer Japan), pp. 83-91.

509 Rehman, F., Medley, G. J. D., Bandulasena, H., and Zimmerman, W. B. J. (2015). Fluidic

510 oscillator-mediated microbubble generation to provide cost effective mass

511 transfer and mixing efficiency to the wastewater treatment plants. Environ. Res.,

512 137, 32-39.

513 Ryu, H. J., Oh, K. K., and Kim, Y. S. (2009). Optimization of the influential factors for

514 the improvement of CO2 utilization efficiency and CO2 mass transfer rate. J. Ind.

515 Eng. Chem., 15, 471-475.

516 Schultze, L. K. P., Simon, M.-V., Li, T., Langenbach, D., Podola, B., and Melkonian, M.

517 (2015). High light and carbon dioxide optimize surface productivity in a Twin-

518 Layer biofilm photobioreactor. Algal Res., 8, 37-44.

23
519 Singh, S. K., Rahman, A., Dixit, K., Nath, A., and Sundaram, S. (2016). Evaluation of

520 promising algal strains for sustainable exploitation coupled with CO2 fixation.

521 Environ. Technol., 37, 613-622.

522 Singh, S. P., and Singh, P. (2014). Effect of CO2 concentration on algal growth: A review.

523 Renewable Sustainable Energy Rev., 38, 172-179.

524 Talukdar, J., Bhadra, B., Dattaroy, T., Nagle, V., and Dasgupta, S. (2020). Potential of

525 natural astaxanthin in alleviating the risk of cytokine storm in COVID-19. Biomed.

526 Pharmacother., 132, 110886.

527 Tjahjono, A. E., Kakizono, T., Hayama, Y., Nishio, N., and Nagai, S. (1994). Isolation of

528 resistant mutants against carotenoid biosynthesis inhibitors for a green alga

529 Haematococcus pluvialis, and their hybrid formation by protoplast fusion for

530 breeding of higher astaxanthin producers. J. Ferment. Bioeng., 77, 352-357.

531 Tocquin, P., Fratamico, A., and Franck, F. (2012). Screening for a low-cost

532 Haematococcus pluvialis medium reveals an unexpected impact of a low N/P ratio

533 on vegetative growth. J. Appl. Phycol., 24, 365-373.

534 Tsygankov, A. A. (2001). Laboratory Scale Photobioreactors. Appl. Biochem. Microbiol.,

535 37, 333-341.

536 Vega-Estrada, J., Montes-Horcasitas, M. C., Domínguez-Bocanegra, A. R., and

537 Cañizares-Villanueva, R. O. (2005). Haematococcus pluvialis cultivation in split-

538 cylinder internal-loop airlift photobioreactor under aeration conditions avoiding

539 cell damage. Appl. Microbiol. Biotechnol., 68, 31-35.

540 Wan, M., Zhang, J., Hou, D., Fan, J., Li, Y., Huang, J., and Wang, J. (2014). The effect

541 of temperature on cell growth and astaxanthin accumulation of Haematococcus

542 pluvialis during a light–dark cyclic cultivation. Bioresour. Technol., 167, 276-283.

24
543 Wang, J., Sommerfeld, M. R., Lu, C., and Hu, Q. J. A. (2013). Combined effect of initial

544 biomass density and nitrogen concentration on growth and astaxanthin production

545 of Haematococcus pluvialis (Chlorophyta) in outdoor cultivation. Algae, 28, 193-

546 202.

547 Wu, K., Ying, K., Liu, L., Zhou, J., and Cai, Z. (2020). High irradiance compensated with

548 CO2 enhances the efficiency of Haematococcus lacustris growth. Biotechnol.

549 Rep., 26, e00444.

550 Xi, T., Kim, D. G., Roh, S. W., Choi, J.-S., and Choi, Y.-E. (2016). Enhancement of

551 astaxanthin production using Haematococcus pluvialis with novel LED

552 wavelength shift strategy. Appl. Microbiol. Biotechnol., 100, 6231-6238.

553 Yamashita, T., Yamamoto-Ikemoto, R., Sakurai, E., Aikawa, K., and Kaneko, E. (2010).

554 Treatment of municipal wastewater using an anaerobic-anoxic-oxic biological

555 filter reactor packed with carbon fibers and aerated with microbubbles. J. Environ.

556 Eng. Manage., 20, 205-211.

557 Yao, K., Chi, Y., Wang, F., Yan, J., Ni, M., and Cen, K. (2016). The effect of

558 microbubbles on gas-liquid mass transfer coefficient and degradation rate of COD

559 in wastewater treatment. Water Sci. Technol., 73, 1969-1977.

560 Ying, K., Al-Mashhadani, M. K. H., and , J. O. H. (2013a). Enhanced mass transfer in

561 microbubble-driven airlift bioreactor for microalgal culture. Eng., 5(9), 1947-

562 3931.

563 Ying, K., Gilmour, D. J., Shi, Y., and Zimmerman, W. B. (2013b). Growth enhancement

564 of Dunaliella salina by microbubble induced airlift loop bioreactor (ALB)—the

565 relation between mass transfer and growth rate. J. Biomater. Nanobiotechnol., 4

566 (2A), 1-9.

25
567 Ying, K., Zimmerman, W., and Gilmour, D. (2014). Effects of CO2 and pH on growth of

568 the microalga Dunaliella salina. J. Microb. Biochem. Technol., 6, 167-173.

569 Yoo, J. J., Choi, S. P., Kim, B. W., and Sim, S. J. (2012). Optimal design of scalable

570 photo-bioreactor for phototropic culturing of Haematococcus pluvialis.

571 Bioprocess Biosyst Eng, 35, 309-315.

572 Zhang, X. W., Gong, X.-D., and Chen, F. (1999). Kinetic models for astaxanthin

573 production by high cell density mixotrophic culture of the microalga

574 Haematococcus pluvialis. J. Ind. Microbiol. Biotechnol., 23, 691-696.

575 Zimmerman, W. B., Zandi, M., Hemaka Bandulasena, H. C., Tesař, V., James Gilmour,

576 D., and Ying, K. (2011). Design of an airlift loop bioreactor and pilot scales

577 studies with fluidic oscillator induced microbubbles for growth of a microalgae

578 Dunaliella salina. Appl. Energy., 88, 3357-3369.

579

580

26
581 FIGURES AND LEGENDS

582

583 Fig. 1: MDPBR setup for H. pluvialis culture. a) Schematic diagram showing the

584 MDPBR culture; b) Photograph taken at the 1st day of culture. In general, the MDPBR is

585 made of acrylic material, with the dimension of 70 cm in height and 10.8 cm in diameter.

586 The working volume is about 5 L, which gives the liquid height of 54.2 cm. The height

587 of degasser zone is about 5.4 cm. The internal draught tube (43.4 cm in height and 8.6 cm

588 in diameter) is 5.4 cm hung from the reactor bottom. A micropore-diffuser (6.8 cm in

589 diameter) is fixed at the centre of the bottom.

590

27
591

592 Fig. 2: The growths and carbon consumptions of H. pluvialis under different carbon

593 concentrations (2 mM, 4 mM and 8 mM). a) H. pluvialis growth curve; b) The overall

594 specific growth rate at each stage of time. Data points are represented as the mean ±

595 standard deviation (SD) for triplicate measurements (n=3). The different letters indicate

596 a significant difference at P<0.05 level. c) Daily carbon concentrations during the

597 cultures. The dotted lines represent the carbon concentrations in the control sets where

598 microalgae were excluded. d) Relation between carbon consumption and biomass (dry

599 weight) increase. The slope of the linear regression stands for the carbon consumption

600 per unit of biomass increase.

601
28
602

603 Fig. 3: Simulated H. pluvialis growth at optimal specific growth rate and the daily

604 carbon consumption. The dotted line stands for the simulation of the H. pluvialis growth

605 in a conventional PBR culture, while the solid line with circles is the prediction of H.

606 pluvialis growth under the circumstance that daily carbon supply meets or exceeds the

607 consumption demand. The bar demonstrates the daily carbon consumption.

608

29
609

610

611 Fig. 4: The CO2 mass transfer coefficients in (a) 5 L and (b) 10 L water under

612 different flowrates, H/D ratios, and bubble sizes. Due to lab limitations, the error bars

613 shown in this Fig. were obtained from the triplication of each average bubble size under

614 the conditions of 0.3L min-1 dosing rate, 5L liquid volume and 3:1 H/D ratio. Data points

615 are presented as the mean ± standard deviation for triplicate measurements (n=3).

616

30
617

618 Fig. 5: The mathematical relationship between KLa and its relevant parameters. a)

619 Schematic diagram demonstrating the relationship between mass transfer

620 coefficient and its relevant parameters. b) plot of KLa versus the item

621 Q(H/D)2/3/(VL2/3dB3). The KLa values were obtained from the experiments, while the

622 values of the item Q(H/D)2/3/(VL2/3dB3) were calculated based on the practical values. c)

623 the comparison between practical and theoretical values of KLa. The theoretical values of

624 KLa were calculated using Eq. 12.

625

31
626

627

628 Fig. 6: The biomass growth of H. pluvialis in both the conventional PBR and the

629 MDPBR. The red and black dotted line represent the theoretical growth of H. pluvialis

630 in the 5 L MDPBR and in the 5 L conventional PBR, respectively. The solid circles and

631 triangles demonstrate the experimental data of H. pluvialis growth in the 5 L MDPBR

632 and in the 5 L conventional PBR, respectively. Data points are presented as the mean ±

633 standard deviation for triplicate measurements (n=3).

634

635

32
636 TABLES

637 Table 1: List of equations describing the H. pluvialis growth and carbon

638 consumption.

First order kinetic model for H. pluvialis growth and nutrients consumptions
Equations Representatives
dw
   w  w  w0 e t Biomass growth, g L-1
dt
dC   w   w0 e t w0
   C  (e t  1) Carbon consumption, mol L-1 OR g L-1
dt Yw/ C Yw/ C Yw/ C

C Specific growth rate under carbon

  max ( )
C  KC
limitation, d-1
w0 Instantaneous concentration of carbon,
C  C0  C  C0  (e t  1)
Yw / C
mol L-1 OR g L-1
Key parameters Values
μmax 0.68 d-1
Kc 0.002 mol L-1 OR 0.024 g L-1
Yw/c 142.9 g mol-1 OR 11.9 g g-1

639

640

641

642

33
643 Table 2: Estimations of mass transfer rates achievable under various combinations.

644 Set.1-5 are the simulations for MDPBR, while Set.6 is the optimistic estimation for

645 conventional PBR. Lower flowrate means lower energy input. Larger bubble size

646 indicates less pressure for gas injection. The set being star marked was considered as

647 optimal when balancing the mass transfer with energy cost.

VL, dB, Q, KLa, Daily mass transfer rate,

No. of Set H/D
L μm L min-1 min-1 (10-3) mol L-1 d-1 (10-3)
1 5 1 350 0.05 2.4 1.4

2 5 1 450 0.1 2.3 1.3

3 5 3 450 0.05 2.3 1.4

4 5 3 550 0.1 2.6 1.5

5* 5 5 550 0.05 1.8 1.1

6 5 5 3000 0.25 0.056 0.032

648

649

34
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