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1. Chemical Engineering Journal 183 (2012) 192– 197 Contents lists available atSciVerse ScienceDirect Chemical Engineering Journal journal hom epage: www.elsevier.com/locate/cej Autotrophic production cultivation of for CO2fixation and phycocyanin Spirulina platensis Zenga, Danquahb, Zhanga, Zhanga, Wua, Chena,b, Xianhai I-Son Michael K. Shiduo Xia Mengyang Xiao Dong Nga, Jinga, Lua,∗ Keju Yinghua aDepartment bDepartment ofChemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China of Chemical Engineering, Monash University, Clayton, VIC 3800, Australia a r t i c l e i n f o a b s t r a c t Article Received Received 11 Accepted history: Microalgae products resources parametrically CO2fixation the achieved mum (that 20 crude mization 20 specific highest CO2fixation The biotechnology provides a new industrial paradigm that simultaneously yields various vital 8September 2011 and captures CO2in a single process. Development of optimal strategies to harness microalgal inrevised form iscritical to full-scale production and application of microalgae-derived products. This work December 2011 investigated the autotrophic cultivation of for enhanced simultaneous Spirulina platensis 14December 2011 and phycocyanin production using conical flasks and newly designed photobioreactors. In absence ofculture aeration in the conical flasks, a maximum biomass concentration of3.20 g/L was Keywords: Microalgae Autotrophic CO2fixation Phycocyanin Photobioreactor under alkaline conditions with initial pH of ∼9.In the presence of culture aeration, a maxi- biomass concentration of 5.96 g/L was obtained under intermittent CO2supply at 20 mM/(L 2 d) cultivation is 20 mM/L every other day). Continuous sparging of 0.1 L/min compressed air in combination with mM/(L d) intermittent CO2supply into a customarily designed photobioreactor resulted in biomass and production phycocyanin concentrations of 5.92 g/L and 1.06 g/L respectively. Further photobioreaction opti- resulted in biomass and phycocyanin concentrations of 7.27 g/L and 1.22 g/L respectively using mM/L d CO2intermittent aeration supplemented with continuously supplied compressed air under flow conditions. The highest volumetric titre of phycocyanin obtained in this work isamongst the reported in literature. These results indicate that high biomass, phycocyanin concentration and rate could be obtained with minimal extra effort simply by optimizing bioprocess conditions. growth results of cultivation fitted well to the logistic © 2011 Elsevier B.V. All rights reserved. rate equation. S.platensis 1. Introduction into to lipids in (CCM) grow by high Inmicroalgae, hence Microalgae ity they ucts simultaneous is with [15]. can and owing chemical energy. Simultaneously, CO2is fixed and transferred carbon-containing high value products, such as carbohydrates, In recent years, increase inatmospheric CO2concentration, and proteins [2]. Therefore, the CO2fixation capacity reflects which great [1,2]. capture or tion one Generally, ation as terrestrial and optimized is the main component of greenhouse gas emissions, poses the biomass productivity. Carbon concentrating mechanisms challenges toworldwide pro-environment and sustainability are present inmany microalgal species, enabling them to At present, reducing the use of fossil fuels orpromoting CO2 and accumulate carbon under variable concentrations ofCO2 and sequestration seem tobe the most feasible way tocut utilizing bicarbonate transporters and CO2receptors togenerate mitigate CO2emissions [3]. Amongst the CO2emission reduc- intracellular concentrations of dissolved inorganic carbon [8]. approaches, biosequestration using microalgae isconsidered CCM acts as anenhancer for higher growth rate and of the most effective one tofix CO2[4]. preferred tocarbon sequestration [8,9]. microalgae have higher growth rates, higher CO2fix- have been employed inmany studies fortheir abil- efficiency and larger quantities of high value products, such toutilize dissolved inorganic carbon [8,10–12].Furthermore, dietary supplements for human, animals and aquaculture, than can also be used for producing high value biological prod- plants [5–7].Significantly, temperature, pH, CO2,light, [13,14]. However, work focused on process optimization for inorganic salts, which are indispensable, can be monitored and generation of multiple products from microalgae for maximum biomass. Microalgae convert solar energy limited. As reported earlier, has ahigh growth rate Spirulina as high as 65% (w/w) protein concentration inthe biomass mciroalgal protein, particularly phycocyanin which Spirulina make as high as 25% of the cell mass, is of great interest, can be used as anutraceutical for both humans and animals ∗Corresponding E-mail author. Tel.: +86 592 2186038; fax: +86 592 2184822. toits immunomodulatory and anti-cancer activities [16,17]. ylu@xmu.edu.cn (Y. Lu). address: 1385-8947/$ doi:10.1016/j.cej.2011.12.062 –see front matter © 2011 Elsevier B.V. All rights reserved.

2. 193 X.Zeng etal. /Chemical Engineering Journal 183 (2012) 192– 197 Aparametric mization capture phycocyanin sented study of key process factors essential for the opti- with Photobioreactor cates noaeration but the same process conditions was investigated. of cultivation system tosimultaneously cultivations were maintained for 18dand 3dupli- Spirulina platensis high levels of CO2and produce high density biomass and ofeach batches of cultivation were conducted. using acustomized photobioreactor system ispre- inthis study. 2.4. Analytical methods 2. Experimental Biomass Samples measured US). RIUS, a weight [20–22]. microalgae sequent was 2.4.1. concentration and pHanalysis of 8mL were taken daily and the OD560nmvalues were 2.1.Algae strain, growth medium and cultivation conditions with a UV3100 spectrophotometer (Thermo Electron, The pHvalues were measured with PB11 pHmeter (SARTO- strain used inthis study was obtained from the platensis Diatom sity, production was Japan) a ture an cycle sity (PGX-350 10hof CO2was odically containing every conditions S. Germany). The biomass concentration was estimated using Laboratory inthe School of Life Sciences, Xiamen Univer- calibration curve relating the OD560nmvalues todry biomass China. The growth medium employed inboth the seed and concentrations [Biomass (g/L) =0.8236·OD560 nm+0.023] cultures was the Zarrouk medium [18,19].The medium The dry biomass weight was obtained byweighing the initially sterilized at121◦CinaHV-50 autoclave (HIRAYAMA, cells after 2times washing with distilled water and sub- for 15min. The microalgae seed cultivation was conducted in drying inanoven at80◦Covernight until aconstant weight 2Lconical flask containing 500 mL medium for 10d. The seed cul- achieved. was used toinoculate the production culture medium toyield initial microalgal concentration of above 0.18 g/L. The cultivation Crude Crude ified microalgae supernatant mixing buffer ator centrifuged for Bogorad 2.4.2. phycocyanin extraction and analysis employed was 14hof light period with illumination inten- phycocyanin extraction was performed using the mod- ?mol/m2sgenerated of 200 using an illumination incubator method previously described in[23,24].Briefly, 1mL of the D, Ningbo SAFE Laboratory Apparatus Co. Ltd., China) and culture was centrifuged for 5min at 10,000 ×gand the dark period. The cultivation temperature was 30 0.5◦C. ± discarded. Extraction was carried out bythoroughly fed into the conical flask via asterile rubber tube peri- the microalgae cells with 4mL of autoclaved potassium PBS (every 1or2d) during the light phase. The conical flask (pH 7.0) and the mixture was stored ina −20◦Crefriger- the microalgae culture was manually agitated for 1min for 12 h. The mixture was thawed toroom temperature and 8h. Culture sampling was performed every 24 hunder sterile for 5min at 10,000 ×g. The supernatant was analysed and the cultivation was terminated after 16 d. crude phycocyanin using the method according toBennett and [24,25]. 2.2. Batch cultivation with CO2aeration inaconical flask 2.5. Kinetic model Three different approaches of CO2aeration were employed to investigate lized the aeration ture intermittent where the CO2fixation ability of S.platensis. Approach 1uti- The basic logistic equation indifferential form was adopted in different levels of intermittent CO2aeration applied daily from this [26,27]. research topredict the kinetic behaviour of microalgal growth start of cultivation. Approach 2utilized different levels of CO2 intermittently applied daily from the point where the cul- established apHof 10. Approach 3utilized different levels of C0e?t C(t) (1) = CO2aeration applied every other day from the point 1−(C0/Cm)(1 −e?t) the pHof the culture reached 10. where biomass time and C0isthe initial biomass (g/L), Cmis the maximum S.platensis (h−1)and (g/L), ?isthe specific growth rate tiscultivation Photobioreactor cultivation 2.3. (h). The modelling work was conducted with MATLAB R2009b Origin Pro 8.0 softwares. Inthis customarily inner thickness glass the exit, in clamped in dong Photon ing inner the ically Lantai Three investigated experiment The pressed both pressed research, the photobioreactor systems employed were designed by the authors. The bioreactor vessel, with 3. Results and discussion diameter 3.4 cmand length 30 cm, was made of glass with of 0.12 cm. Rubber stoppers were fixed on top of the bottles for sealing. Three openings were allowed on top of 3.1. Effect of initial medium pHonS.platensis growth bioreactor for sampling, air/CO2sparging and effluent flue gas respectively. Aflowrator was fixed inthe air/CO2pipeline Cultivation medium investigate pH HCl. (pH imum was 9 3.09 continuous Eq. be increases Cultivation gal sp. 8–10 experiments with different initial pHof growth order toregulate the gas flow. The sampling pipe was always (pH range of 7–11) were performed in the conical flasks to except during sampling. The bioreactor system was placed the extent of pHeffect on growth. Medium S.platensis an LRH-250S incubator (Medical Apparatus Factory of Guang- was varied by adding calculated amounts of either NaOH or Province, China) and the temperature was set to30.0 0.5◦C. Growth medium with unaltered pHwas used as the control ± exposure was supplied bymeans of 8Philips energy sav- value 9.05–9.10 approximately). As shown inFig. 1A, the max- white fluorescent lamps (each having 7Wpower), fixed on the biomass concentration obtained inthe control experiment walls ofthe incubator. The distance between the lamps and about 3.15 g/L, whilst experiments with pHvalues of 8and bioreactors was 16 cm. The illumination intensity was period- displayed alittle bit lower biomass concentrations of 3.01 and determined using aLX9626 illuminance meter (Guangzhou g/L, respectively. The growth curves (shown inFig. 1Aas the Instruments Co. Ltd., China). line connecting the data) were modelled by means of different scenarios of photobioreactor cultivation were (1) and the results of and Cmare shown inTable 1. Itcan ? under key specific conditions. The first case was the seen that the maximum achievable biomass concentration Cm with 20 mM/(L d) CO2aeration intermittently applied. with increasing initial pHof growth medium uptopH∼ 9. second case was with 0.1 L/min continuous aeration using com- experiments with pH11 showed aggregated microal- air fed into the culture, and the third case was aerated with cells at the bottom ofthe flasks. Laboratory cultures of Spirulina intermittent 20 mM/(L d) CO2combined with 0.1 L/min com- have been reported toshow awide range ofoptimum pH of air fed continuously. For comparison, acontrol scenario [28,29], and hence can readily tolerate progressive changes in

3. 194 X. Zeng etal. /Chemical Engineering Journal 183 (2012) 192– 197 Fig. 1.Effect of initial pH of growth medium on growth. S. platensis Fig. growth 2.Effect of intermittent CO2aeration atthe start of cultivation on S.platensis (approach 1). pH. ple, changed are Fig. pH ture reported observed alkalescent, pH uptake to tion implies [30]. When molecules directly hand, ammonia S. the the surate does result During increased reported gen in into However, the cells may rapidly deteriorate through for exam- electromotive burst orpHinduced agglomeration, when pHis former leading being incorporated and utilized as carbon source for growth, abruptly, and this may happen ingrowth systems which topHincrease [8]. not well buffered [29]. 1shows the cell growth profiles for the different initial 3.2. Effect ofCO2onS.platensis growth cultures, and the maximum growth was observed for the cul- with initial pH∼ 9. This observation isinaccordance with that Inorder and 2.2,was the compared with mittently) It during run, tures. acid The biomass CO2aeration reached toevaluate the effect ofCO2on growth S. platensis by De Oliveira et al. [28] and Richmond etal. [29],who the CO2fixation ability, approach 1, as explained inSection that the most suitable pHfor cultivation is S.platensis performed and the results are shown inFig. 2.Generally, mostly within the range of 8.5–9.5. As shown inFig. 1B, cultures with CO2aeration displayed better microalgal growth of all the cultures increased during the cultivation process. The tothe control run with noCO2aeration. The culture of nitrate byS.platensis cells requires preliminary reduction CO2aeration of 20 mM/(L d) (that is20mM/L per day inter- ammonia, and this contributes topHincrease during the cultiva- showed the highest biomass concentration of4.62 g/L. process, whereas the uptake of ammonia from NH4Cl directly was observed that high CO2aeration resulted inlow culture pH the release of hydrogen ions, thus acidifying the medium the cultivation process. However, compared tothe control the pHprofiles were much more stable inthe CO2aerated cul- the pHvalue was around 9inthe culture, S.platensis This isbecause CO2dissolves inthe medium and the carbonic of ammonia entered the cells bydiffusion, and were formed nullifies the pHincrease during the microalgal growth. taken up. Higher initial growth medium pH, on the other results of approach 2are shown inFig. 3. The highest results inhigher NH3concentrations, hence greater loss of concentration of 5.41 g/L was obtained with 20 mM/(L d) tothe off-gas stream. At the optimum initial culture pH, (started atthe point where the pHof the culture NH4+preferentially NO32−,possibly utilizes over due to platensis 10). Figs. 2–3 show that the highest biomass concentration NH4+represses fact that the presence of nitrate utilization, and NH4+as consumption of anitrogen source does not commen- NH4+utilization energy expenditure [30–32]. Furthermore, not require nitrate reductase (NR) biosynthesis, and could inacompetitive inhibition bynitrate utilization [30–32]. the cultivation, the pHvalues of the different cultures except the culture with initial medium pH11. Ithas been that the microalgae consumes nitro- Cyanidium caldcrum H+,leaving with 1or2moles OH−inthe culture and this results pHincrease [33,34].Also bicarbonate isactively transported the cells and converted tocarbon dioxide and carbonate, the Table Estimated different 1 kinetic parameters by the logistic equation inS. cultivation of platensis initial medium pH. ?(×10−3h−1) 11.55 12.00 13.81 10.03 8.35 Cm(g/L)a pH 7 8 9 10 11 ±2.19 2.74 2.89 3.08 1.91 1.74 ±0.39 ±1.07 ±0.24 ±0.94 ±0.54 ±1.12 ±0.21 ±0.85 ±0.17 Fig. the 2). 3. Effect of intermittent CO2aeration daily on growth. CO2was fed at S.platensis Control 13.90 ±1.05 3.24 ±0.24 point where the pH of the culture reached 10 (pointed by the arrows) (approach aThe correlation coefficient of regression R>0.98.

4. 195 X.Zeng etal. /Chemical Engineering Journal 183 (2012) 192– 197 Table Kinetic 3 modelling ofS.platensis inthe customized photobioreactors. Air/CO2areationa(L/min/mM/L ?(×10−3h−1) 13.10 13.75 ± 14.28 17.75 d) Cm(g/L) Control Air CO2 Air/CO2 ±1.57 3.05 4.33 ± 5.92 6.71 ±0.18 0.99 0.54 ±1.24 ±0.58 ±1.38 ±0.74 Control 20 continuous intermittent aThe was on behalf of CO2and airaeration absence (control). CO2was on behalf of mM/(L d) intermittent CO2aeration (the first case); air was onbehalf of 0.1 L/min air aeration (the second case); airwith CO2was on behalf of20mM/(L d) CO2combined with 0.1 L/min continuous air aeration (the third case). correlation coefficient of regression >0.99. R For approaches 2and 3, Cmand ?showed somewhat similar trends. 1and with approach aeration increase growth every were longer of However, Cmof approach 2was higher than that of approach this could be ascribed tothe exhaustion of carbon source simultaneous low CO2aeration and concentration levels in 2when the culture pHreached 10. Thus increasing CO2 atthis point supplements carbon concentration levels to the maximum biomass concentration Cmand the specific Fig. was (approach 4.Effect of CO2aeration applied every other day on growth. CO2 S. platensis fed atthe point where the pH of the culture reached 10 (the arrows pointed) rate ?. Inapproach 3, where CO2aeration was conducted 3). other day after the culture pHreached 10, Cmand results ? higher than that of approaches 1and 2,and this is due tothe CO2aeration interval, which causes partial carbon starvation of higher the ashorter Asshown biomass (that The around itcan 40 To ferent data 1, 20 20 the higher 5.41 g/L, achieved when CO2aeration started from pH10, was the microalgal cells, resulting infaster growth. than that obtained when CO2aeration commenced with cultivation process. However, CO2fixation only occurred for 3.3. Cultivation of S. platensis for phycocyanin production duration. inFig. 4, data corresponding toapproach 3, the highest Based on the earlier results, the customized photobioreactor concentration of 5.96 g/L was achieved with 40 mM/(L 2d) systems under capacity. of achieved 0.1 air the microalgal concentration to 240 sharply, respondingly, unable denaturation From Section the results cell were used for further microalgal cultivation experiments is40 mM/L per every other day intermittently) CO2aeration. key specific conditions toexplore phycocyanin production pHof all the cultures with CO2aeration fluctuated succinctly As shown inFig. 5, the highest biomass concentration pH10. From the experimental results shown inFigs. 2–4, 5.92 g/L and crude phycocyanin concentration of 1.06 g/L were be inferred that under the conditions of20 mM/(L d) and when 20mM/(L d) intermittent CO2was combined with mM/(L 2d) CO2aeration, the culture pHwas well controlled. L/min continuous air aeration. The presence ofboth CO2and study the kinetic behaviour of microalgal growth under dif- simultaneously enhanced the growth ofS.platensis, because levels of CO2aeration, Eq. (1) was used tofit the experimental carbon source gaseous bubbles established the nutritional and and Cmand ?results are presented inTable 2. With approach metabolin equidistribution inthe culture, and the CO2 Cm increased as CO2 aeration increased from 0(control) to augments toexhibit carboxylase activity inrubisco mM/L d, and decreased when CO2aeration increased beyond fix CO2and produce biomass [8,35]. Fig. 5Ashows that after mM/(L d). However, displayed an inverse characteristic. From ? hof cultivation, inthe control case, the culture pHincreased underlying Eqs. (2)–(4) of Cmand ?, higher values of Cmresult in and the crude phycocyanin concentration decreased cor- values of for agiven cultivation duration (constant C0>0). ? and this could the reason why cells were S.platensis 1 Ct ·dCt dt tosurvive under pH∼ 12conditions, with possible protein (2) ? = (Fig. 5B). Table 3, the Cm value ofthe first case, as explained in dCt dt ≈Ct− C0 (3) 2.3, ishigher than that of the control, and this isbecause t constant flow of CO2creates auniform culture system which ? ? 1 1 ≈1 ·dCt dt ·Cm−C0 1−C0 ineffective mass transfer within the culture forenhanced (4) ? = ≈ Cm Cm t t Cm growth. Cmof the third case was higher than that ofthe first Table Modelling 2 parameters of biomass production under various levels of CO2aeration. S.platensis CO2aerationa Approach 1 Approach 2 Approach 3 ?(×10−3h−1) 13.38 14.62 15.49 13.87 13.45 ?(×10−3h−1) 13.43 14.44 15.76 14.36 13.80 ?(×10−3h−1) 13.02 – – – – 16.49 18.10 15.74 Cm(g/L) Cm(g/L) Cm(g/L) 0(control) 10 20 40 80 20 40 60 ±1.37 3.31 4.14 4.90 4.77 4.30 ±0.24 ±1.57 3.14 5.09 5.83 5.73 4.44 ±0.27 1.57 3.16 ±0.14 – – – – 6.70 7.50 5.59 ± mM/L d ±0.49 ±0.48 ±0.99 ±0.29 mM/L d ±1.14 ±0.48 ±1.24 ±0.24 mM/L d ±1.38 ±0.73 ±1.80 ±0.48 mM/L d ±0.98 ±0.38 – – – ±1.07 ±0.48 – – – mM/L 2d – – – – – – 0.92 ±0.80 ± mM/L 2d 1.24 ±0.48 ± mM/L 2d 1.8 ±0.38 ± Approach Approach Approach aThe 1:with different levels of intermittent CO2aeration (mM/L d) applied daily atthe start of cultivation. 2: with different levels of intermittent CO2aeration (mM/L d) applied daily from the point where the culture pHwas 10. 3: with different levels of intermittent CO2aeration (mM/L 2d) applied every 2dfrom the point where the culture pHwas 10. correlation coefficient of regression R>0.96.

5. 196 X. Zeng etal. /Chemical Engineering Journal 183 (2012) 192– 197 Fig. intermittent CO2combined 5.Cultivation profiles of inthe customized photobioreactors. Control was on behalf of CO2and Air aeration absence (control). CO2was on behalf of 20 mM/(L d) S.platensis CO2aeration (the first case); Air was on behalf of 0.1L/min continuous air aeration (the second case); Air with CO2was on behalf of 20mM/(L d)intermittent with 0.1 L/min continuous air aeration (the third case). (A) Cultivation growth curve; (B) phycocyanin production profiles for the different cases. Fig. cultivation 6. Cell growth and phycocyanin production of inthe customized photobioreactor. The compressed air flow rate was increased to0.1 L/min and 0.15 L/min at S. platensis time points of 120 h(the left arrow pointed) and 240 h(the right arrow pointed). h−1respectively. and carbon Experiments fixation platensis tion with 200 equidistribution pressed for shown biomass crude vation. second cases, and this isbecause ofCO2aeration as a kind of rate to the CO2incompressed CO2levels in reversible much [36]. internally, equation were 7.12 g/L and 0.017 Compared source supplement. Fig. 5A, the pHprofile was much more stably controlled because were designed tocorroborate growth rate, CO2 CO2concentration inthe system increased, partly due tothe ability as well as the production of phycocyanin from S. air. Ithas previously been reported that high using the customized photobioreactor systems. A cultiva- support carbon fixation activity of the enzyme rubisco strategy of 20 mM/(L d) intermittent CO2aeration combined microalgal cells. Rubisco facilitates the utilization of CO2through 0.05 L/min compressed air supplied continuously at30◦Cand conversion ofCO2into bicarbonate ion as CO2escapes ?mol/m2slight conditions was employed. Inorder toestablish more easily through the cell membrane than bicarbonates of cells and nutrients inthe culture, the com- Microalgae can survive and enable the accumulation of CO2 air flow rate was increased to0.1 L/min and 0.15 L/min reaching upto50 mM after CCM induction [8,37,38]. cultivation durations of 120 hand 240 h(refer tothe arrows inFig. 6) respectively. As shown inFig. 6, the microalgal 4. Conclusion concentration obtained was as high as 7.27 g/L, while the phycocyanin concentration was 1.22 g/L at the end of culti- In this work, asequential optimization scheme for S.platen- Results for Cmand after fitting the data with the logistic cultivation for co-production of biomass and phycocyanin with sis ?

6. 197 X.Zeng etal. /Chemical Engineering Journal 183 (2012) 192– 197 CO2fixation the compressed S. in pressed microalgal equation. duce such diagnostic effectively option was developed. The influence ofculture initial pHon O.Ciferri, 551–578. [16]S.Boussiba, the [17]R.Sarada, of tionmethods 795–801. [18] teursphysiques maxima [19] E.O. thelaboratory 5 [20] semimicroscopic (1977) [21] A. Spirulina Bioresour. [22] X. s [23] terization IO9201, [24] phycocyanin sour.Technol. [25] blue-green [26] mentation Hydrogen [27] Modeling under [28]M.A.C.L. ical temperatures, [29] A. the Appl. [30] cultivation niumchloride 4491–4498. [31]D.Soletto, cultivations gensources, [32] of ammonium (2004) [33] acidophilic [34] PlantSci. [35] 1039–1040. [36]M.R. Rev. [37] carbonfluxes Scenedesmus [38]H. Ccml, mechanism Natl.Acad. [15] Spirulina, the edible microorganism, Microbiol. Rev. 47(1983) batch cultivation process was investigated alongside CO2and A.E. 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