Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1186-5

ORIGINAL PAPER

Mixotrophic cultivation of oleaginous Chlorella sp. KR-1 mediated by actual coal-fired flue gas for biodiesel production Ramasamy Praveenkumar • Bohwa Kim • Eunji Choi • Kyubock Lee • Sunja Cho • Ju-Soo Hyun • Ji-Yeon Park • Young-Chul Lee • Hyun Uk Lee • Jin-Suk Lee • You-Kwan Oh

Received: 23 January 2014 / Accepted: 26 March 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Flue gases mainly consist of CO2 that can be utilized to facilitate microalgal culture for bioenergy production. In the present study, to evaluate the feasibility of the utilization of flue gas from a coal-burning power plant, an indigenous and high-CO2-tolerant oleaginous microalga, Chlorella sp. KR-1, was cultivated under mixotrophic conditions, and the results were evaluated. When the culture was mediated by flue gas, highest biomass (0.8 g cells/ L
d) and FAME (fatty acid methyl esters) productivity (121 mg/L
d) were achieved in the mixotrophic mode with 5 g/L glucose, 5 mM nitrate, and a flow rate of 0.2 vvm. By contrast, the photoautotrophic cultivation resulted in a lower biomass (0.45 g cells/L
d) and a lower FAME productivity (60.2 mg/L
d). In general, the fatty acid profiles of Chlorella sp. KR-1 revealed meaningful contents ([40 % of saturated and mono-unsaturated fatty acids)

Electronic supplementary material The online version of this article (doi:10.1007/s00449-014-1186-5) contains supplementary material, which is available to authorized users. R. Praveenkumar
B. Kim
E. Choi
K. Lee
J.-S. Hyun
J.-Y. Park
J.-S. Lee
Y.-K. Oh (&) Biomass and Waste Energy Laboratory, Korea Institute of Energy Research (KIER), Daejeon 305-343, Republic of Korea e-mail: [email protected] S. Cho Department of Microbiology, School of Natural Science, Pusan National University, Busan 609-735, Republic of Korea Y.-C. Lee Department of BioNano Technology, Gachon University, Seongnam 461-701, Republic of Korea H. U. Lee Division of Materials Science, Korea Basic Science Institute (KBSI), Daejeon 305-333, Republic of Korea

under the mixotrophic condition, which enables the obtainment of a better quality of biodiesel than is possible under the autotrophic condition. Conclusively then, it was established that a microalgal culture mediated by flue gas can be improved by adoption of mixotrophic cultivation systems. Keywords Mixotrophic culture
Coal-fired flue gas
Chlorella sp. KR-1
Biodiesel

Introduction The finitude and insecurity of fossil fuel supplies, not to mention their detrimental effects on the environment, encourage the development of biofuels such as biodiesel [1, 2]. Biodiesel can be produced from neutral lipid via the catalytic (trans)esterification reaction in the presence of methanol, yielding fatty acid methyl esters (FAME) with glycerol as a by-product [3]. Biodiesel, crucially, does not contribute to net carbon dioxide (CO2) accumulation in the atmosphere, and produces lower levels of harmful gas emissions compared with conventional petroleum diesel [3–5]. Aside from plant-based lipid sources such as soybean, rapeseed, and palm, recently microalgae have been proposed as a promising biodiesel feedstock, owing to their higher lipids content per cell and areal lipid productivity, their utilization of non-arable land, and the cultivation availability of various wastewaters [6–11]. In general, in the presence of light, microalgae can grow using CO2 via autotrophic metabolism. By contrast, without light, some microalgae can use organic substrates as the sole source of energy and electrons, growing via a form of heterotrophic metabolism [12, 13] in which not only is the requirement for light eliminated, but also the culture

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process can be easily controlled in a conventional fermentor system. With sugars and organic acids such as glucose and acetate, higher cell densities and productivities have been reported [14]. Under the heterotrophic growth condition as compared to the autotrophic one moreover, intracellular lipid synthesis can be considerably enhanced [15]. However, the limited availability of organic resources in place of CO2 and light, and the relative costliness of the necessary fermenter system, could result in the incurring of significantly higher overall operating and capital costs in the biodiesel production process [12, 13]. A mixotrophic cultivation system combining CO2 and an organic carbon substrate in the presence of light can be a more practical means of developing cost-effective mass production of microalgae for biodiesel feedstocks [14, 16]. This growth mode, additionally, could hold an economic advantage associated with the utilization of low-priced inorganic carbon (CO2), especially from industrial exhaustgas sources, and could also prevent the reduction in the overall cell-growth rate by nighttime respiration metabolism using organic carbon. The synergistic effect of light and organic carbon can induce higher biomass and lipid productivities in mixotrophic cultures [17]. Wan et al. [18] reported that the growth rate of Chlorella sorokiniana under the mixotrophic condition was 4.2-fold higher than under the autotrophic condition. Furthermore, mixotrophic cultivation of C. vulgaris with glucose has resulted in higher biomass (254 mg/L
d) and lipid (54 mg/L
d) productivities than under either autotrophic or heterotrophic cultivation [12]. Utilization of industrial exhaust gases, as a rich CO2 resource and cost-reducing carbon-source alternative, recently has gained a lot of attention. However, direct supply of CO2 from the atmosphere is not an efficient means of obtaining high biomass productivity, due to the low CO2 content of air (380 ppmv) [19]. Direct use of flue gases, on the other hand, imposes extreme conditions on microalgae, such as high concentrations of CO2 (for example, 5–6 % for natural gas burning and 10–15 % for coal burning) and inhibitory compounds including NOx, SOx, and CO [19, 20]. Some microalgae species show little growth inhibition under high CO2 percentages (10–15 %) in flue gas [21–23]; some other studies, though, have reported significant inhibition of microalgal growth under CO2 concentrations above 5 % [24, 25]. The SOx and NOx in flue gas, especially from coal-fired power plants, more seriously inhibit microalgae [19–21]. Moreover, SOx causes a sharp decline of culture-medium pH, resulting in the reduction of microalgal growth rates [26]. Although many autotrophic cultures based on CO2 utilization from flue gas have been reported [22, 23, 27–29], research on mixotrophic culture to improve microalgal lipid productivity from actual coal-burned flue gas is very limited.

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The purpose of this study was to examine the possibility of enhancing the biomass and lipid productivities of Chlorella sp. KR-1 using real coal-fired flue gas collected from a 2 MW demonstration-scale coal-firing thermal plant [30] and supplementing glucose as a model organic carbon in the presence of light (i.e., in the mixotrophic mode). Cell growth and lipid production under the photoautotrophic and mixotrophic conditions were compared. Next, the effects of the glucose and nitrate concentrations and fluegas flow rates were evaluated. Finally, the fatty acid profiles of Chlorella sp. KR-1 corresponding to the cultivation conditions were analyzed in terms of biodiesel fuel quality.

Materials and methods Microalga and pre-culture conditions Chlorella sp. KR-1 was isolated from a freshwater stream close to the thermal power plant, located in Young-wol, Korea [20, 31], and cultivated in modified N8 medium. The per-liter compounds in the modified N8 medium were as follows: 505.5 mg KNO3, 740 mg KH2PO4, 259.8 mg Na2HPO4, 50 mg MgSO4
7H2O, 17.5 mg CaCl2
2H2O, 11.5 mg FeNaEDTA
3H2O, 3.2 mg ZnSO4
7H2O, 13 mg MnCl2
4H2O, 18.3 mg CuSO4
5H2O, and 7 mg Al2(SO4)3
18 H2O. The medium was sterilized by filtration through a 0.2 lm pore membrane, and the pH was adjusted to 6.5. One agar-plate-grown colony of Chlorella sp. KR-1 was cultured in a 250 mL Erlenmeyer flask (working volume, 100 mL) to produce an inoculum for the photo-bioreactors (PBRs). The flasks were incubated in a shaking incubator (IS-971RF, Lab Companion, Korea) at 25 °C and 150 rpm. Light was supplied continuously for 7 days by four white fluorescent lamps with ca. 40 lmol/m2
s. Batch experiment using PRBs A batch culture was performed in a Pyrex glass bubblecolumn photo-bioreactor (b-PBR) (length, 35 cm; inner diameter, 3.7 cm; working volume, 500 mL). The phosphate concentration in the N8 medium was enhanced to 30 mM to prevent significant pH drop in supplying CO2, SOx, and NOx. The initial pH was maintained at 6.5. The inoculum concentration was adjusted constantly to the initial optical density (OD) of 0.2 at 660 nm. The b-PBR was continuously supplied with 10 (%, v/v) CO2 in air or real flue gas from the bottom of the reactor. The supplied gas was passed through a 0.2 lm PTFE venting filter (Minisart 2000, Satorius Stedium Biotech., Germany) and controlled by mass-flow controllers (MKP, Korea) and flow meters (Dwyer Instruments Inc., USA). For mixotrophic

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cultivation, glucose was added as a model organic source after 0.2 lm syringe filtration (Minisart High-Flow, 16532-K, Sartorius Stedium Biotech., Germany). The b-PBR was maintained under continuous illumination (white fluorescent lamps, *170 lmol/m2
s) in a temperature-controlled room (28–31 °C). Coal-burning thermal plant and flue-gas transfer The flue gas used in this study was obtained from a 2 MW demonstration-scale coal-burning power plant located at the Korea Institute of Energy Research (KIER) in Daejeon, Korea [32]. The plant consists of a coal feeder, a circulating fluidized bed combustor with the coal-firing capacity of 1.2 ton/h, a steam turbine and generator, and an airpollutants treatment system including a bag filter, selective catalytic reduction, flue-gas desulfurization and an activated carbon tower. Bituminous coal was imported from China. In addition, limestone (about 90 % content of CaCO3) and urea (produced from swine urine) were utilized for SOx and NOx removal, respectively. The raw flue gas was collected from the middle position of stack of the plant and treated, in series, by a sediment trap, a water trap, and a gas cooler to avoid compressor corrosion and lower the gas temperature. The flue gas was then transferred through an SUS 304 pipeline using an air-cooled rotary vane compressor (Mapro, Italy), and stored in a gas tank (material, SUS 400; volume, 4.0 m3; pressure, 0.25 MPa) before feeding to the microalgal b-PBR.

collected in TedlarÒ gas-sample bags (Sigma-Aldrich, USA) for 1 h. The compositions (CO, NO, NO2, SO2, CO2, and O2) of the flue gas were analyzed twice per day during the final 3 days of cultivation using a portable flue-gas analyzer (Vario Plus, MRU, Germany). The fatty acid content was determined by the direct transesterification method [34]. After 70 h of cultivation, cells were harvested by centrifugation (3,8009g, 10 min), washed three times with distilled water and freeze-dried for 4 days or longer in a lyophilizer (FD5512, IlShin BioBase Co., Korea). Approximately 10 mg of dried cells was mixed vigorously with 2 mL of freshly prepared chloroform/methanol (2:1 v/v) for 10 min. Sequentially added were 1 mL chloroform, heptadecanoic acid as an internal standard (500 mg/L), 1 mL methanol, and 300 lL sulfuric acid (95 %), which were then mixed well for 5 min and incubated at 100 °C for 10 min. After additional, vigorous mixing, this time with 1 mL distilled water, the mixture was cooled to room temperature and centrifuged at 4,0009g. The lower organic phase was extracted and filtered using a plastic syringe with a 0.2 lm PTFE filter (Minisart SRP15, Satorius Stedium, Germany). FAME were analyzed using a gas chromatograph (GC) equipped with a flame-ionization detector and a 0.32 mm (ID) 9 30 m HP-INNOWax capillary column (Agilent Technologies, USA). The detailed GC conditions are available in the literature [33].

Results and discussion Analytical methods The biomass concentration was calculated by multiplying the OD660 values by the factor of 0.2244, which was derived from a plot of the OD660 values versus the variation of the dry-cell weight (DCW). The OD was measured at 660 nm by UV–Vis spectrophotometry (Optizen 2120UV, Mecasys Co., Korea) [33]. To determine the DCW, fresh samples according to different growth times were filtered through pre-weighed GF/C filters (Whatman, UK) that had been twice rinsed with distilled water, dried at 105 °C for 2 h and weighted. The DCW was determined by subtraction of the filter weight. The elemental C, H, N, and S compositions of the dried microalgal biomass were examined using an Element Analyzer (FLASH 2000 series, Thermo Scientific, USA). The sample pH was measured with an HM-30R pH meter (DKK-TOA Co., Japan). The nitrate concentration was analyzed by the chromotropic acid method using the D5030-11 nitrate analysis kit with a portable HS-1000 water analyzer (Humas Co., Korea). The glucose concentration was determined using an AceChem glucose analysis kit (YD-Diagnostics, Korea). Outlet samples of coal-fired flue gas from the b-PBRs were

Cell growth and lipid production under photoautotrophic and mixotrophic conditions using synthetic CO2 The feasibility of enhancing the cell growth and lipid production of Chlorella sp. KR-1 under mixotrophic culture conditions versus autotrophic ones in a batch-culture mode was investigated. Figure 1 shows batch cultivations of Chlorella sp. KR-1 in b-PBRs under autotrophic and mixotrophic conditions. The autotrophic cells grew slowly; the biomass productivity was estimated to be 0.56 g cell/ L
d at 70 h. Addition of glucose to the photosynthetic N8 medium significantly accelerated the growth of Chlorella sp. KR-1; exponential cell growth from the beginning, with a maximum specific growth rate of 0.098 h-1, was observed. Under this cultivation condition, glucose was steadily consumed; the utilization efficiency was as high as 93 % after 70 h incubation (Fig. 1a). As for the mixotrophic culture, it showed higher biomass productivity (0.8 g cells/L
d) than obtained under the autotrophic condition (0.56 g cells/L
d). This result, significantly, indicated the ability of Chlorella sp. KR-1 to effectively

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Fig. 1 a Time-dependent biomass productivity and glucose consumption and b FAME contents and productivities for photoautotrophic and mixotrophic Chlorella sp. KR-1 cultivation. For the autotrophic culture, 10 % CO2 in air, as the inorganic carbon source, was continuously fed to the b-PBR. By contrast, for the mixotrophic

culture, in addition to 10 % CO2, 5 g/L glucose was added to the N8 medium as an organic carbon substrate. The initial nitrate concentration and gas flow rate were 5 mM and 0.6 vvm, respectively. The initial pH was maintained at 6.5

assimilate glucose as the carbon source for cell growth. Under both culture conditions, the nitrate nutrient was almost completely consumed within 45 h (data not shown). Figure 1b shows the FAME contents and productivities for the cells grown for 70 h under the autotrophic and mixotrophic conditions. Under the mixotrophic culture condition, the FAME content (*268 mg/g DCW) and productivity (*214 mg/L
d) were 1.9- and 2.7-fold higher, respectively, than under the autotrophic condition. This implies that the mixotrophic culture with glucose, compared with the autotrophic one, is effective for obtaining higher lipids content and productivity. Many Chlorella sp. are known to possess an inducible membrane-bound hexose transport system [12]. However, the characteristics of cell growth and cellular lipid synthesis with hexose substrates in the presence of light are largely dependent on the microalgal species and strains employed. Enhanced cell growth and lipid synthesis have been reported for C. sorokiniana CCTCC M209220 with glucose [18]. For C. vulgaris 2714, a significantly higher biomass concentration (1.4 g/L) in a mixotrophic culture with glucose was obtained compared with an autotrophic culture (0.40 g/L), but the cellular lipid content from the former (13.8 wt%) was only a half that of the latter (27.4 wt%) [35]. In the presence of organic carbon sources, and in the mixotrophic mode, C. protothecoides do not utilize CO2 for cell growth [35]. Contrastingly, Chlorella sp. KR-1, in the present study, could grow and accumulate a high level of cellular lipids under light, by assimilating both CO2 and glucose simultaneously.

Effect of use of flue gas on glucose concentration

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Figure 2a shows the effect of glucose concentration (3–10 g/L) on biomass productivity and glucose consumption at 70 h cultivation. Under all of the test conditions, Chlorella sp. KR-1 showed linear growth patterns without reaching the stationary phase. Under photoautotrophic cultivation, a low biomass productivity (0.45 g cells/L
d) was obtained; addition of glucose at 3 g/L, as expected, enhanced the growth significantly, enabling a high biomass productivity (0.66 g/L
d). However, for glucose additions over 5 g/L, significant amounts could not be utilized after 70 h of cultivation (glucose removal efficiencies: 53 and 19 % for 5 and 10 g/L, respectively), while the biomass productivity did not markedly differ from that with 3 g/L glucose. Considering the higher glucose removal efficiency, 93 %, at 5 g/L with synthetic CO2 gas, it seems that the metabolic activity of Chlorella sp. KR-1 is affected considerably by the toxicity of the coalburning flue gas. The nitrate supplied was almost completely (96–100 %) consumed, and the culture pH was maintained at 6.2–6.6 under all of the tested conditions. As shown in Fig. 2b, the maximal lipid content (168 mg FAME/g cells) and lipid productivity (118 mg FAME/L
d) were observed at the 3–5 g/L glucose levels, which values were 1.3- and 2.0-fold higher than their autotrophic counterparts, respectively. But a higher glucose concentration, 10 g/L, did not improve the lipid productivity of Chlorella sp. KR-1. A significant decrease in chlorophyll content was observed (data not shown), leading to heterotrophic metabolism of glucose [12, 36, 37].

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Fig. 2 a Effects of initial glucose concentration on biomass productivity and glucose consumption and b FAME contents and productivities. Actual CO2-rich (13 %, v/v) flue gas from a coal-burning power was applied. The initial pH and nitrate concentration were

fixed at 6.5 and 5 mM, respectively. The flow rate of the flue gas was adjusted to 0.2 vvm in consideration of its potential toxicity to microalgal cell growth

Fig. 3 a Effects of initial nitrate concentration on biomass productivity and glucose consumption and b FAME contents and productivities. The glucose concentration was 5 g/L. The initial pH and flue-gas flow rate were fixed at 6.5 and 0.2 vvm, respectively

Another reason for the higher lipid content under the mixotrophic condition might have been the earlier nitrogen deprivation experienced by the cells, which occurred at 60 h, compared with 70 h under the autotrophic condition (data not shown). The effect on lipid productivity of nitrogen deprivation, which produces a stressful microenvironment and the resultant accumulation of triacylglyceride, has been well studied [11]. Effect of nitrate concentration using flue gas The effect of nitrate concentration (3–10 mM) on the cell growth and lipid production of Chlorella sp. KR-1 was

tested (Fig. 3). As shown in Fig. 3a, as the nitrate concentration increased from 3 to 8 mM, the residual glucose concentrations gradually decreased from 2.55 to 0.51 g/L, and the biomass productivity, correspondingly, increased from 0.58 to 0.80 g cell/L
d. But at a higher, 10 mM nitrate concentration, neither glucose consumption efficiency nor biomass productivity was improved. This indicates that although the microalgae could completely consume the nitrate, they could only partially convert it to organic nitrogen inside the cell [38]. Nitrogen remains the major constituent of protein, and nitrogen-rich conditions improve the rate of protein synthesis [38]. It could also be assumed that increasing nitrate concentrations facilitate

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Bioprocess Biosyst Eng Table 1 Elemental compositions of Chlorella sp. KR-1 under different culture conditions

Chlorella sp. KR-1

Compositions (wt%) C

H

N

S

Photoautotrophic

46.53 ± 0.45

6.88 ± 0.11

3.62 ± 0.83

0.29 ± 0.05

Mixotrophic

47.54 ± 1.44

7.15 ± 0.26

3.64 ± 0.88

0.31 ± 0.11

Photoautotrophic

46.06 ± 0.37

7.12 ± 0.12

3.40 ± 0.44

0.28 ± 0.01

Mixotrophic

47.38 ± 1.20

7.27 ± 0.24

3.52 ± 0.88

0.33 ± 0.10

Synthetic CO2

Flue gas

transporter protein synthesis and, thereby, glucose uptake. At a higher, 10 mM nitrate concentration, cell inhibition might be attributed to the stimulation of nitrate reductase activity and the resultant NH4? accumulation. And in fact, excess NH4? can inhibit the metabolic activities of microalgal cells [39]. In an elemental (C, H, N, and S) analysis of the dried biomass (Table 1) for 0 g/L glucose in the photoautotrophic mode and 5 g/L glucose in the mixotrophic mode, 5 mM nitrate, and flow rates of 0.6 vvm for synthetic CO2 and 0.2 vvm for flue gas at an approximate pH of 6.5 for 70 h (see Supplementary Information Table S2), the nitrogen content (wt%) of Chlorella sp. KR-1 cultivation with the flue-gas supply was slightly reduced relative to the synthetic CO2 cases, indicating slight changes to the biochemical composition of the KR-1. Figure 3b shows the effects of nitrate concentration on FAME content and productivity. The FAME contents were as high as 161–176 mg/g DCW in the range of 3–5 mM nitrate, and gradually decreased with increasing nitrate concentration. Maximal FAME productivities (118–121 mg/L
d) were observed for 5–8 mM nitrate, based on the high lipid content and biomass concentration. It is well known that the nitrogen-deficient conditions can improve lipid synthesis in microalgae. In the present study, at a lower, 3 mM nitrate concentration, relatively low lipid productivity was observed due to lower biomass productivity, despite the high lipid content. Chiu et al. [29] reported a biomass productivity of 0.48 g cell/L
d and a lipid productivity of 120.5 mg/L
d for Chlorella sp. MTF-7 from coke-oven flue gas. Recently, Arbiba et al. [40] demonstrated the cultivation of Scenedesmus obliquus SAG276 from a natural gas power plant using real flue gas, achieving a biomass productivity of 0.35 g cell/L
d and a lipid productivity of 82.3 mg/L
d. Effect of flue-gas flow rate The effects of different flue-gas flow rates (0.1–0.6 vvm) on the mixotrophic mode of Chlorella sp. KR-1 cultivation over 70 h were studied (Fig. 4). Maximal biomass productivity (0.68 g cell/L
d) was observed at 0.2 vvm, but

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with increasing flow rate, the productivity decreased (Fig. 4a). As the flow rate was increased from 0.1 to 0.6 vvm, the glucose consumption efficiency was very greatly reduced, from 72 to 6 %. This clearly indicated the toxic effects of flue gas on microalgae at higher flow rates. This might be attributable to the increasing dissolution of toxic gases such as SOx, NOx, and CO compounds at high flow rates. Notwithstanding, Chlorella sp. KR-1 has been reported to tolerate high concentrations of NOx (100 ppm) and SO2 (60 ppm) in air [20]. However, significant cellgrowth inhibition might possibly be due to the combinatorial effects of toxic gas mixtures of, for example, SOx, NOx, and CO. Further, it was noted that at a high flow rate, the glucose-utilization capacity of Chlorella sp. KR-1, in comparison with photosynthetic CO2, was markedly inhibited, to only 6 % glucose consumption. Additionally to the issue of unspent glucose, during growth, microalgal cells might release various organic substances such as polysaccharides, proteins, organic acids, vitamins, and others [41]. The extracellular discharge of the biomolecules has any of a number of specific functions such as resource acquisition, defense, or communication (e.g., extracellular enzymes, siderophores etc.); secretion might also result from photorespiration/photosynthetic overflow, passive diffusion or mere leakage from cells [42]. Considering the economic and environmental impacts, both residual glucose and secretory organics should be recovered if a medium is recycled for further cultivation. Recently, HadjRomdhane et al. [41] reported the repeated use of a culture supernatant for subsequent, 63-day cultivation of C. vulgaris. Usually, flue-gas bubbling results in a pH drop; therefore, suitable buffering is warranted in flue-gas experiments. Maintaining a pH above 6 prevents the transformation of SO2 to toxic species such as bisulphites [19]. The buffering capacity of phosphates in media has been well studied [43]; in the present study accordingly, the phosphate buffer was enhanced to stabilize the pH at around 6–6.5. Figure 4b shows the effects of flue-gas flow rates on FAME content and productivity. As can be seen, with an increasing flow rate, the FAME content gradually decreased. As already noted, flue-gas compounds such as

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NOx, SOx and CO can have toxic effects on microalgae, which might induce peroxidation of membrane lipids, blocking of membrane transport systems, bleaching of chlorophyll, or other disadvantageous outcomes [19]. Higher flue-gas flow rates can increase the rate of dissolution of NOx and SOx into a medium, which can impair cell growth and energy storage. This mechanism, in fact, could have lowered the FAME contents of the KR-1. Indeed, a higher FAME productivity was observed, at the flow rate of 0.2 vvm, due to the higher biomass productivity (Fig. 4b). Chlorella sp. KR-1 was shown to be capable of growing rapidly and accumulating lipid in significant amounts when mediated by actual coal-fired flue gas in the mixotrophic mode with glucose. However, for the practical application

of mixotrophic cultivation with KR-1, extensive studies including scale-up, process optimization such as fed-batch and continuous strategies, outdoor culturing and utilization of waste organic substrates, should be carried out. In the present case, mixotrophic fed-batch cultivation of Chlorella sp. KR-1 with 3 g-glucose/L under the indoor condition resulted in a 1.3-fold increase in lipid productivity compared with that of a batch-culture system (unpublished data). Removal of flue-gas compounds Figure 5 shows the flue-gas compositions and their removal by Chlorella sp. KR-1 at different flow rates. Flue gas released from the coal-burning power plant had very low

Fig. 4 a Effects of flue-gas flow rates on biomass productivity and glucose consumption and b FAME contents and productivities. The initial pH was adjusted to 6.5, and the initial glucose and nitrate concentrations were maintained at 5 g/L and 5 mM, respectively

Fig. 5 a Compositions of flue gases from 2 MW coal-burning power plant and b removal (%) of flue-gas components at different flow rates

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123

25.49 ± 0.11

100

Others

Total

nd Not detected

36.74 ± 0.94

nd

11.74 ± 0.05

C20:0

Arachidate

8.10 ± 0.89

Poly-unsaturated

C18:3n3

Linolenate

0.39 ± 0.05

28.25 ± 0.38

Mono-unsaturated

C18:3n6

c-Linoleate

26.04 ± 0.96

C18:2n6c

Linoleate

11.36 ± 0.03

2.34 ± 0.39

0.37 ± 0.03

23.16 ± 0.78

Saturated

C18:0

C18:1n9c

Stearic acid

C16:1

Palmitoleate

Oleate

C16:0

Palmitate

nd

0.11 ± 0.16

C14:0

C15:0

Myristic acid

Pentadecanoate

nd

0.43 ± 0.03

C10:0

C13:0

Caprate

0.05 ± 0.08

100

16.86 ± 0.19

32.77 ± 0.09

19.13 ± 0.29

31.23 ± 0.33

0.11 ± 0.15

5.46 ± 0.29

0.33 ± 0.02

26.98 ± 0.44

18.94 ± 0.39

5.73 ± 0.53

0.19 ± 0.02

24.87 ± 0.21

0.05 ± 0.07

0.23 ± 0.01

0.20 ± 0.01

nd

100

19.97 ± 1.26

35.17 ± 2.27

13.97 ± 1.09

30.89 ± 3.28

0.92 ± 0.25

7.51 ± 1.11

0.45 ± 0.05

27.21 ± 1.11

13.72 ± 1.08

6.44 ± 1.95

0.25 ± 0.01

22.67 ± 0.92

nd

0.36 ± 0.09

0.51 ± 0.07

100

20.18 ± 1.01

34.58 ± 0.66

16.33 ± 1.47

28.92 ± 0.10

nd

6.86 ± 0.17

0.40 ± 0.05

27.32 ± 0.71

16.09 ± 2.04

4.98 ± 0.26

0.23 ± 0.04

23.33 ± 0.36

nd

0.31 ± 0.05

0.30 ± 0.00

nd

D—0.2 vvm

C—0.1 vvm

A— Photoautotrophic

B— Mixotrophic

Flue-gas flow rate

Synthetic CO2

Ratio (%) of fatty acids to total FAME (mean ± STD)

Tridecanoate

Fatty acid

100

22.74 ± 0.21

41.67 ± 0.12

10.86 ± 0.09

24.73 ± 0.69

1.22 ± 0.08

11.17 ± 0.02

0.50 ± 0.01

29.99 ± 0.09

10.64 ± 0.08

2.11 ± 0.13

0.22 ± 0.01

20.62 ± 0.46

nd

0.53 ± 0.02

0.25 ± 0.01

nd

E—0.4 vvm

100

22.86 ± 0.19

41.35 ± 0.27

10.91 ± 0.54

24.88 ± 0.93

1.18 ± 0.10

12.23 ± 0.12

0.49 ± 0.02

28.63 ± 0.13

10.66 ± 0.54

1.86 ± 0.04

0.25 ± 0.00

21.33 ± 0.67

nd

0.43 ± 0.03

0.09 ± 0.09

nd

F—0.6 vvm

100

26.99 ± 3.64

42.52 ± 0.49

8.13 ± 1.28

22.36 ± 1.78

nd

10.61 ± 0.42

0.46 ± 0.01

31.45 ± 1.10

8.13 ± 1.81

2.15 ± 1.10

nd

19.70 ± 1.48

nd

0.11 ± 0.16

0.13 ± 0.19

0.26 ± 0.04

G—Auto

100

20.77 ± 3.64

37.49 ± 0.49

13.62 ± 1.28

28.12 ± 1.78

0.70 ± 0.09

7.92 ± 0.09

0.46 ± 0.01

29.11 ± 0.37

13.51 ± 0.37

5.01 ± 0.10

0.11 ± 0.11

21.24 ± 0.44

nd

0.55 ± 0.01

0.54 ± 0.04

0.08 ± 0.08

H—3 g/L

Glucose concentration

100

21.59 ± 0.60

38.97 ± 0.63

12.39 ± 0.78

27.05 ± 1.06

1.21 ± 0.29

8.85 ± 0.04

0.49 ± 0.02

29.62 ± 0.57

12.29 ± 0.68

3.87 ± 0.37

0.11 ± 0.11

20.63 ± 0.28

nd

0.63 ± 0.03

0.62 ± 0.00

0.09 ± 0.09

I—10 g/L

Table 2 FAME compositions of Chlorella sp. KR-1 under photoautotrophic and mixotrophic culture conditions using synthetic CO2 and flue gas

100

25.65 ± 1.89

32.07 ± 1.43

13.04 ± 0.57

29.23 ± 1.28

0.81 ± 0.11

12.94 ± 0.97

0.58 ± 0.01

18.55 ± 0.45

12.77 ± 0.53

4.21 ± 0.38

0.27 ± 0.03

22.54 ± 0.67

0.22 ± 0.00

0.46 ± 0.04

0.79 ± 0.06

0.20 ± 0.02

J—3 mM

20.05 ± 0.53 3.60 ± 0.05

100

26.89 ± 1.07

31.98 ± 1.99

12.38 ± 0.96

28.76 ± 0.45

0.84 ± 0.03

13.06 ± 1.15

0.59 ± 0.01

18.32 ± 0.83

12.07 ± 0.91

100

33.49 ± 3.75

25.96 ± 9.52

15.02 ± 7.01

25.53 ± 1.41

0.81 ± 0.04

8.55 ± 1.61

0.69 ± 0.00

16.72 ± 7.90

14.45 ± 6.83

3.07 ± 0.39

0.58 ± 0.18

22.72 ± 0.17 0.31 ± 0.05

0.18 ± 0.18

0.58 ± 0.04

0.71 ± 0.10

0.13 ± 0.13

L—10 mM

0.23 ± 0.00

0.47 ± 0.04

0.79 ± 0.04

0.11 ± 0.11

K—8 mM

Nitrate concentration

Bioprocess Biosyst Eng

5.17 ± 0.28

0.31 ± 0.09

2.78 ± 0.03 2.91 ± 0.71 4.68 ± 0.02 4.91 ± 0.15

D—5 mM NO3

K—8 mM NO3

L—10 mM NO3

1.95 ± 0.49

I—10 g/L glucose

J—3 mM NO3

2.39 ± 0.29 2.91 ± 0.71

H—3 g/L glucose

D—5 g/L glucose

–

F—0.6 vvm

G— photoautotrophic

93 ± 4

90 ± 1

53 ± 13

52 ± 1

19 ± 5

53 ± 13

85 ± 14

–

6±2

18 ± 5

53 ± 13

72 ± 5

93 ± 6

–

Glucose consumption efficiency (%)

568.1 ± 0.1

466.0 ± 2.2

284.2 ± 4.5

180.2 ± 8.4

285.9 ± 4.7

284.2 ± 4.5

251.9 ± 41.8

270.5 ± 3.4

217.9 ± 10.5

266.9 ± 20.1

284.2 ± 4.5

263.6 ± 29.8

287.7 ± 2.5

281.1 ± 2.5

Nitrate consumed (mg/L)

100

100

100

100

96 ± 1

100

99 ± 1

99 ± 1

71 ± 2

86 ± 9

100

100

100

100

Nitrate consumption efficiency (%)

0.79

1.86

1.27

1.01

0.49

1.27

0.83

1.19

na

na

1.27

0.63

–

–

*CCO2

0.98

0.94

0.58

0.56

0.39

0.58

0.48

0.00

0.06

0.17

0.58

0.73

–

–

*Cglucose

na Not available (see Supplementary Information Table S1)

0.80

1.99

2.19

1.82

1.25

2.19

1.73

700 ± 70.6

795 ± 7.2

675 ± 7.4

578 ± 6

639 ± 18.5

675 ± 7.4

662 ± 17.8

415 ± 20.9 453 ± 1.2

na

463 ± 3.1

675 ± 7.4

598 ± 8

800 ± 28.3

561 ± 1.2

Biomass productivity (mg/L
d)

?

na

2.19

0.87

–

–

*CCO2/ **Cglucose

Carbon consumed (g)

* CCO2 and Cglucose were calculated based on the CO2 and glucose consumption data, respectively (see Supplementary Information Table S1)

Flue gas – nitrate concentrations at 5 g/L glucose

Flue gas – glucose concentrations

2.91 ± 0.71 0.84 ± 0.22

D—0.2 vvm

E—0.4 vvm

3.63 ± 0.16

C—0.1 vvm

B—5 g/L glucose

Flue gas—flow rates at 5 g/L glucose

–

A— photoautotrophic

Synthetic CO2

Glucose consumed (g/L)

Treatments

Experiments

Table 3 Results of nutrient consumption and yield parameters by Chlorella sp. KR-1 under different culture conditions

88.7 ± 19.2

121.3 ± 6.1

118.38 ± 8.2

92.9 ± 6.7

101.25 ± 2.8

118.38 ± 8.2

116.63 ± 2.1

60.02 ± 2.9

58.58 ± 1.2

68.43 ± 2.9

118.38 ± 8.2

109.45 ± 20.4

214.41 ± 6.4

79.17 ± 0.9

FAME productivity (mg/L
d)

Bioprocess Biosyst Eng

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Bioprocess Biosyst Eng

levels (*5 ppm) of NO and SO2, with 13.2 % CO2 (Fig. 5a). The flue-gas concentrations changed after passing through the microalgal culture. The flue-gas removal percentage was maximal at the flow rate of 0.1 vvm, and decreased with increasing flow rates thereafter. Chlorella sp. KR-1 showed a high SO2 removal efficiency (Fig. 5b). This could be explained by the low input levels and effective oxidation into sulfates that can be taken up by microalgae for metabolism in strongly buffered environments. The CO2 removal rate was only 4.7 % at the flow rate of 0.1 vvm. Only trace amounts of the other flue gases were removed. A low NOx-removal rate was suspected, due to its poor solubility in a pH-stabilized system [19], whereas the continuous flow and shorter retention time of flue gas in the reactor would have affected the removal of CO2. NOx present in flue gas can be a nitrogen source for microalgae. However, considering its solubility and lower concentration in the flue gas supplied, the effect of NOx versus KNO3, as added to a liquid medium, might be negligible. Studies that have reported high CO2 removal rates either adopted intermittent flue-gas flow [29] or lower flow rates [23, 44] for efficient mitigation of flue-gas compounds. These strategies could also be considered when KR-1 cultures are utilized in masscultivation systems. Again, CO gas can have a toxic, growthretarding effect on microalgae. Although there is as yet no direct evidence of CO removal by microalgae, the bacterial communities present in mixotrophic cultures can oxidize CO to supply microalgae with CO2 [45]. Chlorella sp. KR-1 can grow well only at temperatures up to 40 °C [31]. Therefore, the flue gas employed in this study was pre-treated to room temperature by a sediment trap, a water trap, and a gas cooler in series, after which it was stored in a tank. For practical applications, the high temperature of flue gas can be brought down by developing an inexpensive cooling system using cooling water (20–35 °C; unpublished data from KIER) for the steam turbine of the power plant, which can be directly supplied to microalgal cultures. Fatty acids profiles The fatty acid profiles of Chlorella sp. KR-1 under different cultivation conditions are provided in Table 2. Under those conditions, significant variations in the fatty acid compositions were observed. However, under most of the conditions, even after flue-gas mediation, the fatty acids produced met the requirement of the European EN14214 (2008) standard [46] for biodiesel production. According to EN14214, the permissible level for C18:3 (linolenic acid) is B12 %. In this study, the C18:3 levels, again under almost all of the conditions, were \12 %. The saturated and mono-unsaturated fatty acid contents accounted for more than 40 % of the total fatty acids under most of the

123

mixotrophic conditions, whereas under the photoautotrophic conditions, the saturated and mono-unsaturated fatty acids accounted just above 30 %. The important biodiesel properties, in view of the cetane number, iodine number, heat of combustion, NOx emission, oxidative stability, lubricity, viscosity, and cold flow point, are principally dependent on the fatty acid profiles [47]. Usually, the presence of poly-unsaturated fatty acids in biodiesel leads to lowering of the cetane number and increased NOx emission. Poly-unsaturated fatty acids are prone to further oxidation, which affects biodiesel lubricity [47]. In this regard, it is desirable that biodiesel has high levels of saturated and mono-unsaturated fatty acids and low levels of poly-unsaturated fatty acids [48]. The fatty acid profiles of Chlorella sp. KR-1 under mixotrophic cultivation using flue gas satisfy the biodiesel quality requirements outlined in the literature [33].

Conclusions As summarized in Table 3, oleaginous Chlorella sp. KR-1 was cultured mixotrophically, yielding, at 5 g/L glucose, both higher biomass and lipid contents compared with the autotrophic condition. As mediated by flue gas, the optimal conditions for biomass and lipid productivities were a flow rate of 0.2 vvm, 5 g/L glucose, and 5 mM nitrate, corresponding to a ratio of *CCO2/**Cglucose of 2.19. Under all of the optimal conditions at a[1.0 ratio, inorganic carbon from flue gas was utilized more than organic glucose carbon. In addition, the FAME profiles under the mixotrophic conditions revealed the suitability of the biomass for biodiesel production. It is believed that mixotrophic cultivation of oleaginous Chlorella sp. KR-1, as mediated by a supply of real flue gas, can be considered a practical and efficient means of biomass production, without any subsequent deterioration of biodiesel quality. Acknowledgments This work was conducted under the framework of the Research and Development Program of the Korea Institute of Energy Research (KIER) (B4-2434-01). Further support was received from the Advanced Biomass R&D Center (ABC) of the Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2012M3A6A205388), and by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and Ministry of Knowledge Economy (MKE) of Korea as a part of the Project of ‘‘Process demonstration for bioconversion of CO2 to high-valued biomaterials using microalgae’’(2012-T-100201516) in ‘‘Energy Efficiency and Resources R&D project’’.

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