J. Cent. South Univ. (2013) 20: 730736 DOI: 10.1007/s11771-013-1541-8

Effects of simulated flue gases on growth and lipid production of Chlorella sorokiniana CS-01 XIA Jin-lan(夏金兰), GONG San-qiang(巩三强), JIN Xue-jie(金雪洁), WAN Min-xi(万民熙), NIE Zhen-yuan(聂珍媛) School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China © Central South University Press and Springer-Verlag Berlin Heidelberg 2013 Abstract: To study the abilities of Chlorella sorokiniana CS-01 on using CO2 from flue gases to produce biodiesel, the microaglae was cultured with different simulated flue gases containing 5%–15% (volume fraction) of CO2. The results show that strain CS-01 could grow at 15% CO2 and grow well under CO2 contents ranging from 5%–10%. The maximal biomass productivity and lipid productivity were obtained when aerating with 10% of CO2. The lipids content ranged from 28% to 43% of dry mass of biomass. The main fatty acid compositions of strain CS-01 were C14–C18 (>72%) short-chain FAMEs (known as biodiesel feedstocks). Meanwhile, the biodiesel productivity was over 60%, suggesting that Chlorella sorokiniana CS-01 has a great potential for CO2 mitigation and biodiesel production. Furthermore, differential expression of three genes related to CO2 fixation and fatty acid synthesis were studied to further describe the effect of simulated flue gases on the growth and lipid accumulation of strain CS-01 at molecular level. Key words: microalgae; Chlorella sorokiniana; flue gases CO2 mitigation; biodiesel

1 Introduction Greenhouse gas emissions and uncertainties about the cost and supply of oil have received great concern in recent years [1–2]. Compared with other plants, microalgae have relatively higher photosynthetic efficiency, higher biomass production, faster growth rate, and higher lipids content and so on, Therefore, usage of microalgae for CO2 mitigation and biodiesel feedstock production has attracted great attention [3–5]. Chlorella is a genus of unicellular green algae. It can be easily cultured and attain high cell density and high oil content under photoautotrophic or mixotrophic conditions. Therefore, it has been considered as one of the most promising microalgal candidates for flue gases CO2 mitigation and commercial biodiesel feedstock production [6–7]. Chlorella sorokiniana CS-01 was isolated from a freshwater sample taken from Inner Mongolia Province, Northern China. This alga has a conspicuous ability to synthesize and accumulate lipids. The total lipids content of this strain may reach over 60% under mixotrophic cultivation, indicating that it has a great potential in biodiesel production [8]. In this work, the ability of strain CS-01 for mitigating CO2 in flue gases and producing biodiesel feedstock was investigated. Furthermore,



differential expressions of genes related to CO2 fixation and lipid synthesis were also examined.

2 Experimental 2.1 Strain and culture conditions Chlorella sorokiniana CS-01 was recently isolated from a freshwater sample from Baotou, Inner Mongolia Province, China, and conserved in China Center for Type Culture Collection, Wuhan, China (the collection number; CCTCC M209220). It was cultured in a 2.4 L air-lift photobioreactor. The culture medium was BG11[9]. The photobioreactor was incubated at (25±1) ºC and aerated by filter-sterilized air at 2.4 vvm. Illumination of 70 µmolm−2s−1 photons was provided by white fluorescent lamps with 12:12 of light/dark ratio. The strain was firstly adapted to air (containing 0.02%–0.03% (volume fraction) of CO2), and then cultivated with aeration of simulated flue gases containing 5%, 10% and 15% of CO2 under the same condition, respectively. Table 1 lists the compositions of the simulated flue gases, which were composed according to the typical composition of flue gases of thermal power plant of China. Cultivation with aeration of air was adopted as the control. The schematic diagram of the experiment apparatus is shown in Fig. 1. Triplicates of assays were performed for each experiment.

Foundation item: Project(50621063) supported by the National Natural Science Foundation for Distinguished Group of China; Projects(2010bsxt05, 2010ssxt246) supported by the Innovation Foundation of Science and Technology of Central South University, China. Received date: 2012–01–15; Accepted date: 2012–03–13 Corresponding author: XIA Jin-lan, Professor, PhD; Tel: +86–73188836944; Email: [email protected]

731

J. Cent. South Univ. (2013) 20: 730736

Fig. 1 Schematic diagram of cultivation system Table 1 Composition (volume fraction, %) of simulated flue gases Experiment No.

CO2

O2

N2

1

5.00±0.50

5.00±0.50

90.00±0.50

2

10.00±0.50

5.00±0.50

85.00±0.50

3

15.00±0.50

5.00±0.50

80.00±0.50

2.2 Determination of cell concentration and growth parameters Cell concentration was determined with platelet counter under optical microscopy (Olympus CX21FS1). The pH values of culture media were detected by pH meter (Sartorius, PB-10). During the exponential phase, the specific growth rate μ is calculated by Eq. (1). The cells were collected by centrifugation (6 000 r/min, 10 min), followed by vacuum-freeze drying for over 8 h, and then kept in a desiccator for further use. The productivity of microalgal biomass gL1d1 is determined using Eq. (2). According to the method described by Ref. [10], the carbon dioxide biofixation rate RCO (gL–1d–1) is calculated as Eq. (3). 2

  (ln P

X2 1 ) X1 t

(1)

We  Wb  100% V d

RCO2  CC P 

M CO2 MC

(2) (3)

where X1 and X2 the concentrations of cells growing at t1 and t2 of exponential phase, and the time (d) between t2

and t1, respectively; Wb and We are the dry mass of cells harvested at the beginning and end of cultivation, respectively; P, V and d are productivities of microalgal biomass (gL–1d–1), culture volume (L), and time of culture (d), respectively; CC is the average carbon content (%, mass fraction), which was measured by an element analyzer (Elementar, Germany); RCO2 and MC are the masses of weights of CO2 and elemental carbon, respectively. 2.3 Analysis of lipids content and composition of fatty acids The total lipids were extracted from microalgal cells according to LEE’s method with a little modification [11]. Microalgal cells were harvested by centrifugation at 6 000 r/min for 10 min and washed three times with distilled water. After dried by vacuum freeze drier (LGJ25, China), the samples were disrupted by sonication (JY 92-II, China) for 3 min, and the total lipids were then extracted by V(chloroform): V(methanol)=2:1 from the samples. About 30 mL of mixed solvents were used for every gram of dried sample in each extraction step and the process was repeated more than three times. The samples were centrifuged at 8 000 r/min for 10 min. The solvent phase was transferred by pipette and evaporated in a rotary evaporator under vacuum at 60 ºC. Then the total lipids were weighed using analytical balance. Fatty acids composition was analyzed by gas chromatography (GC) with GC-9A (Shimadzu). Prior to GC analysis, acidic trans-esterification of the lipid extract was performed to transform the fatty acids to methyl esters by a mixture of methanol, chloroform and HCl with V(methanol):

732

V(chloroform):V(HCl)=10:1:1 in 10 mL crimped vials at 90 ºC for 2 h, the fatty acid methyl esters (FAMEs) were then extracted using V(hexane):V(chloroform)=4:1 and analyzed via GC. The operating conditions of GC were as follows: Injection temperature was 240 ºC, column temperature was held at 210 ºC, PEG-20M capillary column (2.6 m×3.2 mm) and over 20 fatty acid methyl esters were used for external standard quantitative analysis. 2.4 Real-time PCR assays 2.4.1 Total RNA extraction and cDNA synthesis The differential expressions of accD, acc1, and rbcL genes were examined in response to different levels of simulated flue gases containing 5%–15% of CO2. Those genes code key enzymes related to CO2 fixation and lipid synthesis of rbcL (coding ribulose 1, 5-bisphosphate carboxylase oxygenase (RuBisCO) large subunit), accD (coding heteromeric/bacterial acetyl-CoA beta subunit), and acc1 (coding homomeric/eukaryotic ACCase) [12–14]. Microalgal cells were collected when the culture was ventilated continuously with air or simulated flue gases for 2, 4, and 6 h, respectively. Total RNA was isolated and purified with SV Total RNA Isolation System (Z3150-USA), and quantified at OD260/OD280 with NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA). The integrity of total RNA was detected by 1% agarose gel electrophoresis and ethidium bromide staining. After confirming the removal of DNA by checking PCR amplification result of 18S rRNA, total RNA was used as the template to synthesize cDNA with RevertAidTM H Minus First Strand cDNA Synthesis Kit and random primers (Fermentas, China). 2.4.2 Primers design According to the gene sequences obtained from strain CS-01, real-time PCR primers were designed by Primer 5.0 and then synthesized by Sagon Biotech, Shanghai, China. All primer pairs are listed in Table 2. The 18S rRNA gene of C. sorokiniana was used as the internal control. The primers were diluted to obtain 10 mmoL of working stock solutions, and their quality was checked by sequencing the corresponding PCR products. 2.4.3 Real-time PCR assay Real-time PCR was performed in an iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, USA) using SYBR® Green real-time PCR Master Mix (Toyobo Co. Ltd, Osaka, Japan) according to the manufacturer’s instructions: 1 cycle of 95 ºC for 30 s, and then 40 cycles of 95 ºC for 15 s each, followed by 55 ºC for 15 s, and 72 ºC for 30 s. In order to normalize the

J. Cent. South Univ. (2013) 20: 730736 Table 2 Primers for Real-time PCR detection of expression of CO2 fixation and lipid accumulation-related genes in Cherella sorokiniana CS-01 Base pairs Annealing number of Gene Primer sequence temperature/°C amplicon F: TTT GGT TTG TGC TTC TGG TG 66 149 accD R: CAC CAC CAG TTG TTG GAG AA F: TGA CCG TGA AAA 68 AG CATC TG 163 acc1 69 R: CGA CAT ATT CGC CTG ATT GA F: ATA CCG TGT GGA 70 GGA CCT TG 201 rbcL 68 R: AGC CAG TTC CAG GTG AAG AA F: CCT GCG GCT TAA 68 TTT GAC TC 18S 192 68 rRNA R: GCG AAC CAA CCG TGA CTA TT

amount of transcripts, the relative abundance of 18S rRNA was also determined and used as the internal control. At the end of each cycle, melting curves were determined by raising the temperature stepwise by 0.5 ºC from 55 to 95 ºC. The specificity of the PCR amplification was checked by melting temperature (Tm), symmetry, and non-specific peaks of the melting curve. The gene expression data was analyzed by 2-ΔΔCt method [15]. Positive control, negative control, and blank control were also conducted in the experiment. The gene expression ratio was recorded as the fold difference in quantity from samples which were grown in the media ventilated with natural air. The results were normalized against the expression of the control gene 18S rRNA to correct sample-to-sample variation.

3 Results and discussion 3.1 Effect of simulated flue gases on growth of Chlorella sorokiniana CS-01 The effects of simulated flue gases on the growth of the microalgae are shown in Table 3 and Fig. 2. The results show that strain CS-01could grew well under aeration of the simulated flue gases containing 5% and 10% CO 2 . It can grow under aeration of the simulated flue gases containing 15% CO2 (Fig. 2). As shown in Table 3, strain CS-01 shows higher maximum biomass productivity and maximum specific growth rate under the CO2 contents in simulated flue gases ranging from 5% to 10%. The highest biomass productivity (0.17 gL–1d–1) and maximum specific growth rate (3.42 d–1)

733

J. Cent. South Univ. (2013) 20: 730736

Table 3 Growth rates, specific growth rates (μ), biomass productivity (P), and RCO2 under aeration of simulated flue gases containing different concentrations of CO2 Flue gas (CO2)/%

0–6 d growth rate/ (107 mL–1d–1)

610d growth rate/ (107 mL–1d–1)

μ/d–1

P/(gL–1d–1)

RCO2 /(gL–1d–1)

0.02±0.01 (Control)

1.05±0.00

0.24±0.00

0.29±0.01

0.04±0.02

0.07±0.00

5.00±0.05 (Flue gas 1)

2.28±0.01

2.57±0.00

0.31±0.04

0.16±0.02

0.23±0.00

10.00±0.05 (Flue gas 2)

1.88±0.00

3.42±0.01

0.35±0.02

0.17±0.01

0.24±0.01

15.00±0.05 (Flue gas 3)

0.65±0.00

1.30±0.00

0.24±0.01

0.06±0.00

0.09±0.02

Fig. 2 Effect of simulated flue gases containing different contents of CO2 on growth of Chlorella sorokiniana CS-01

were obtained at 10% CO2. In previous study, the C. pyrenoidosa SJTU-2 isolated by TANG et al [16] in the presence of 10% CO2 after 14 d cultivation showed the maximum specific growth rate of 0.99 d–1 and S. obliquus isolated by de MORAIS and COSTA [10] in the presence of 12% after 21 d cultivation showed the maximum specific growth rate of 0.33 d–1. Compared with the C. pyrenoidosa SJTU-2 isolated by TANG et al [16] and the S. obliquus isolated by de MORAIS and COSTA [10], the specific growth rate of Chlorella sorokiniana CS-01 showed 4–10 times higher. These results indicate that strain CS-01 has a good adaptation to flue gas containing high levels of CO2. [9–11]. Furthermore, this phenomenon may be due to the different CO2 biofixaton mechanisms. The decreases in pH of the medium are resulted from entrance of CO2, which made the cells of strain need more time to adapt the lower pH in the medium. Figure 3(a) shows the changes in pH during 10 d cultivation with applying simulated flue gases containing different levels of CO2. The results reveal that the pH of the culture medium remains slightly basic (8.2–8.5) when aerated with air. However, the pH values of the culture media decreased dramatically from 8.3 to 6.8, 5.8 and 5.6, when aerated with simulated flue gases containing 5%, 10%, and 15%

Fig. 3 Changes of pH during cultivation of Chlorella sorokiniana CS-01 under aeration of different levels of simulated flue gases (a), and effect of initial pH on growth of C. sorokiniana CS-01(b)

CO2, respectively. Fig. 3(b) shows obviously that the growth rates decrease with the decrease of pH from 10.0 to 5.0. It was obvious that when air was applied at the control case, the CO2 in the medium was limited, and the more basic the medium is, the more the CO2 would be absorbed and fixed by Calvin cycle, and thus enhanced the growth of the cells. Compared to the control case, CO2 was not a limited factor in the media when the simulated flue gases containing high levels of CO2 were applied. In these cases, the CO2 fixation and the growth of the cells may mainly depend on both the pH and the concentration of free CO2 of the medium, it is well

734

known that CO2 is equilibrated with other inorganic carbon species, such as H2CO3, HCO3–, and CO32–, which is dependent on the pH in the water or medium. On the basis of this theory and experimental results, the efficiency of CO2 maybe depends on the pH in the medium. Figure 2 shows that the growth of strain CS-01 aerated with 10% of CO2 is very different from experimental group aerated with 15% of CO2, but from Fig. 3(a), we can see that the pH values of the two groups are close to each other, therefore pH is not an effective factor of growth in this case. It may be caused by differential concentration of free CO2 in the medium. On the basis of previous researchers studies, it is indicated that carbonate and carbon dioxide can both pass into cells and be utilized by Chlorella. And carbon dioxide can more easily transfer into cells through membrane [17–18]. From this work, it is found that strain CS-01 can survive at a wide range of pH from 5 to above 10, and the growth of strain CS-01 can be promoted when simulated flue gas containing 5% to 15% of CO2 are supplied, indicating a potential application of strain CS-01 to treatment of flue gases of typical power plants that normally emit flue gases containing 5%–15% of CO2. 3.2 Effect of simulated flue gases on lipids content, lipids productivity and fatty acids composition of Chlorella sorokiniana CS-01 The total lipids content and lipids productivity are shown in Fig. 4. The results show that the total lipids content, described as percentage of dry mass, of strain CS-01 ranged from 28% for the control to 43% for the simulated flue gases containing 15% CO2, which was higher than the reported lipids content of Chlorella (25%–32%) [6]. According to Fig. 4, the total lipids content in Chlorella CS-01 increased under the aeration of simulated flue gases with higher levels of CO2. TANG et al [16] also found that the total lipids content increased with the increase of CO2 contents from 0.03% to 50%. Lipids productivity increased with the increase in CO2 contents, such as 5% and 10% CO2 in the flue gases, and decreased in higher CO2 content (15% CO2). It is indicated that CO2 in flue gases was an important factor for lipids accumulation, and increasing CO2 content within a proper degree could significantly promote lipid production of Chlorella CS-01. The profiles of fatty acids and fatty acid methyl esters (FAMEs) are listed in Tables 4 and 5, respectively. The results indicate that trans-esterification of fatty acids yielded over 60% of FAMEs, and more than 72% of fatty acids of strain CS-01 were main short-chain fatty acids (C 14 –C 18 ), these short-chain fatty acids have been

J. Cent. South Univ. (2013) 20: 730736

Fig. 4 Dry mass of biomass, total lipids masss fraction and total lipids productivity of Chlorella. Sorokiniana CS-01 under aeration of simulated flue gases containing different contents of CO2

reported to be favourable for biodiesel production [7]. The unsaturated fatty acids were the main profiles for strain CS-01 (>55%), suggesting that the biodiesel derived from the biomass of this microalgae would have low viscosity. The unsaturated, especially polyunsaturated fatty acid methyl esters have lower melting temperature, which were of vital importance for the improvement of low-temperature properties of biodiesel [19]. These results suggested that Chlorella CS-01 has great potential as a feedstock for biodiesel production. 3.3 Effect of simulated flue gases on expression of three genes related to CO2 fixation and fatty acid synthesis of Chlorella sorokiniana CS-01 It was observed that different kinds of simulated flue gases have a significant influence upon carbon dioxide fixation and lipids accumulation, meanwhile, the highest amount of the total lipids content was observed in the cultures under aeration of simulated flue gases containing 15% CO2, and the highest amount of biomass and the highest lipids productivity were observed in the cultures under aeration of simulated flue gases containing 5% and 10% CO2, respectively. Thus, the differential expressions of three genes related to CO2 fixation and fatty acid synthesis (rbcL, accD and acc1) were studied under aeration of the simulated flue gases containing 5%–15% of CO2, with aeration of air as the control. As listed in Table 6, the expression of accD in strain CS-01 was up-regulated significantly when the cultures were aerated with flue gases containing 10% and 15% of CO2, but little or no effect was found in the case aerated with flue gas containing 5% of CO2. Expression of accD

735

J. Cent. South Univ. (2013) 20: 730736

Table 4 Fatty acid compositions of Chlorella sorokiniana CS-01(percentage of total fatty acids) grown under aeration of simulated flue gases containing different contents of CO2(%)

(CO2)=0.02±0.01

 (CO2)= 5.00±0.05

 (CO2)= 10.00±0.05

 (CO2)=15.00±0.05

(Control)

(Flue gas 1)

(Flue gas 2)

(Flue gas 3)

C12:0

0.20±0.00

0.20±0.00

0.80±0.10

1.20±0.10

C12:1

0.00

0.00

0.00

0.00

C12:2

0.00

0.00

0.00

0.00

Parameter

C14:0

5.2± 0.63

5.90±0.68

7.90±0.72

10.80±0.92

C14:1

12.00±2.12

11.30±1.30

12.40±1.22

10.60±0.86

C14:2 C16:0 C16:1

0.00 6.40±1.00 0.00

0.00 2.30±0.98 0.00

0.00 6.20±0.64 0.00

0.00 6.20±1.20 0.00

C16:2

18.20±2.34

21.30±3.83

13.00±1.22

25.80±4.50

C18:0

16.00±0.09

18.00±1.64

19.00±3.50

10.70±1.22

C18:1

0.00

0.00

0.00

0.00

C18:2

0.00

0.00

0.00

0.00

C18:3

32.90±2.89

35.00±4.69

33.00±2.65

8.10±0.97

C20:1

3.20±0.05

2.20±0.09

3.80±0.00

10.90±1.29

C20:2

0.00

0.00

0.00

0.00

C20:5

0.00

0.00

0.00

0.00

C22:0

0.00

0.00

0.00

0.00

C22:6

0.00

0.00

0.00

0.00

Others

5.2±0.00

3.80±0.29

3.90±0.55

15.70±3.50

Table 5 Comparison of contents of saturated, unsaturated fatty acids to total fatty acids and contents of FAMEs to total lipids of Chlorella. sorokiniana CS-01 under aeration of simulated flue gases containing different contents of CO2. Flue gas (CO2)/%

w(SFA)/%

w(UFA)/%

w(Yield of FAMEs) /%

0.02±0.01 (Control)

27.70±1.75

66.30±7.80

62.25±5.80

5.00±0.05 (Flue gas 1)

26.40±3.30

69.80±9.90

64.00±2.45

10.00±0.05 (Flue gas 2)

33.90±4.75

62.20±5.09

70.21±3.26

15.00±0.05 (Flue gas 3)

28.90±3.44

55.40±7.55

68.70±1.90

SFA—Saturated fatty acids; UFA—Unsaturated fatty acids; FAMEs—Fatty acid methyl esters

Table 6 Differential expressions of genes related to lipid synthesis and CO2 fixation in Chlorella sorokiniana CS-01 grown in culture aerated with simulated flue gases containing different levels of CO2 Genes

accD

acc1

rbcL

(CO2)/%

Culture time/h 2

4

6

0.02±0.01

1.00

1.00

1.00

5±0.05

1.20±0.04

0.82±0.00

0.83±0.01

10±0.05

5.02±0.02

5.45±0.05

5.6±0.03

15±0.05

6.42±0.23

7.42±0.00

6.60±0.13

0.02±0.01

0.00

0.00

0.00

5±0.05

0.00

0.00

0.00

10±0.05

0.00

0.00

0.00

15±0.05

0.00

0.00

0.00

0.02±0.01

1.00

1.00

1.00

5±0.05

3.15±0.00

4.48±1.14

3.13±0.14

10±0.05

4.25±0.05

6.60±0.00

8.45±0.40

15±0.05

0.87±0.08

1.00±0.00

2.08±0.11

736

J. Cent. South Univ. (2013) 20: 730736

is crucial to the amount of heteromeric ACCase, thus one of the key enzymes was required for the synthesis of fatty acids. As the lipids content increased with the increase of the content of CO2 in simulated flue gases (Fig. 4), it suggested that the expression level of accD might be used to estimate lipid content. The expression of acc1 gene was not detected in all experiments (Table 6). The acc1 gene, encoding homomeric ACCase, is responsible for the synthesis of long-chain fatty acids, flavonoids, and anthocyanins in the cytosol, and it could replace heteromeric ACCase in chloroplast in some plants. It suggests that lipids might be mainly synthesized by heteromeric ACCase in strain CS-01. This phenomenon was also observed when strain CS-01 was cultured under mixotrophy and heterotrophy [8]. The rbcL gene encodes the catalytic large subunit of RuBisCO, which is an enzyme required to catalyze carbon fixation in the first reaction of the Calvin cycle, which is often the rate-limiting step for photosynthesis [10, 20]. Table 6 shows that when flue gases containing 5% and 10% of CO2 were respectively ventilated, the expression of rbcL gene was significantly up-regulated, compared to that of control. Significant up-regulation in the expression of rbcL gene was obtained after 6 h of cultivation in the case of aeration with flue gas containing 15% of CO2. These results are in consistent with the trends of growth and CO2 fixation as shown in Fig. 2.

4 Conclusions 1) Chlorella sorokiniana CS-01 has a good adaptation to a wide range of pH and a good utilization of 5%–15% of CO2 in the simulated flue gases. 2) The maximal CO2 biofixation rate and lipids productivity were obtained under aeration of simulated flue gases containing 10% CO2. Higher contents of CO2 in the simulated flue gases could lead to higher content of the total lipids, but decrease in lipid productivity because of obvious decrease in biomass. 3) All the results indicated that Chlorella CS-01 has a great potential for CO2 mitigation and biodiesel production.

[4]

GOUVEIA L. Microalgae as a raw material for biofuels production [J]. Journal of Industrial Microbiology and Biotechnology, 2009, 36(2): 269–274.

[5]

YOO C, JUN S Y, LEE J Y, AHN C Y, OH H M. Selection of microalgae for lipid production under high levels carbon dioxide [J]. Bioresoure Technology, 2010, 101(1): 71–74.

[6]

LV J M, CHENG LH, HUA X, ZHANG L, CHEN HL. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions [J]. Bioresource Technology, 2010, 101(17): 6797–6804.

[7]

CHISTI Y. Biodiesel from microalgae [J]. Biotechnology Advances,

[8]

WAN MX, LIU P, XIA J L, ROSENBERG J N, OYLER G A,

2007, 25(3): 294–306. BETENBAUGH M J, NIE Z Y, QIU G Z. The effect of mixotrophy on microalgal growth, lipid content,and expression levels of three pathway genes in Chlorella sorokiniana [J]. Applied Microbiology and Biotechnology, 2011, 91(3): 835–844. [9]

KUHL A, LORENZEN H. Handling and culturing of Chlorella [M].

[10]

de MORAIS, M G, COSTA J A. Biofixation of carbon dioxide by

New York: Academic Press, 1964: 152–187. Spirulina sp. and Scenedesmus obliquus cultivated in a three-stage serial tubular photobioreactor [J]. Journal of Biotechnology, 2007. 129(3): 439–445. [11]

LEE J Y, YOO C, JUN S Y, AHN C Y, OH H M. Comparison of several methods for effective lipid extraction from microalgae [J]. Bioresoure Technology, 2010, 101(1): 75–77.

[12]

CRONAN J E. Multi-subunit acetyl-CoA carboxylases [J]. Progress in Lipid Research, 2002, 41(5): 407–435.

[13]

IIDA S, MIYAGI A, AOKI S, ITO M, KADONO Y, KOSUGE K. Molecular adaptation of rbcL in the heterophyllous aquatic plant Potamogeton [J]. PLoS One, 2009, 4(2): 4633.

[14]

SASAKI Y, NAGANO Y. Plant acetyl-CoA carboxylase: Structure, biosynthesis, regulation, and gene manipulation for plant breeding [J]. Bioscience,

Biotechnology

and

Biochemistry,

2004,

68(6):

1175–1184. [15]

KENNETH J L, THOMAS D S. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCt method [J]. Methods, 2001, 25(4): 402–408.

[16]

TANG D H, HAN W, LI P L, MIAO X L. CO2 biofixation and fatty acid

composition

of

Scenedesmus

obliquus

and

Chlorella

pyrenoidosa in response to different CO2 levels [J]. Bioresoure Technology, 2011, 102(3): 3071–3076. [17]

SORENSEN B H, NYHOLM N, BAUN A. Algal toxicity tests with volatile and hazardous compounds in Air-tight test flasks with CO2 enriched headspace [J]. Chemosphere, 1996, 32(8): 1513.

[18]

WIDJAJA A, CHIEN C C, JU Y H. Study of increasing lipid production from freshwater microalgae Chlorella vulgaris [J].

References

Journal of the Taiwan Institute of Chemical Engineers, 2009, 40(1): 13–20.

[1]

ALPER H, STEPHANOPOULOS G. Engineering for biofuels:

[19]

[3]

“Designer”

biodiesel:

Optimizing

fatty

ester

22(2): 1358-1364.

potential [J]. Nature Reviews: Microbiology, 2009, 7(10): 715–723. DEMIRBAS A. Use of algae as biofuel sources [J]. Energy

G.

composition to improve fuel properties [J]. Energy and Fuels, 2008,

Exploiting innate microbial capacity or importing biosynthetic [2]

KNOTHE

[20]

RATLEDGE C, WYNN J P. The biochemistry and molecular biology

Conversion and Management, 2010, 51(12): 2738–2749.

of lipid accumulation in oleaginous microorganisms [J]. Advances in

WANG B Y, LI N, LAN C Q. CO2 bio-mitigation using microalgae

Applied Microbiology, 2002, 51(1): 1–44.

[J]. Applied Microbiology and Biotechnology, 2008, 79(5): 707–718.

(Edited by DENG Lü-xiang)