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African Journal of Microbiology Research Vol. 6(7), pp. 1489-1496, 23 February, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.

1349 ISSN 1996-0808 2012 Academic Journals

Full Length Research Paper

Enhancement of biomass and hydrocarbon productivities of Botryococcus braunii by mixotrophic cultivation and its application in brewery wastewater treatment
Wei-Bao Kong1,2*, Hao Song2, Shao-Feng Hua2, Hong Yang1, Qi Yang1 and Chun-Gu Xia2
2

College of Life Science, Northwest Normal University, Lanzhou, 730070, China. State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, 730000, China.
Accepted 9 December, 2011

Successful production of mixotrophic algae allows the integration of both photosynthetic and heterotrophic components during the diurnal cycle. This reduces the impact of biomass loss during dark respiration and decreases the amount of organic substances utilization during growth; these features infer that mixotrophic production can be an important part of the microalgae to biofuels process. Mixotrophic mode provides an effective way to cultivate Botryococcus braunii with high cell density and short cultivation cycle because exogenous carbon sources, in especial, organic carbon sources, such as sodium acetate, glucose and sucrose can stimulate the biomass production of the alga remarkably. The biomass and hydrocarbon volumetric productivities were promoted significantly by the mixotrophic cultivation of B. braunii compared with the photoautotrophic group, even though the hydrocarbon contents under mixotrophic conditions were not increased in pace with the biomass contents. The present study suggested that coupling the cultivation of mixotrophic microalgae and organic wastewater treatment is a potential way to produce microalgae biomass, accumulate hydrocarbon and remove organic and inorganic salts. Key words: Botryococcus braunii, mixotrophic cultivation, growth characteristics, biomass production, hydrocarbon accumulation, brewery wastewater treatment.

INTRODUCTION Microalgal cells can trap light energy as the energy source and assimilate CO2 as the carbon source; moreover, organic substrates can also be utilized as the carbon and energy sources by many microalgae (Yang et al., 2000). Many algal organisms are capable of using either metabolism process (autotrophic or heterotrophic) for growth, meaning that they are able to photosynthesis as well as ingest prey or organic materials (Zhang et al., 1999). Growth is influenced by the media supplement with glucose during the light and dark phases; hence, there is less biomass loss during the dark phase (Andrade and Costa, 2007). Chojnacka and Noworyta (2004) compared Spirulina sp. growth in photoautotrophic, heterotrophic and mixotrophic cultures. They found that mixotrophic cultures reduced photoinhibition and improved growth rates over both autotrophic and heterotrophic cultures. Successful production of mixotrophic algae allows the integration of both photosynthetic and heterotrophic components during the diurnal cycle. This reduces the impact of biomass loss during dark respiration and decreases the amount of organic substances utilization during growth. These features infer that mixotrophic production can be an important part of the microalgae-to-biofuels process (Brennan and Owende, 2010).

*Corresponding author. E-mail: kwbao@163.com or cgxia@lzb.ac.cn. Tel: +86 931 7971414. Fax: +86 931 7971207.

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Table 1. Carbon sources employed for the autotrophic and mixotrophic cultivation of B. braunii.

Code 1 2 3 4 5 6

Carbon sources Control NaHCO3 CH3COONa Na3-Citric acid Glucose Sucrose

Content (g/L) 1 1 1 1 1

Code Control SA10 SA20 SA40 SA80 SA100

Carbon sources Air

Sodium acetate

Content (mM) 10 20 40 80 100

Code Control G1 G5 G10 G15 G20

Carbon sources Air

Glucose

Content (g/L) 1 5 10 15 20

The green alga Botryococcus braunii has high hydrocarbon content, ranging from 15 to 75% of dry weight, as long chain unsaturated hydrocarbons (Sawayama et al., 1994). This characteristic has attracted increasingly more attention in the last two decades in attempts to exploit B. braunii for renewable biofuel (Kojima and Zhang, 1999). Many studies have been conducted to determine the culture conditions for B. braunii biomass production and hydrocarbon accumulation (Metzger and Largeau, 2005). Recently, the impact of carbon, nitrogen, phosphorus and other nutrients on the growth of B. braunii CHN 357 have been studied (Wang et al., 2003; Yang et al., 2004a) and the growth and lipid content of three B. braunii strains from China, United Kingdom and Japan were also compared at three temperatures (20, 25 and 30 C), three 2 irradiances (60, 100 and 300 Wm ) and four salinities (0, 0.15, 0.25, and 0.5 M NaCl), the study showed that the environmental requirement varied among the three B. braunii strains (Li and Qin, 2005). The effect of nitrogen limitation in a medium on the composition of intracellular lipids in the alga B. braunii Ktz IPPAS H-252 indicated that under nitrogen limitation, the alga cells accumulated lipids in the form of oleic acid-rich triacylglycerols (Zhila et al., 2005); however, its industrial cultivation has not been realized due to the economical and technical barriers, especially its slow growth rate and low biomass concentration (Yang et al., 2004b; Banerjee et al., 2002). In this study, the effects of carbon sources, sodium acetate and glucose content on the biomass production and hydrocarbon accumulation of B. braunii under mixotrophic cultivation were investigated and particularly, the growth characteristics and hydrocarbon production of these green algae in brewery wastewater medium were analyzed.

illumination (2500 lux; 12:12 h light:dark cycle) and shaking under 100 rpm in an illumination incubator for 15 days. For mixotrophic culture, the medium was supplemented with different inorganic and organic carbon sources (Table 1). B. braunii was also cultured in untreated BWW (brewery wastewater) medium (obtained from Gansu Resources Breweries Co., Ltd., pH=7.6), or BWW supplied with 0.50 g/L potassium nitrate as the nitrogen source (triplicate were performed for both cultures), respectively. Algal stock culture was inoculated to the medium to give a 10% (v/v) concentration. All solutions were autoclaved at 121 for 20 min prior to use. C

Determination of algal growth and biomass concentrations The cultures were harvested and the cells were washed with distilled water after centrifugation at 5000 rpm for 10 min; then the pellet was dried at 60 for 48 h to give the dry cell weight. The dry C weight of algal biomass was determined gravimetrically and growth was expressed in terms of dry weight. At the end of each run, specific growth rate (, day-1) of B. braunii at the exponential phase was calculated according to the equation: = (lnXt lnX0) / (tx t0), The biomass doubling time (G, day) calculated using: G = (ln 2) / , The maximum biomass concentration (Xmax, mg/l) was recorded and the productivity (P, mg/L/day) calculated from the equation: P = (XtX0) / (txt0), Where Xt and X0 are the dry cell weight concentration (mg/L) at time tx and t0, respectively (Andrade and Costa, 2007). Hydrocarbon extraction and determination Dried algal biomass was homogenized and pestle with n-hexane for 30 min and centrifuged. The supernatants were taken into preweighed glass vial. The extraction process was repeated three times and the solvents were pooled and evaporated under 40 C, then the glass vial was dried completely at 70 Th e quantity of C. residue was measured gravimetrically (Dayananda et al., 2005).

MATERIALS AND METHODS Strain and cultivation B. braunii, obtained from the Culture Collection of Algae from the Institute of Hydrobiology (Chinese Academy of Sciences), was grown on Chu 13 2 medium (Yamaguchi et al., 1987). Cultivation of B braunii was carried out in 500 ml Erlenmeyer flasks containing 150 ml Chu 13 2 medium (pH 7.2) at 25 2 with light C

Carbohydrate estimation The occurrence of dissolved polysaccharides in BWW and the spent medium was checked by phenol-sulphuric acid method (Rao and Pattabiraman, 1989).

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0.28 0.24 0.20

Biomass (g/L)

Control Sodium bicarbonate Sodium acetate Sodium citrate Glucose Sucrose

0.16 0.12 0.08 0.04 0.00 0 3 6 9 12 15

Culture time (days)


Figure 1. Effect of carbon sources on the growth and the biomass content of B. braunii.

Table 2. Effect of carbon sources on the growth parameters (, G, Xmax, P) of B. braunii

Parameters -1 , day G, day Xmax, mg/L P, mg/L/day

Control a 0.2220.008 e 3.130.11 a 22.332.52 a 1.520.18

NaHCO3 b 0.2500.004 d 2.780.05 a 33.002.00 a 2.290.14

CH3COONa d 0.3400.001 b 2.040.01 c 117.331.53 c 8.310.11

Na3-Citric acid c 0.3120.002 c 2.220.02 b 79.332.52 b 5.600.18

Glucose f 0.6130.004 a 1.130.01 e 250.0010.00 e 17.790.71

Sucrose e 0.5950.004 a 1.170.01 d 211.007.00 d 15.000.50

Values are mean SD, n=3; mean values in the same line with different letters in the superscript are significantly different (p<0.05).

Nitrite analysis Nitrite was determined according to the standard methods for the examination of water and wastewater (Rand et al., 1975). Statistical analysis Data were expressed as mean standard deviation (SD) from three independent parallel experiments. The analysis of variance was performed by ANOVA and significant differences among the means of samples were analyzed by Turkeys test with a 95% confidence level.

RESULTS AND DISCUSSION Effect of carbon sources and content on the biomass and hydrocarbon production of B. braunii To determine the utilization of carbon source types, the effect of different inorganic and organic carbon sources on the growth of B. braunii were investigated. B. braunii was grown in Chu 13 2 medium containing the different

carbon sources at 1.0 g/L concentration, under an irradiance of 2500 Lux. Over the course of each culture, the biomass concentrations were determined. The experimental results are summarized in Figure 1 and Table 2; the algal cells specific growth rates and biomass concentrations were significantly enhanced by the addition of glucose, sucrose, sodium acetate, and sodium citrate at 1.0 g/L concentration, respectively, but except to sodium bicarbonate. Furthermore, the cultures added with glucose and sucrose reached exponential and stationary phases early. The specific growth rates, maximum biomass concentrations and productivities of B. braunii cultured under supplemented with glucose, sucrose, sodium acetate, sodium citrate and sodium bicarbonate were much higher than that of the control, meanwhile, the doubling times were shortened by the addition of carbon sources. The facilitation effects caused by organic carbon sources were higher than inorganic carbon sources. The maximum biomass concentration and productivity of B. braunii were 250 mg/L and 17.79 mg/L/day for cells cultured under glucose concentration of 1.0 g/L, while 22.33 mg/L and 1.52 mg/L/day for control

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0.20 0.16

Biomass (g/L)

0.12 0.08 0.04 0.00 0

Control 10 mM 20 mM 40 mM 80 mM 100 mM

12

15

18

Culture time (days)


Figure 2. Effect of sodium acetate content on the growth and biomass content of B. braunii.

Table 3. Effect of sodium acetate content on the growth parameters (, G, Xmax, P) of B. braunii.

Parameters -1 , day G, day Xmax, mg/L P, mg/L/day

Control a 0.1910.004 d 3.630.07 a 42.332.52 a 2.520.16

SA10 e 0.2810.001 a 2.470.01 e 178.332.52 e 11.020.16

SA20 d 0.2690.002 b 2.580.02 d 147.674.51 d 9.100.28

SA40 cd 0.2650.002 b 2.620.02 c 138.003.61 c 8.500.23

SA80 bc 0.2590.001 bc 2.680.01 b 125.672.08 b 7.730.13

SA100 b 0.2550.002 c 2.720.02 b 118.673.06 b 7.290.19

Values are mean SD, n=3; mean values in the same line with different letters in the superscript are significantly different (p<0.05).

cells culture. These results indicated that the B. braunii can utilize variety of carbon sources, especially, the organic carbon sources such as saccharide and acetate better than the inorganic carbon sources. To investigate the effect of the organic carbon source content on the growth behaviour of B. braunii, sodium acetate was added in Chu 13 2 medium containing different concentration (from 10 to 100 mM). The laboratory findings are showed in Figure 2 and Table 3; in all cultures, the algal cells specific growth rates and biomass concentrations were significantly promoted by the addition of sodium acetate from 10 to 100 mM compared with the control group. The maximum specific growth rate, biomass concentration and productivity of -1 0.281 day , 178.33 mg/L and 11.02 mg/L/day were obtained at 10 mM (0.82 g/L), respectively. The higher sodium acetate concentrations (20-100 mM) at the initial culture period resulted in drastic lowering in biomass concentrations and growth rates, which might be due to the inhibition of high substrate concentration and the rising of pH values in culture media. The results from Figure 1 and Table 2 indicated that

glucose was the best carbon source for B. braunii growth, thus the influence of glucose content (from 1 to 20 g/L) on the biomass production and growth parameters of B. braunii were investigated. As shown in Figure 3 and Table 4, the supplement of glucose could enhance the B. braunii growth. The algal cells reached stationary phase -1 rapidly and obtained the specific growth rate (0.336 day ) and productivity (9.69 mg/L/day) under the initial glucose content in medium at 1 g/L. The growth of B. braunii was vigorous and strong at the glucose content from 5 to 15 g/L, but the results indicated that there were no significant differences among the growth parameters (, G, Xmax, P) at p<0.05 level. The specific growth rates, maximum biomass contents and productivities of B. braunii under mixotrophic cultivation (supplement with glucose and illumination) were higher than the values of control (0.201 -1 day , 25 mg/L, 1.23 mg/L/day, respectively), while, the doubling times of the algal cells were shortened by the addition of glucose. However, higher glucose content (20 g/L) resulted in cell growth inhibition of B. braunii, with specific growth -1 rate of 0.345 day and maximum biomass concentration

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0.30 0.25

Biomass (g/L)

0.20 0.15 0.10 0.05 0.00 0

Control 1 g/L 5 g/L 10 g/L 15 g/L 20 g/L

12

15

18

Culture time (days)


Figure 3. Effect of glucose content on the growth and biomass content of B. braunii.

Table 4. Effect of glucose content on the growth parameters (, G, Xmax, P) of B. braunii.

Parameters -1 , day G, day Xmax, mg/L P, mg/L/day

Control a 0.2010.005 b 3.450.09 a 25.002.00 a 1.230.16

G1 b 0.3360.001 a 2.060.01 b 215.334.51 b 9.690.83

G5 c 0.3510.001 a 1.970.01 d 275.334.51 b 14.812.52

G10 c 0.3510.001 a 1.970.01 d 276.335.13 b 15.382.09

G15 c 0.3500.002 a 1.980.01 d 272.006.56 b 15.292.06

G20 b 0.3450.001 a 2.010.01 c 248.004.00 b 13.561.69

Values are mean SD, n=3; mean values in the same line with different letters in the superscript are significantly different (p<0.05).

Table 5. Biomass and hydrocarbon content and productivity of B. braunii under photoautotrophic and mixotrophic cultivation with different carbon sources.

Parameters Biomass, g/L Biomass productivity, mg/L/day Hydrocarbon, % Hydrocarbon productivity, mg/L/day

Control (Chu 13 2) a 0.120.02 a 7.781.02 c 19.850.58 a 1.550.22

Sodium acetate (5 g/L) b 0.280.03 b 18.891.68 b 14.581.00 b 2.770.43

Glucose (5 g/L) d 0.580.02 d 38.671.33 a 11.030.80 c 4.260.16

Sucrose (5 g/L) c 0.510.03 c 33.781.68 a 9.141.00 b 3.080.19

Values are mean SD, n=3; mean values in the same line with different letters in the superscript are significantly different (p<0.05).

of 248 mg/L. Above results suggested that glucose is more suitable for B. braunii culture with the purpose of high density. The results also indicated that stepwise addition of glucose can avoid the inhibitory action of high substrate content at the initial culture period. The influence of cultivation modes (photoautotrophic and mixotrophic) and glucose content on the biomass production and hydrocarbon content and productivity of B. braunii were investigated. The cultures were carried out in 500-ml Erlenmeyer flask with shaking at 100 rpm,

25C, light flux density 2500 Lux, light-dark cycle 12 h /12 h, inoculation at 10% (v/v), and cultured 15 days. As shown in Table 5, the biomass concentrations were significantly enhanced by the addition of glucose, sodium acetate and sucrose compared with control (0.12 g/L), and the maximum values of 0.28, 0.58 and 0.51 g/L were obtained under the carbon sources contents at 5 g/L, respectively; however, the hydrocarbon contents for B. braunii under mixotrophic cultivation were not grew in pace with the biomass enhancement. The hydrocarbon

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Table 6. Effects of glucose content on the biomass and hydrocarbon content and productivity of B. braunii under mixotrophic cultivation.

Parameters Biomass, g/L Biomass productivity, mg/L/day Hydrocarbon, % Hydrocarbon productivity, mg/L/day

0 a 0.150.02 a 10.221.02 c 17.951.01 a 1.840.29

1 a 0.340.03 a 22.441.68 b 14.790.47 b 3.310.15

Glucose content (g/L) 5 10 b c 1.060.10 1.500.09 b c 70.676.67 100.225.67 ab ab 12.630.60 12.461.20 c d 8.900.42 12.440.49

15 bc 1.190.17 bc 79.5611.00 a 11.791.44 c 9.270.17

20 b 1.160.18 b 77.3312.00 a 11.761.22 c 9.000.48

Values are mean SD, n=3; mean values in the same line with different letters in the superscript are significantly different (p<0.05).

content of control (19.85%) was higher than the mixotrophic groups, and the culture added with sucrose content at 5 g/L obtained the lowest hydrocarbon content of 9.14 %; However, taking into account the productivity of biomass and hydrocarbon, the mixotrophic cultivation displayed its superiority. The biomass and hydrocarbon productivities were highly enhanced by the mixotrophic cultivation compared with the control of 7.78 and 1.55 mg/L/day, respectively. The maximum productivity of biomass (38.67 mg/L/day) and hydrocarbon (4.26 mg/L/day) were obtained at 5 g/L glucose content. As shown in Table 6, the addition of glucose in Chu 13 2 medium could enhance the growth of B. braunii. The biomass contents were increased by the glucose concentrations in cultures, and the maximum value of 1.50 g/L was obtained at 10 g/L glucose concentration. However, higher glucose concentration (15 to 20 g/L) could inhibit the biomass rising. While, the hydrocarbon contents of B. braunii were not increased in the same pace with the supply of glucose. On the contrary, addition of glucose reduced the accumulation of hydrocarbon in the algal cells. The hydrocarbon content was decrease to 11.76% at the 20 g/L glucose concentration compared with the control (17.95%); however, the productivity of biomass and hydrocarbon in cultures supplied glucose were also displayed the advantages of mixotrophism. Compared with the control group, the biomass and hydrocarbon productivities were significantly promoted by the mixotrophic cultivation supplemented with glucose. The maximum productivity of biomass (100.22 mg/L/day) and hydrocarbon (12.44 mg/L/day) were obtained at 10 g/L glucose concentration. Thus, supply of a sufficient level of organic carbon source is required for high cell density culture and an increase in hydrocarbon production of B. braunii. Strategies to improve the microalgal biomass production for reducing the cost of cultivation involved mixotrophic, photoheterotrophic or heterotrophic growth of algae. Mixotrophy and photoheterotrophy allow microalgal cells to synthesize simultaneously characteristic compounds of both heterotrophic and photosynthetic metabolisms at high production rates. Chen et al. (2006) reported that supplementation of acetate to the mixotrophic culture of Spirulina platensis

led to a significant enhancement in biomass concentration, chlorophyll a, lutein, -carotene, phycocyanin and allophycocyanin production when compared to the photoautotrophic culture, and the optimal acetate concentration of 4.0 g/L was tested. Although heterotrophic growth and photosynthesis has been reported to occur simultaneously and independently in mixotrophic Spirulina cultures (Marquez et al., 1993), the presence of organic carbon can alter both the photosynthetic and heterotrophic metabolism of Chlorella (Villarejo et al., 1995) and decreases production of photosynthetic pigments as compared with the amounts present in the absence of organic carbon source (Ogbonna and Tanaka, 1998). In the present study, the experiments indicated that the utilization variety of carbon sources for B. braunii and organic carbon sources such as saccharide and acetate were better than inorganic carbon sources for biomass production. The specific growth rates, maximum biomass concentrations and productivities of B. braunii cultured under mixotrophic conditions were much higher than that of autotrophic groups. The optimal sodium acetate concentration of 10 mM (0.82 g/L) for mixotrophic cultivation of B. braunii was obtained. The growth characteristics and the hydrocarbon content of B. braunii were very well under mixotrophic cultivation when the glucose content ranged from 5 to 20 g/L; although the biomass production not increased in pace in such condition. Thus, the productivity of biomass and hydrocarbon for B. braunii under mixotrophic cultivation indicated the superiority of mixotrophism.

Biomass production and hydrocarbon accumulation of B. braunii in BWW Figure 4 shows the growth and hydrocarbon production of B. braunii in BWW during batch culture for 20 days. As shown in Figure 4A, with an initial potassium nitrate concentration of 0.50 g/L, the algal growth was enhanced due to the appropriate concentration of nitrate. After 20 days of cultivation, a biomass of 0.109 g/L and hydrocarbon content of 9.36 mg/L obtained in crude BWW (Figure 4B). Dissolved polysaccharides concentration of 1,250 mg/L in crude BWW was removed by the algae and the final polysaccharides concentration was 135 mg/L

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0.24

27

A
Hydrocarbon content (mg/L)
0 4 8 12 16 20 0.20 0.16 0.12 0.08 0.04 0.00

24 21 18 15 12 9 6 3

Biomass (g/L)

12

16

20

Culture time (days)

Culture time (days)

Figure 4. Changes of biomass and hydrocarbon content of B. braunii in BWW. A: biomass, B: hydrocarbon, --untreated BWW, -addition of 0.50 g/L KNO3 in BWW.

after 20 days, showing that 89.20 % of the initial carbohydrate was utilized by microalgal cultivation. In BWW supplemented with nitrate, the final biomass of 0.205 g/L and hydrocarbon content of 24.44 mg/L were obtained, respectively (Figure 4A). Nitrate of 512.2 mg/L was utilized by the algae. The final nitrate concentration was 2.8 mg/L after 20 days, indicating that 99.46% of the initial nitrate was utilized by the microorganism; meanwhile, the results from the Figure 4B indicated that 97.76 % of the carbohydrate in BWW supplemented with potassium nitrate was utilized, which higher than the remove rate in crude BWW group (p<0.05). The productivity of biomass and hydrocarbon for B. braunii in the presence of nitrate (0.50 g/L) showed a 1.88-fold and 2.61-fold increase over that in BWW without any modification at the end of cultivation (20 days), respectively. The above results indicated that the untreated nitrogen-free BWW was not suitable for B. braunii culture directly. Wastewater treatment by microalgal culture has several major advantages: it rests on the principles of natural ecosystems and is therefore not environmentally hazardous; it causes no secondary pollution, as long as the biomass produced is reused; and it allows efficient recycling of nutrients (Martnez et al., 2000). B. braunii was able to grow well in secondarily treated sewage (STS) in a batch system. The growth in STS was as good as in the common artificial medium of modified Chu 13 and its hydrocarbon contents were high enough at 53 and 40% compared with 58% in the case of Chu 13 medium (Sawayama et al., 1992). Their results showed the possibility of using STS as a medium to grow B. braunii and for removal of nitrogen and phosphorus by algal consumption in STS. B. braunii

also grew well in piggery wastewater pretreated by a membrane bioreactor with acidogenic fermentation (An et al., 2003). A dry cell weight of 8.5 mg/L and hydrocarbon level of 0.95 g/L were obtained, and nitrate was removed at a rate of 620 mg/L. These results indicated that pretreated piggery wastewater provides a good culture medium for the growth and hydrocarbon production by B. braunii. The present case shown the algal cells grew better, produced hydrocarbons, and removed carbohydrates with the addition of 0.50 g/L potassium nitrate in crude BWW, which suggested the growth of B. braunii in BWW was limited to the availability of nitrogen source. These results also indicated good prospects for being able to cultivate B. braunii to reduce the amount of organic nutrients in BWW. Moreover, to improve hydrocarbons productivity, microalgae can be harvested and inoculated in nitrogenfree media, especially in the later phase of growth, resulting in the ccumulation of reserve hydrocarbons under optimal culture conditions (Su et al., 2011); although nitrogen-free conditions can reduce the algal biomass production. Thus, stepwise addition of nitrogen in fed batch cultivation can avoid the problems of growth restriction and hydrocarbons accumulation of algae. For inorganic and organic nutrients consumption, B. braunii can utilize soluble carbohydrate and nitrate effectively as long as it was cultured in BWW, which demonstrated that the productivity of biomass and hydrocarbons can be improved by cultivating the green algae in BWW supplied with appropriate level of nitrogen. The present work indicated that it is a potential way to cultivate B. braunii under mixotrophic conditions with the purposes of biomass production, hydrocarbons accumulation and

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reduction the amount of organic nutrients in BWW.

Conclusion Mixotrophic mode provides an effective way to cultivate B. braunii with high cell density and short cultivation cycle because exogenous carbon sources, in especial, organic carbon sources, such as sodium acetate, glucose and sucrose can stimulate the biomass production of the alga remarkably. The biomass and hydrocarbon volumetric productivities were promoted significantly by the mixotrophic cultivation of B. braunii compared with the photoautotrophic group, even though the hydrocarbon contents under mixotrophic conditions were not increased in pace with the biomass contents. The present study suggested that coupling the cultivation of mixotrophic microalgae and organic wastewater treatment is a potential way to produce microalgae biomass, accumulate hydrocarbon and remove organics and inorganic salts from BWW.

ACKNOWLEDGEMENTS Financial support was provided by National Science Found for Distinguished Young Scholars of China (Grant No. 20625308) and Research Fund for Young Teachers of Northwest Normal University (Grant No. NWNU-LKQN10-30).
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