Bioresource Technology 102 (2011) 101105

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Lipid production of Chlorella vulgaris cultured in articial wastewater medium

Yujie Feng a,*, Chao Li a, Dawei Zhang b
a b

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, No 73 Huanghe Road, Nangang District, Harbin 150090, PR China Department of Environmental Science & Engineering, Harbin Institute of Technology (Weihai), Weihai 264200, China

a r t i c l e

i n f o

a b s t r a c t
Chlorella vulgaris was used to study algal lipid production with wastewater treatment. Articial wastewater was used to cultivate C. vulgaris in a column aeration photobioreactor (CAP) under batch and semicontinuous cultivation with various daily culture replacements (0.5 l1.5 l per 2 l reactor). The cell density was decreased from 0.89 g/l with the daily replacement of 0.5 l to 0.28 g/l with 1.5 l replacement. However, C. vulgaris culture achieved the highest lipid content (42%, average value of the phase) and the lipid productivity (147 mg/l d1) with daily replacement of 1.0 l. And then the nutrient removal efciency were 86% (COD), 97% (NH 4 ) and 96% (TP), respectively. Analyses of energy efciency showed that the net energy ratio (NER) for lipid production with daily replacement of 1.0 l (1.25) was higher than the other volume replacement protocols. And cost analyses showed that the algal biomass can be competitive with petroleum at US$ 63.97 per barrel with the potential credit for wastewater treatment. According to the above results, it is concluded that the present research will lead to an economical technology of algal lipid production. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 31 March 2010 Received in revised form 1 June 2010 Accepted 2 June 2010

Keywords: Chlorella vulgaris Lipid content Articial wastewater Energy and cost analyses

1. Introduction Global demand for food is expected to double within 50 years, and the demand for transportation fuels is expected to increase even more rapidly (Hill et al., 2006). Diversion of food crops to biofuels would not be right approach to solve the problems because they compete with food production for high-grade arable land (Rittmann, 2008). There is a great need for renewable energy supplies that do not cause signicant environmental harm and not competed with food supply. Because of their higher photosynthetic efciency, higher biomass production and faster growth compared with other energy crops, microalgae have been receiving attentions as candidates for fuel production (Minowa et al., 1995). Microalgae can be used to produce various forms of biofuel including biodiesel (Converti et al., 2009; Gao et al., 2010), ethanol (Shirai et al., 1998), bioelectricity (Powell et al., 2009), hydrogen (Ghirardi, 2006; Hemschemeier et al., 2009), and methane (Stucki et al., 2009). Biodiesel is produced from plant oils or animal fats, and biodiesel industries are expanding rapidly both in the United States and in Europe with soybean or rapeseed oils as the feedstock. However, the potential market for biodiesel far surpasses the availability of plant oils, waste cooking oil and animal fats. Therefore, microalgae have been studied as alternative feedstock for biodiesel production
* Corresponding author. Tel.: +86 451 86283068; mobile: +13069891017; fax: +86 451 87162150. E-mail address: yujief@hit.edu.cn (Y. Feng). 0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.016

recently. Use of microalgae to produce biodiesel would not compromise production of food, fodder and other products derived from crops (Chisti, 2007). Many microalgae accumulate lipids as storage materials and their accumulation is stimulated under environment stress, such as nutrient deciency (Dunahay et al., 1996) or salt stress (Takagi et al., 2006). Widjaja et al. (2009) reported that maximum lipid content of Chlorella vulgaris was only 26% under normal nutrition medium with nitrogen (NaNO3) content of 70.02 mg/l. However, after normal nutrition cultivation, the medium was changed into nitrogen depletion(0.02 mg/l) continued for 7 d and 17 d, and the lipid contents were 36% and 43%, respectively. Furthermore, according to the results obtained by Converti et al. (2009), a threefold increase (from 5.9% to 15.3%) in lipid content took place with NaNO3 concentration decrease from 1.5 to 0.375 g/l. Hsieh et al. (2009) used urea as the nitrogen source at concentrations of 0.025, 0.050, 0.100, 0.150, and 0.200 g/l. After 6 days of cultivation, the lipid contents of Chlorella sp. were 66%, 60%, 52%, 37%, and 33% respectively. Microalgal biomass can be produced through autotrophic cultivation in open ponds or photobioreactors by using solar energy and xing carbon dioxide. Alternatively they are cultivated heterotrophically or mixotrophically using organic compounds as energy and carbon sources. Due to the reduction in light penetration (Chaumont, 1993) in autotrophic culture, the cell density is usually less than 1 g/l (Borowitzka, 1994). So far as we know, there is no effective cultivated method to increase cell density in the autotrophic cultivation processes. Therefore, the downstream

102

Y. Feng et al. / Bioresource Technology 102 (2011) 101105

processing costs are relatively high (Shi et al., 1997). For the heterotrophic or mixotrophic cultivation, organic carbon compounds such as glucose are responsible for higher production costs. Glucose used in this process comprises about 80% of the total costs (Li et al., 2007). In this work C. vulgaris was used to produce algal lipid using wastewater as medium. In order to simply investigate the factors related with algal lipid production during wastewater treatment, preliminary results were obtained here using synthetic wastewater instead of real wastewater. The use of wastewater as feedstock for algal lipid is economically attractive since the production costs can be reduced with credits for wastewater treatment as well as with reduction in the greenhouse gas emission. 2. Methods 2.1. Algal strain and culture medium C. vulgaris (FACHB1068) was purchased from Freshwater Algae Culture Collection, Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The strain was preserved in the BG11 medium containing following chemicals: NaNO3 (1.5 g/l), K2HPO43H2O (0.04 g/l), MgSO47H2O (0.075 g/l), CaCl22H2O (0.036 g/l), Na2CO3 (0.02 g/l), citric acid (0.006 g/l), Ferric ammonium citrate (0.006 g/l), EDTA (0.001 g/l), and A5 + Co solution (1 ml/l) that consists of H3BO3 (2.86 g/l), MnCl2H2O (1.81 g/l), ZnSO47H2O (0.222 g/l), CuSO45H2O (0.079 g/l), Na2MoO42H2O (0.390 g/l) and Co(NO3)26H2O (0.049 g/l). C. vulgaris was inoculated at 20% (v/v) in 250 ml Erlenmeyer asks containing 100 ml BG11 medium. The asks were incubated under stationary condition at 30 C with 3000 lx continuous cool-white uorescent light illumination, and were hand shaken three to ve times daily to avoid sticking. The algal cells which just reached the stationary phase were used to inoculate the column aeration photobioreactor (CAP). 2.2. Articial wastewater The articial wastewater was prepared dissolving following chemicals; glucose (0.4125 g/l), NH4Cl (0.078 g/l), KH2PO4 (0.018 g/l), MgSO47H2O (0.013 g/l), CaCl22H2O (0.043 g/l), FeSO7H2O (0.005 g/l), and A5 + Co solution (1 ml/l). The initial pH was adjusted to 7.08.0 and sterilized at 121 C for 20 min before inoculation. The initial N-NH4+, total phosphate (TP), and COD concentration were 20, 4, and 400 mg/l, respectively. 2.3. Reactor design and its operation Four 2.2 l CAPs were consructed with 2 l effective volume (10 cm diameter and 25 cm height) using polymethyl methacrylate (PMMA). The CAPs containing 1.5 l sterilized articial wastewater were inoculated with 0.5 l ask culture of C. vulgaris. The culture pH decreased due to NH4+ assimilation, which was observed in our previous study (data not shown). Therefore, the pH of culture was maintained between 8 and 10 during Day 2 to 14. All the experiments were carried out at 30 C and 3000 lx continuous cool-white uorescent light illumination. The reactors were aerated with sterilized air at 0.5 vvm (volumes of air per total volume of bioreactor per minute) to provide mixing and CO2, as well as O2 to the algae. Before the cultures reach stationary phase various volume was replaced daily with fresh medium to operate the reactors in the semi-continuous mode. When the cell density reached about 0.8 g/l on Day 4 after the inoculation, the culture was operated in the rst phase of semi-continuous cultivation for 3 d by replacing

0.5 l of the culture with fresh articial wastewater everyday. After that, the culture was operated in the second and third phases of semi-continuous mode by replacing 1.0 and 1.5 l of the culture with fresh medium everyday, respectively. Both these phases were maintained for 4 days each. The batch culture under the same condition except for medium replacement was used as positive control. It was operated for 14 days. All the experiments were carried out in duplicate and average values are reported. 2.4. Lipid extraction Algal cells were harvested by centrifugation at 10,000 rpm, 4 C for 10 min. Supernatant was decanted and cell pellets were washed with distilled water and then freeze-dried under 80 C. Thereafter, the total lipids were extracted from microalgal biomass using a modied method of Bligh and Dyer (1959). Fifty mg of lyophilized microalgal biomass was placed into a 15 ml test tube and 1.6 ml water, 4.0 ml methanol and 2.0 ml chloroform were added. The solution was mixed for 30 s. Thereafter, an additional 2.0 ml of chloroform and 2.0 ml water were added and the content of the test tube was mixed for 30 s. The test tubes were centrifuged at 5000 rpm for 10 min. The upper layer was withdrawn by using a pipette and the lower chloroform phase containing the extracted lipids was transferred into a 30-ml culture tube. The solid material left at the bottom of extraction tube was extracted with the same procedure two more times and the chloroform phases were mixed together and then evaporated in a nitrogen evaporator until obtaining dry lipid. Thereafter, the total lipids were measured gravimetrically, and then lipid content and lipid yields were calculated. 2.5. Analyses Samples were taken from CAPs each day for analyses. Optical density (OD) of the algae culture at 658 nm was measured daily as the cell density indicator using a spectrophotometer (752 Grating Spectrophotometer, Shandong Gaomi Caihong Analytical Instrument Factory, China). A linear relationship between OD658 and dry weight (DW, g/L) of algal biomass was determined previously for this strain:

Dry weight g=l 0:4818 OD658 ;

R2 0:9962

Samples were centrifuged at 10,000 rpm for 10 min to determine NH 4 , TP and COD concentrations in the supernatants. COD was determined by a Multi-Function Reactor (ET3150B Multi-Function Reactor, Euro Tech, China), Nash reagent photometry was used for measuring NH 4 concentration. TP was determined by molybdenumantimony anti-spectrophotometric method. For the energy analysis, the values of lipid content, cell density, and hydraulic retention time were obtained from the results of the semi-continuous cultivation. According to Jorqueras (2010) research, a production scale of 100 ton of algal biomass per year was set as the basis to calculate energy balance. And the energy consumption term included only the energy required for air pumping that was used to maintain appropriate culture mixing and liquid/gas mass transfer. Thereafter, volumetric productivity, reactor volume required for a biomass production of 100 ton/year, net lipid yield, energy consumption required for a biomass production of 100 ton/year, total energy consumption, energy produced as lipid, and NER for lipid production were calculated. Because the structure and the operational mechanism of CAP were similar to at-plate photobioreactor, the energy consumption of CAP was assumed equal to at-plate photobioreactor (53 W/m3).

Y. Feng et al. / Bioresource Technology 102 (2011) 101105

103

3.1. Algal growth and lipid content CAPs inoculated by the test organism were operated for 14 days in a batch mode monitoring algal growth and lipid content (Fig. 1). As shown in Fig. 1, the alga grew up to 7 days of cultivation to the cell density of 1.581.72 g/l with a lag phase of one day. On the other hand the lipid content increased to 37% from 15% in Day 2, and decreased to 7.8% during the growth phase. However, the lipid content increased to 34.1% in day 10 but did not increase further at the stationary phase. The high lipid content at Day 2 is probably because the culture was under heterotrophic/mixotrophic conditions. Organic carbon in the culture was quickly consumed and was exhausted on Day 2 (see Section 3.2), and then C. vulgaris switched from mixotrophic metabolism to autotrophic metabolism. It had been reported (Miao and Wu, 2004) that the autotrophic microalgae had low lipid content in comparison with those under heterotrophic and mixotrophic conditions. Therefore, the switch of metabolism on Day 2 resulted in the signicantly decrease of lipid content. The lipid content in the stationary phase was up to 37%. Li et al. (2008) reported that the nitrogen deciency would result in more metabolic ux generated from photosynthesis to be turned to lipid accumulation in Neochloris oleoabundans. The reason may be that under nitrogen deciency or limitations the synthetic rate of essential cell structures including proteins and nucleic acids becomes low. Therefore, the major part of carbon xed is converted into carbohydrate or lipid (Richardson et al., 1969). The articial wastewater used in these experiments contains only 20 mg/l nitro gen in form of N NH 4 . Although N NH4 was depleted on Day 3 (see Fig. 2c), the growth rate of algal cell was not limited signicantly. The reason may be that nitrate has been introduced to the culture at inoculation (The initial N NO 3 concentration in BG11 medium is 247 mg/l). Only ammonium is utilized when cultures containing both nitrate and ammonium (Ahmad and Hellebust, 1990). Therefore, ammonium was depleted rst in the culture, and then algal cell grew with nitrate as nitrogen source until nitrate was depleted. Thus, the lipid accumulation was not enhanced up due to most metabolic ux generated from photosynthesis was still used for cell synthesis from Day 3 to 8. After Day 8, the growth of algae was nearly ceased, thus resulting in the increased lipid synthesis. The relatively high lipid content at Day 1 is believed due to the fact that cells in this phase are similar to those in the stationary phase culture in BG11 medium which was used as the inoculums. CAPs was operated in semi-continuous mode at Day 4 by replacing different volume of the culture with fresh medium every day; 0.5 l (rst phase), 1.0 l (second phase) and 1.5 l (third phase). The cell density and lipid content were determined, and the lipid

300 200 100 0 0 2

80
semi-continuous batch removal efficiency in semi-continuous

60 40 20

6 8 10 12 Days of cultivation

14

16

3 2 1 0 0 2 4

semi-continuous batch removal efficiency in semi-continuous

80 60 40 20 0 16

6 8 10 12 Days of cultivation

14

20 15 10 5 0 0 2 4

semi-continuous batch removal efficiency in semi-continuous

80 60 40 20 16 0

6 8 10 12 Days of cultivation

14

Fig. 2. Removal efciency and mean concentration of nutrients for C. vulgaris growing in the batch and semi-continuous cultivation. (A) COD; (B) TP; (C) NH 4.

2 Cell density (g/l) 1.6 1.2 0.8 0.4 0 0 2 4 6 8 10 12 Days of cultivation 14

cell density lipid content

40 30 20 10 0 16 Lipid content (%)

productivity was calculated based on the results. As shown in Table 1, cell density decreased from 0.89 g/l in the rst phase to 0.28 g/l in the third phase with the increase in daily changed culture volume during the semi-continuous cultivation. As for the lipid content, it increased signicantly from 20% in the rst phase to 42% in the second phase, and then decreased slightly to 38% in the third phase. According to the results of cell density and lipid content, lipid productivity was calculated. The highest lipid productivity (147 mg/l d1) was achieved during the second phase compared with the rst (44 mg/l d1) and the third (79 mg/l d1) phases. The main reason for the reduction of cell density is due to the reduced algal cell retention time in the reactor with the increased daily changed volume. As discussed earlier, the lipid content is dependent on the nitrogen limitations and on the trophic conditions. Higher lipid content is expected in the cells facing nitrogen limitation under mixotrophic conditions. In a semi-continuous

Table 1 Cell density and lipid content of C. vulgaris in different phases of the semi-continuous cultivation. First Daily change medium (l/2 l reactor) Cell density (g/l) Lipid content (%) Lipid productivity (mg/l d1)
*

Second 1.0 0.69 42 147

Third 1.5 0.28 38 79

0.5 0.89 20 44

Fig. 1. Cell density and lipid content of C. vulgaris culture in the batch cultivation.

Cell density, lipid content and productivity were average value in the phase.

Removal efficiency (%)

+ NH4 concentrations (mgN/l)

25

100

Removal efficiency (%)

B
TP concentrations (mgP/l)

100

Removal efficiency (%)

3. Results and discussion

COD concentrations (mg/l)

400

100

104

Y. Feng et al. / Bioresource Technology 102 (2011) 101105 Table 2 Comparative analyses of algal biomass and lipid production in different phases of the semi-continuous cultivation. Variable Cell density (g/l) Hydraulic retention time (d) Volumetric productivity (g/l d1) Reactor volume required for a biomass production of 100 ton/yeara (m3) Lipid content (%) Net lipid yieldb (m3/year) Energy consumption (W/m3) Energy consumption required for a biomass production of 100 ton/yearc (W) Total energy consumptiond (GJ/year) Energy produced as lipide (GJ/year) NER for lipid production First 0.89 4 0.223 1246 20 22 53 66,038 2054.05 772.93 0.38 Second 0.69 2 0.346 803 42 47 53 42,550 1323.48 1651.27 1.25 Third 0.28 1.3 0.21 1323 38 42 53 70,119 2180.99 1475.60 0.68

culture system the nitrogen availability and trophic conditions are determined by the volume of daily changed medium. The lipid content was low in the rst phase with low volume change. This is believed due to the fact that organic carbon was not enough to support C. vulgaris grown in mixotrophic metabolism for long time to maintain a high lipid synthesis rate. And nitrate may have been introduced to the culture at inoculation, thus resulting in abundant nitrogen in culture in the rst phase. On the other hand the highest lipid content of 42% was achieved during the second phase of the semi-continuous cultivation. This result suggests that the culture was supplied with enough organic carbon to maintain mixotrophic metabolism with nitrogen limitation. However, further increase in daily changed volume during the third phase caused a slight decrease in lipid content (38%). These results suggest that the culture had abundant nutrient during the third phase with high changed volume, therefore, the algae grew vigorously and more assimilated organic carbon was used for cell growth. Thus, the lipid content obtained a slight decrease. 3.2. Nutrients removal efciency The culture supernatant was analyzed for COD, TP, and NH4+ to determine the process performance of the system (Fig. 2). As expected the nutrients removal efciency was poor at the beginning of the reactor operation (Day 01) due to low cell density (0.05 g/l). It is expected that the higher the cell density, the better the nutrient removal efciency (Lau et al., 1995). Thereafter, the removal efciency of nutrient achieved higher level during the growth phase, due to the higher cell density and vigorous growth. On Day 2 the removal efciencies of COD, TP, and NH4+ were 87%, 94%, and 90%, respectively. It was interesting to note that COD in the supernatant prepared from the batch cultivation was higher than those from the semi-continuous cultivation during Days 2 to 14, although the higher cell density and longer HRT in batch cultivation. The possible reason for this result is that C. vulgaris secretes extra cellular substances during the growth process (Babel et al., 2002; Paralkar and Edzwald, 1996), which was hardly degradable by the alga. In the semi-continuous cultivation, the extra cellular substances were removed by medium replacement. Therefore, the removal efciency of COD in semi-continuous cultivation increased from 85% to 88% along with the increasing daily changed medium. As for TP and NH4+, the batch cultivation had good removal efciency which was 96% and 97%, respectively. The removal efciency of TP in the third phase of semi-continuous cultivation was 92% that was lower than that of batch cultivation. This might be due to low cell growth. The removal efciency of NH 4 was high (97%) in the whole semi-continuous processes. 3.3. Energy and cost analysis The net energy ratio (NER) for lipid production was dened as the ratio of the energy produced as lipid over the total energy consumption. As shown in Table 2, the second phase not only achieved the highest value of energy produced as lipid, but also the lowest total energy consumption among the three phases. Therefore, the NER for lipid production in the second phase (1.25) was higher than the others. It suggests that the semi-continuous in the second phase was the most efciency energy production system among others. A large part of the production cost of algal lipid is downstream processing costs including cell harvest and lipid extraction costs that are dependent considerably on the cell density and lipid content (Li et al., 2008). High cell density reduces the cell harvesting cost, so does high lipid content to lipid extraction cost. Therefore, the downstream processing costs of algal lipid can be calculated based on the following equation:

a Determined by dividing the annual biomass production by the volumetric productivity. b Determined by dividing the product of annual biomass and lipid content by the density of lipid (assumed to be 0.9 kg/l). c Determined by multiplying the energy consumption by the reactor volume required. d Determined by multiplying the energy consumption by the number of hours of air pumping (it was 24 h of one day). e Determined by multiplying the net lipid yield by energy content of lipid (assumed value of 35, 133.33 kJ/l).

z x D y=D L

where z, downstream processing cost of algal lipid ($/g); x, algae harvesting cost ($/l), y, lipid extraction cost ($/g); D, cell density (g/l); L, lipid content (%). It was assumed that the harvesting cost is dependent on the volume of cultures, and that the extraction cost dependent on the weight of algae. Cell density and lipid content of each phase were used to calculate the downstream processing cost according to Equation (2). The downstream processing costs of algal lipid were 5.6x + 5y, 3.4x + 2.4y, and 9.5x + 2.6y for the rst, second, and third phases, respectively. Hence, the downstream processing of the second phase was the lowest among three phases. According to Chistis (2008) research, the algal biomass (lipid content of 42%) with the production costs of US$ 217.22/ton becomes competitive with petroleum at US$ 60.00 per barrel. Using a value of US$ 0.22/kWh for the energy consumption, the production cost of 1 ton algal biomass in the second phase was estimated to be US$ 808.79. To produce 1 ton algal biomass, 1443 m3 of wastewater is treated. If the credit for wastewater treatment at US$ 0.4/m3 is counted, the price of 1 ton of biomass would be reduced to US$ 231.59. This gure shows that algal biomass can be competitive when supposing the price of petroleum is US$ 63.97 per barrel. However, since the energy and cost analyses were carried out based on synthetic wastewater, the results could be different when using different real wastewater. 4. Conclusions The results in this study showed that microalgae cultivation with wastewater as medium is a promising method to produce algal lipid. The highest lipid content (42%) and productivity (147 mg/ l d1) were achieved in the semi-continuous cultivation with daily replacement of 1.0 l of the 2.0 l culture. And then the nutrient removal efciencies were 86% (COD), 97% NH 4 and 96% (TP), respectively. These results were used to analyze the energy efciency. The NER for lipid production (1.25) was greater than unity. And cost analysis exhibited that the algal biomass can be competitive with petroleum at US$ 63.97 per barrel with the potential credit for wastewater treatment. Furthermore, this process also reduces the greenhouse gas emission in wastewater treatment.

Y. Feng et al. / Bioresource Technology 102 (2011) 101105

105

Acknowledgements The research is supported by the Scientic Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China. The authors also acknowledge the support of the National Creative Research Groups of China (50821002) and the technical and nancial support of the State Key Laboratory of Urban Water Resource and Environment (2010TS08), HIT, China. Prof. Byung Hong Kim (Water Environment and Remediation Research Center, Korea Institute of Science and Technology) is gratefully thanked for his efforts on this paper. References
Ahmad, I., Hellebust, J., 1990. Regulation of chloroplast development by nitrogen source and growth conditions in a Chlorella protothecoides strain. Plant Physiology 94 (3), 944949. Babel, S., Takizawa, S., Ozaki, H., 2002. Factors affecting seasonal variation of membrane ltration resistance caused by Chlorella algae. Water Research 36 (5), 11931202. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purication. Canadian Journal of Biochemistry and Physiology 37 (8), 911917. Borowitzka, M.A., 1994. Large-scale algal culture systems: the next generation. Australasian Biotechnology 4 (4), 212215. Chaumont, D., 1993. Biotechnology of algal biomass production a review of systems for outdoor mass-culture. Journal of Applied Phycology 5 (6), 593604. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnology Advances 25 (3), 294 306. Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends in Biotechnology 26 (3), 126131. Converti, A., Casazza, A.A., Ortiz, E.Y., Perego, P., Del Borghi, M., 2009. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chemical Engineering and Processing 48 (6), 11461151. Dunahay, T.G., Jarvis, E.E., Dais, S.S., Roessler, P.G., 1996. Manipulation of microalgal lipid production using genetic engineering. Applied Biochemistry and Biotechnology 5758, 223231. Gao, C.F., Zhai, Y., Ding, Y., Wu, Q.Y., 2010. Application of sweet sorghum for biodiesel production by heterotrophic microalga Chlorella protothecoides. Applied Energy 87 (3), 756761. Ghirardi, M.L., 2006. Hydrogen production by photosynthetic green algae. Indian Journal of Biochemistry and Biophysics 43 (4), 201210. Hemschemeier, A., Melis, A., Happe, T., 2009. Analytical approaches to photobiological hydrogen production in unicellular green algae. Photosynthesis Research 102 (23), 523540.

Hill, J., Nelson, E., Tilman, D., Polasky, S., Tiffany, D., 2006. Environmental, economic, and energetic costs and benets of biodiesel and ethanol biofuels. Proceedings of the National Academy of Sciences of the United States of America 103 (30), 1120611210. Hsieh, C.H., Wu, W.T., 2009. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresource Technology 100 (17), 3921 3926. Jorquera, O., Kiperstok, A., Sales, E.A., Embirucu, M., Ghirardi, M.L., 2010. Comparative energy life-cycle analyses of microalgal biomass production in open ponds and photobioreactors. Bioresource Technology 101 (4), 14061413. Lau, P.S., Tam, N.F.Y., Wong, Y.S., 1995. Effect of algal density on nutrient removal from primary settled waste-water. Environmental Pollution 89 (1), 5966. Li, X.F., Xu, H., Wu, Q.Y., 2007. Large-scale biodiesel production from microalga Chlorella protothecoides through heterotropic cultivation in bioreactors. Biotechnology and Bioengineering 98 (4), 764771. Li, Y.Q., Horsman, M., Wang, B., Wu, N., Lan, C.Q., 2008. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Applied Microbiology and Biotechnology 81 (4), 629636. Miao, X.L., Wu, Q.Y., 2004. High yield bio-oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides. Journal of Biotechnology 110 (1), 8593. Minowa, T., Yokoyama, S., Kishimoto, M., Okakura, T., 1995. Oil production from algal cells of Dunaliella tertiolecta by direct thermochemical liquefaction. Fuel 74 (12), 17351738. Paralkar, A., Edzwald, J.K., 1996. Effect of ozone on EOM and coagulation. Journal American Water Works Association 88 (4), 143154. Powell, E.E., Mapiour, M.L., Evitts, R.W., Hill, G.A., 2009. Growth kinetics of Chlorella vulgaris and its use as a cathodic half cell. Bioresource Technology 100 (1), 269 274. Richardson, B., Orcutt, D.M., Schwertner, H.A., Martinez, C.L., Wickline, H.E., 1969. Effects of nitrogen limitation on the growth and composition of unicellular algae in continuous culture. Applied Microbiology 18 (2), 245250. Rittmann, B.E., 2008. Opportunities for renewable bioenergy using microorganisms. Biotechnology and Bioengineering 100 (2), 203212. Shi, X.M., Chen, F., Yuan, J.P., Chen, H., 1997. Heterotrophic production of lutein by selected Chlorella strains. Journal of Applied Phycology 9 (5), 445450. Shirai, F., Kunii, K., Sato, C., Teramoto, Y., Mizuki, E., Murao, S., Nakayama, S., 1998. Cultivation of microalgae in the solution from the desalting process of soy sauce waste treatment and utilization of the algal biomass for ethanol fermentation. World Journal of Microbiology and Biotechnology 14 (6), 839842. Stucki, S., Vogel, F., Ludwig, C., Haiduc, A.G., Brandenberger, M., 2009. Catalytic gasication of algae in supercritical water for biofuel production and carbon capture. Energy and Environmental Science 2 (5), 535541. Takagi, M., Karseno, Yoshida, T., 2006. Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. Journal of Bioscience and Bioengineering 101 (3), 223226. Widjaja, A., Chien, C.C., Ju, Y.H., 2009. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. Journal of the Taiwan Institute of Chemical Engineers 40 (1), 1320.