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1 Bioresoure Tehnology 113 (2012) Contents lists available at SiVerse SieneDiret Bioresoure Tehnology journal homepage: The feasibility of biodiesel prodution by miroalgae using industrial wastewater Li Fen Wu a, Pei Chung Chen b, Ai Ping Huang a, Chi Mei Lee a, a Department of Environmental Engineering, National Chung Hsing University, 250 Kuo-Kuang Rd., 402 Taihung, Taiwan b Institute of Biomedial Nutrition, Hung Kuang University, 34 Chung-Chie Rd., Sha Lu, 443 Taihung, Taiwan artile info abstrat Artile history: Available online 2 January 2012 Keywords: Biodiesel Miroalgae Industrial wastewater This study investigated nitrogen and phosphorus assimilation and lipid prodution of miroalgae in industrial wastewater. Two native strains of freshwater miroalgae were evaluated their biomass growth and lipid prodution in modified BBM medium. Chlamydomonas sp. TAI-2 had better biomass growth and higher lipid prodution than Desmodesmus sp.tai-1. The optimal growth and lipid aumulation of Chlamydomonas sp. TAI-2 were tested under different nitrogen soures, nitrogen and CO 2 onentrations and illumination period in modified BBM medium. The optimal CO 2 aeration was 5% for Chlamydomonas sp. TAI-2 to ahieve maximal lipid aumulation under ontinuous illumination. Using industrial wastewater as the medium, Chlamydomonas sp. TAI-2 ould remove 100% NH 4 + -N (38.4 mg/l) and NO 3 -N (3.1 mg/l) and 33% PO 4 3 -P (44.7 mg/l) and aumulate the lipid up to 18.4%. Over 90% of total fatty aids were 14:0, 16:0, 16:1, 18:1, and 18:3 fatty aids, whih ould be utilized for biodiesel prodution. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introdution The prodution of biodiesel has reently reeived muh attention worldwide beause of the world energy risis. Miroalgae, the third generation biodiesel feedstoks, are a promising andidate for biodiesel prodution beause of their high photosyntheti effiieny ompared to onventional rops (Ahmad et al., 2011). Biodiesel produed from miroalgal lipid is more sustainable and environmentally friendly than petroleum-derived diesel fuels. The advantages of miroalgae as feedstoks for biodiesel inlude they an aumulate large quantities of triaylglyerols, grow at high rates, fix CO 2 from atmosphere, adapt to wide area inluding extreme environment and utilize nutrient from wastewater (Hu et al., 2008). Nevertheless, the miroalgal biodiesel has not been widely ommerialized mainly due to its high osts. Different strategies have been proposed to improve the ost-effetiveness of miroalgal biofuel prodution. The apparent benefits of ombining miroalgal biodiesel prodution and wastewater treatment are to minimize the use of freshwater, redue the ost of nutrient addition for miroalgal ultivation and remove nitrogen and phosphorus from effluents (Li et al., 2008; Pittman et al., 2011). Based on different miroalgae and ulture onditions suh as temperature, nutrient and light intensity, miroalgal oil ontent and omposition are varied (Converti et al., 2009; Solovheno et al., 2008; Li et al., 2009, 2010). The ellular lipid ontent in various lasses of miroalgae was improved signifiantly under stress Corresponding author. Tel.: ; fax: addresses: allywu0903@gmail.om (L.F. Wu), henp@sunrise.hk.edu.tw (P.C. Chen), talk623@hotmail.om (A.P. Huang), mlee@nhu.edu.tw, mlee@ dragon.nhu.edu.tw (C.M. Lee). onditions, suh as nitrogen starvation, phosphate limitation and high Fe 3+ onentration. (Illman et al., 2000; Khozin-Goldberg and Cohen, 2006; Liu et al., 2007). Oil ontent of some miroalgae suh as Senedesmus sp., Chlorella sp., Neohloris oleoabundans an ahieve from 20% to 50% of total ell dry weight (Gouveia and Oliveira, 2009), revealing the signifiant potential of biodiesel prodution. Isolation and sreening of native speies for biodiesel prodution are important beause they are already adapted to the loal environment. Alimation in aordane with the loal environment an failitates the miroalgae growth and oming appliation of native speies (Mansour et al., 2005). Moreover, pratial use with native speies an also avoid the risks of importing foreign speies that might aidentally be released into the environment. In the study of Chinnasamy et al. (2010), it was indiated that 63.9% of the lipid harvested from native onsortium of miroalgae an be onverted into biodiesel when ultivated in wastewater. The omposition of wastewater disharged from industrial faility is omplex. Carbon is defiient but nitrogen and phosphorus are two main omponents in industrial wastewater, whih are apable of supporting algae growth. The aim of this study was to estimate the possibility of removing nitrogen and phosphorus from industrial wastewater and to sreen a promising strain for the exploitation of renewable energy from miroalgae. 2. Methods 2.1. Industrial wastewater olletion Untreated industrial wastewaters used in this study were olleted from a wastewater treatment fatory in the Taihung /$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi: /j.biorteh

2 L.F. Wu et al. / Bioresoure Tehnology 113 (2012) siene industrial park in Taiwan. The wastewater onsists of 2.43 ls/m ondutivity, 42.2 mg O 2 /L COD, 38.4 mg N/L NH + 4 -N, 3.1 mg N/L NO 3 -N, 16.2 mg N/L organi N and 44.7 mg P/L PO 3 4 -P. Wastewater samples were olleted in a 20-L plasti ontainers and stored in a old room at 4 C. Wastewater samples using in the experiments were filtered through a 0.22 lm membrane in advane Miroalgal strain, media and ultivations Desmodesmus sp.tai-1 and Chlamydomonas sp. TAI-2 were isolated from ponds in the middle Taiwan and grown in modified Bold s basal medium (BBM medium) (Sterin, 1973) ontaining the following omponents (per liter) 0.25 g NaNO 3, g MgSO 4 7H 2 O, g CaCl 2 2H 2 O, g KH 2 PO 4, g K 2 HPO 4, g NaCl, g FeSO 4 7H 2 O, 0.01 g Na 2 EDTA, 8.05 mg H 3 BO 3, 1.81 mg MnCl 2 4H 2 O, mg ZnSO 4 7H 2 O, mg CuSO 4 5H 2 O, mg NaMoO 4 5H 2 O and mg Co(NO 3 ) 2.6H 2 O. The miroalgae were ultivated in 100 ml autolaved BBM medium in 250 ml flasks. The flasks were inubated at room temperature with ontinuous illumination of 25 lmol photons m 2 day 1 and shaken at 120 rpm on an orbital shaker Experimental design The miroalgae were ultivated in 300 ml autolaved modified BBM medium in 500 ml flasks. The flasks were inubated at room temperature with ontinuous illumination of 25 lmol photons m 2 day 1 and shaken at 120 rpm on an orbital shaker for 11 days to evaluate their biomass growth and lipid prodution under different nitrogen soures (nitrate, ammonium or urea). The photobioreator was a 6-L glass olumni-form flask with an inner diameter of approximately 13 m and a height of approximately 41 m (Fig. 1). The working volume was 5-L. There are eight light soures (Cold-athode Fluoresent Lamp, CCFL) in the interior of photobioreator. The miroalgae were ultivated in 5-L autolaved modified BBM medium in photobioreator to evaluate their biomass growth and lipid prodution under different nitrogen soure onentrations, CO 2 onentrations and illumination period. The miroalgae were ultivated in 5-L industrial wastewater in photobioreator to evaluate their biomass growth and lipid prodution with ontinuous illumination of 125 lmol photons m 2 day 1 and aeration of 5% CO 2 /air (v/v) mixture at onstant flow rate (165 ml/min) Analysis of lipid ontent and fatty aid omposition The miroalgal ells were harvested from the ultural mixture by entrifugation at 8000 rpm for 15 min (Hima CR22GII, Hitahi, Japan) and the ell pellet was frozen at 20 C. Total lipids were extrated from lyophilized biomass in CHCl 3 MeOH (2:1, v/v) by a modified method of Folh (Christie, 2003). Freeze-dried biomass was suspended in 3 ml CHCl 3 MeOH (2:1, v/v) solution, extrated by soniation for 90 min, and then olleted the extrat by entrifugation at 2000 rpm for 10 min. The pellet was re-extrated in 3 ml CHCl 3 MeOH solution twie. The olleted extrat was evaporated at 40 C, dried at 70 C for 2 h, and subsequently weighed after ooling to the room temperature. Lipid ontent of miroalgae was alulated by dividing the residue weight by the freeze-dried ell weight. Gas hromatography (HP 6890, USA) equipped with 30 m DB- WAXETR (J&W, Agilent) apillary olumn was used for qualitative and quantitative determination of fatty aid omposition. The oven temperature program started at 190 C, inrease at 4 C min 1 until 220 C. Carrier gas, N 2, was kept at a onstant rate of 15 ml/min. Injetor and detetor (flame ionization) temperature were kept at 220 C. The fatty aid methyl ester (FAME) was prepared by adding 2 ml 2 N KOH in methanol solution to the sample for saponifiation. Then 4 ml 2.5 N HCl in methanol solution and 1 ml BF 3 (14%, methanol solution) were added for esterifiation. After methylation, 1 ml saturated NaCl solution and 2 ml n-hexane were added to the vial and the top n-hexane layer was removed and plaed into vials for GC analysis (Christie, 2003). The individual fatty aid ompositions were identified by omparison of their retention time with those of the authenti standards (Sigma), and were quantified by omparing their peak area with that of the standard. Fatty aid omposition was alulated as perentage of the total fatty aids present in the sample Analytial methods The miroalgae onentration was determined by measuring the optial density of algal ulture at 680 nm by spetrophotometer (Hitahi, Japan), and biomass onentration was related to optial density by the equation y = 0.399x 0.02 (R 2 = 0.990) for Desmodesmus sp. TAI-1 and y = 0.716x (R 2 = 0.992) for Chlamydomonas sp. TAI-2, respetively. The dry weight of algal biomass was measured using the method of suspended solid (SS) measurement. Nitrate onentrations in the ulture medium were measured by determining the absorbane at 220 nm and subsequently subtrating the double absorbane at 275 nm to avoid the dissolved organi matter interferene (APHA-AWA-WEF, 2005). Phosphate and ammonium onentrations were determined olorimetrially (APHA-AWA-WEF, 2005). All the analysis were arried out with ulture supernatants obtained after entrifugation at 12,000 rpm for 5 min and filtered through a 0.22 lm membrane. ph Variation during the miroalgae ultivation was measured by ph meter (WTW, Germany). 3. Results and disussion 3.1. Comparision of growth and lipid ontent in two miroalgal strain Fig. 1. Shemati diagram of the photobioreator. Desmodesmus sp. TAI-1 and Chlamydomonas sp. TAI-2 were isolated from ponds in the middle Taiwan. To ompare the growth and lipid ontent of these two strains, the experiments were arried out in a photobioreator with ontinuous illumination and 3% CO 2 aeration in the modified BBM medium. As shown in Fig. 2a, the growth of Chlamydomonas sp. TAI-2 was better than

3 16 L.F. Wu et al. / Bioresoure Tehnology 113 (2012) Desmodesmus sp. TAI-1 under the investigated onditions. The biomass onentration produed from Chlamydomonas sp. TAI-2 was approximately 1.34 g/l at day 10, more than that produed from Desmodesmus sp. TAI-1, whih was approximately 0.93 g/l. Miroalgal biomass was harvested from ulture medium every two days and subjeted to extration for lipid ontent measurement. As shown in Fig. 2b, The biomass obtained from Chlamydomonas sp. TAI-2 has the higher lipid ontent than Desmodesmus sp. TAI-1 during the ultivation period. The maximum lipid ontent of Chlamydomonas sp. TAI-2 was 25.3% at day 10, better than the maximum lipid ontent of Desmodesmus sp. TAI-1, 19.7%. The lipid produtivity obtained from Chlamydomonas sp. TAI-2 was 0.34 g/l, whih was approximately double of that obtained from Desmodesmus sp. TAI-1, 0.18 g/l (Fig. 2). These results suggested that Chlamydomonas sp. TAI-2 was the promising strain for biomass growth and lipid aumulation under the investigation onditions Effet of illumination period on growth and total lipid ontent of Chlamydomonas sp. TAI-2 Light and dark phases are the two phases of photosynthesis. In the light reations the ells onvert light energy into hemial energy, whih is stored in high-energy ompounds for dark reations to fix CO 2 (Jaob-Lopes et al., 2009). In order to investigate the effet of illumination period on ell growth and lipid aumulation of Chlamydomonas sp. TAI-2, ontinuous illumination and 14:10 light and dark yle was studied, respetively. As shown in Table 1, the growth of Chlamydomonas sp. TAI-2 with ontinuous illumination was 1.3 g/l, slightly lower than that obtained from 14:10 light and dark yle, 1.5 g/l. However, the lipid ontent of ells obtained from ontinuous illumination was 25.3%, whih was approximately 1.5 times of that obtained from 14:10 light and dark yle, 16.8%. The lipid produtivity obtained from ontinuous illumination was 0.34 g/l, better than that obtained from 14:10 light and dark yle, 0.25 g/l. As a result of the experiments of various illuminations, ontinuous illumination was hosen as the best ondition with respet to lipid prodution and further experiments were onduted under this ondition Effet of different nitrogen soure onentrations on ell growth and lipid aumulation The effet of nitrogen soure onentrations on ell growth and lipid aumulation of Chlamydomonas sp. TAI-2 was studied in modified BBM medium ontaining 3 and 6 mm sodium nitrate, respetively. The biomass, lipid ontent and lipid produtivity obtained from different sodium nitrate onentration are shown in Table 1. Li et al. (2009) demonstrated that inreased sodium nitrate onentration ould promote the growth of N. oleoabundans signifiantly when the sodium nitrate onentration was below 10 mm. In this study, the biomass of Chlamydomonas ells ultured in modified BBM medium ontaining 6 mm sodium nitrate was 1.8 g/l, whih was approximately 1.4 times of that obtained with 3 mm sodium nitrate. In ontrast to biomass, the lipid ontent of miroalgal ells obtained with 3 mm sodium nitrate, 25.3%, was better than that obtained with 6 mm sodium nitrate, 18%. The redution in nitrogen in the medium inreases the lipid ontent in this miroalgal strain. This result are similar to some Chlorella strains and N. oleoabundans (Illman et al., 2000; Li et al., 2009). Under nitrogen starvation, many algal speies were reported to aumulate lipids (Illman et al., 2000; Solovheno et al., 2008; Li et al., 2009). The nitrogen onentration was depleted at day 3 and day 4 when Chlamydomonas sp. TAI-2 was ultivated in medium ontaining 3 mm sodium nitrate and 6 mm sodium nitrate, respetively. The lipid produtivity of Chlamydomonas ells ultivated in 6 mm sodium nitrate was slightly lower than that ultivated in 3 mm sodium nitrate Effet of different nitrogen soures on growth and lipid aumulation To investigate an optimal nitrogen soure for ell growth and lipid aumulation of Chlamydomonas sp. TAI-2, 41.2 mg N/L of sodium nitrate, ammonium hloride and urea supplied in modified BBM medium were tested, respetively. As shown in Table 2, ammonium an support less growth of Chlamydomonas ells under the investigated onditions than the other ompounds. The maximum biomass onentration obtained with nitrate as the nitrogen soure was approximately g/l. However, the maximum lipid Table 1 Biomass, lipid ontent and lipid produtivity prodution of Chlamydomonas sp. TAI-2 at different illumination period and initial sodium nitrate onentrations. LDC + N (3 mm) CL + N (3 mm) CL + 2N (6 mm) Biomass (g/l) a Lipid ontent (%) a Lipid produtivity (g/l) b Fig. 2. Biomass, lipid ontent and lipid produtivity variations of Desmodesmus sp.tai-1 and Chlamydomonas sp. TAI-2 ultivated in BBM medium with 3% CO 2 aeration under ontinuous illumination during 10 days inubation. a The biomass and lipid ontent were measured at day 10. b The lipid produtivity was obtained at day 10. LDC + N: 10:14 light/dark yle and 3 mm sodium nitrate. CL + N: ontinuous illumination and 3 mm sodium nitrate. CL + 2 N: ontinuous illumination and 6 mm sodium nitrate. All experiments were performed in 6-L photobioreator ontained 5-L modified BBM medium at room temperature with ontinuous illumination of 125 lmol photons m 2 day 1 and aerated with 3% CO 2.

4 L.F. Wu et al. / Bioresoure Tehnology 113 (2012) ontent of Chlamydomonas sp. TAI-2 was 23.2% when urea was the nitrogen soure (Table 2). Compared with these three nitrogen soures, the maximum lipid produtivity of Chlamydomonas sp. TAI-2 was obtained when urea is the nitrogen soure. The results showed that urea was the suitable nitrogen soure for lipid prodution of Chlamydomonas sp. TAI-2 among the three tested nitrogen ompounds under the tested onditions Effet of CO 2 onentration on growth and total lipid ontent In order to investigate the influene of CO 2 onentration on miroalgal growth and lipid aumulation, 3%, 5% and 10% of CO 2 onentrations were studied on Chlamydomonas sp. TAI-2, respetively. The results were shown in Table 3. The biomass prodution of miroalgal ells was very similar in all three ultures at approximately 1.5 g/l. However, the maximal lipid produtivity (0.31 g/l) of miroalgal ells was obtained in 5% CO 2 aeration due to the maximal lipid ontent (20.9%). The optimal CO 2 aeration was 5% for Chlamydomonas sp. TAI-2 to ahieve maximal lipid aumulation under ontinuous illumination for 10 days. Table 3 Biomass, lipid ontent and lipid produtivity prodution by Chlamydomonas sp. TAI-2 at different CO 2 onentrations. 3% CO 2 5%CO 2 10% CO 2 Biomass (g/l) a Lipid ontent (%) a Lipid produtivity (g/l) b a The biomass and lipid ontent were measured at day 10. b The lipid produtivity was obtained at day 10. All experiments were performed in 6-L photobioreator ontained 5-L BBM medium at room temperature with ontinuous illumination of 125 lmol photons m 2 day 1 and 1.5 mm urea Potential of Chlamydomonas sp. TAI-2 for ombined wastewater treatment and lipid aumulation Aording to the previous results, the N/P removal and lipid aumulation of the Chlamydomonas sp. TAI-2 was studied under ontinuous illumination and 5% CO 2 aeration using industrial wastewater. As shown in Fig. 3, the Chlamydomonas ells were able to ahieve omplete NH + 4 -N and NO 3 -N removal from industrial wastewater ontaining 38.4 mg NH + 4 -N/L and 3.1 mg NO 3 -N/L in two days. The rate of ammonium onsumption was 19.2 mg NH + 4 -N/L/d. The removal of P was 33% from industrial wastewater ontaining 44.7 mg/l PO 3 4 -P in 10 days. The organi nitrogen in the wastewater ould not be removed by the Chlamydomonas ells in the investigation ondition. In Fig. 4, the Chlamydomonas ells were well ultured in industrial wastewater. In generally, miroalgal growth rate are lower in many industrial wastewater, due to low N and P onentration and high toxin onentrations (Pittman et al., 2011). In our study, this industrial wastewater was shown to have enough N and P to support miroalgal growth, and there were no inhibitory effets of miroalgal growth in this wastewater. The study of Chinnasamy et al. had shown that two freshwater miroalgae Botryoous braunii and Chlorella saharophila were able to grow well on the untreated arpet industrial wastewater (Chinnasamy et al., 2010). The biomass of the Chlamydomonas ells obtained was onstant after 5 days, the maximal biomass was 1.5 g/l. The maximal lipid produtivity was obtain, 0.28 g/l, at day 10. The fatty aid omposition of typial miroalgal oil is mainly omposed of a mixture of unsaturated fatty aids: palmitolei aid Table 2 Biomass, lipid ontent and lipid produtivity prodution of Chlamydomonas sp. TAI-2 at different nitrogen soure. Urea Sodium nitrate Ammonium hloride 1.5 mm 3 mm 3 mm Biomass (g/l) a Lipid ontent (%) a Lipid produtivity (g/l) b a The biomass and lipid ontent were measured at day 11. b The lipid produtivity was obtained at day 11. All experiments were performed in 500 ml flasks ontained 300 ml modified BBM medium at room temperature with ontinuous illumination of 25 lmol photons m 2 day 1 and shaken at 120 rpm on an orbital shaker for 11 days. Fig. 3. Nitrogen and phosphorus assimilation of Chlamydomonas sp. TAI-2 ultivated in the industrial wastewater with 5% CO 2 aeration under ontinuous illumination during 10 days inubation. (C16:1), olei aid (C18;1), linolei aid (C18:2) and linoleni aid (C18:3) as well as small ontent of saturated fatty aids: palmiti aid (C16:0) and steari aid (C18:0) (Meng et al., 2009). The fatty aid ompositions of the lipid from the Chlamydomonas sp. TAI-2 ultivated in industrial wastewater was determined every two days and the result was listed in Table 4. The fatty aid omposition varied slightly during 10 days and the saturated fatty aids palmiti aid (C16:0), monounsaturated olei (C18:1), and linoleni aid (C18:3) were dominant. The miroalgal lipid was mainly omposed of saturated fatty aid (54 79%) and the saturated fatty aids palmiti aid (16:0) presented a signifiant perentage (44 68%) of total lipid. Aording to the quality standards of biodiesel from European Standards (EN, 2004), the linoleni aid (C18:3) ontent has a limit of 12% for a quality biodiesel. As shown in Table 4, the linoleni aid ontent of miroalgal lipid was lower than 12% at day 6 and day 8. High-quality biodiesel should have similar perentage of saturated and unsaturated fatty aids to maintain high oxidative stability and low-temperature property (Knothe, 2005; Feng et al., 2011). In Chlamydomonas sp. TAI-2, the perentage was almost equal between saturated and unsaturated fatty aids at day 6 and day 8. Moreover, this strain had a high perentage of olei aid (31.6%) onsidered to be an optimal fatty aid to exhibit a ombination of oxidative stability and low-temperature property at day 6 and day 8. Therefore, the lipid produed from the Chlamydomonas sp. TAI-2 at day 6 and 8 might be ompletely ompatible with biodiesel appliation. However, the maximal lipid produtivity of the Chlamydomonas sp. TAI-2 was produed at day 10. In order to onsist with European Standard EN the miroalgal lipid from our isolates at day 10 an be further im-

5 18 L.F. Wu et al. / Bioresoure Tehnology 113 (2012) ahieve maximal lipid aumulation under ontinuous illumination. Using the industrial wastewater as the medium, Chlamydomonas sp. TAI-2 ould remove 100% NH 4 + -N and NO 3 -N and 33% PO 4 3 -P and aumulate the lipid up to 18.4%. The omposition of fatty aids is suitable to be utilized for biodiesel prodution. Therefore, the Chlamydomonas sp. TAI-2 is a promising miroalgae to remove nitrogen and phosphorus in the industrial wastewater and subsequently to produe biodiesel. Aknowledgement This study was supported by a fund from National Siene Counil in Taiwan. Referenes Fig. 4. The variation of biomass, lipid ontent and lipid produtivity of Chlamydomonas sp. TAI-2 ultivated in industrial wastewater with 5% CO 2 aeration under ontinuous illumination during 10 days inubation. Table 4 Fatty aids omposition of lipid aumulated by Chlamydomonas sp. TAI-2 ultivated in the industrial wastewater with 5% CO 2 aeration under ontinuous illumination during 10 days inubation. Fatty aid (% of total fatty aid) Time (day) C14: C16: C16: C18: C18: C18:2 C18: Saturated Unsaturated : Not detetable. proved if mix with other soures of algal oil (Chinnasamy et al., 2010). 4. Conlusion The native strains, Chlamydomonas sp. TAI-2 had higher biomass prodution and lipid aumulation than Desmodesmus sp. TAI-1. The optimal CO 2 aeration was 5% for Chlamydomonas sp. TAI-2 to Ahmad, A.L., Yasin, N.H., Mat, C.J.C., Derek, Lim, J.K., Miroalgae as a sustainable energy soure for biodiesel prodution: A review. Renew. Sust. Energ. Rev. 15, APHA-AWA-WEF, Standard Methods for the Examination of Waters and Wastewater, 21st ed. Washing, DC. Chinnasamy, S., Bhatnagar, A., Hunt, R.W., Das, K.C., Miroalgae ultivation in wastewater dominated by arpet mill effluents for biofuel appliations. Bioresour. Tehnol. 101, Christie, W.W., Lipid analysis: Isolation separation, identifiation and strutural analysis of lipid. Oily Press. Bridgewater, UK. Converti, A., Casazza, A.A., Ortiz, E.Y., Perego, P.D., Borghi, M., Effet of temperature and nitrogen onentration on the growth and lipid ontent of Nannohloropsis oulata and Chlorella vulgaris for biodiesel prodution. Chem. Eng. Proess. 48, European Standard EN Automotive fuels-fatty aid methyl esters (FAME) for diesel engines-requirements and test methods. Feng, D., Chen, Z., Xue, S., Zhang, W., Inreased lipid produtivity of marine oleaginous miro Isohrysis zhangjiangensis (Chrysophyta) by nitrogen supplement. Bioresour. Tehnol. 102, Gouveia, L., Oliveira, A.C., Miroalgae as a raw material for biofuels prodution. J. Ind. Mirobiol. Biotehnol 36, Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., Miroalgal triaylglyerols as feedstoks for biofuel prodution: perspetives and advanes. Plant J. 54, Illman, A.M., Sragg, A.H., Shales, S.W., Inrease in Chlorella strains alorifi values when grown in low nitrogen medium. Enzym. Mirob. Tehnol. 27, Jaob-Lopes, E., Soparo, C.H.G., Laerda, L.M.C.F., Frano, T.T., Effet of light yle (night/day) on CO 2 fixation and biomass prodution by miroalgae in photobioreators. Chem. Eng. Proess. 48, Khozin-Goldberg, I., Cohen, Z., The effet of phosphate starvation on the lipid and fatty aid omposition of the fresh water eustigmatophyte Monodus subterraneus. Phytohemistry. 67, Knothe, G., Dependene of biodiesel fuel properties on the struture of fatty aid alkyl esters. Fuel Proess. Tehnol. 86, Li, Y., Horsman, M., Wu, N., Lan, C.Q., Dubois-Calero, N., Biofuels from miroalgae. Biotehnol. Prog. 24, Li, X., Hu, H.Y., Gan, K., Sun, Y.X., Effet of different nitrogen and phosphorus onentration on the growth, nutrient uptake, and lipid aumulation of a freshwater miroalga Senedesmus sp. Bioresour. Tehnol. 101, Li, Y., Horsman, M., Wang, B., Wu, N., Lan, C.Q., Effets of nitrogen soures on ell growth and lipid aumulation of green alga Neohloris oleoabundans. Appl. Mirobiol. Biotehnol. 81, Liu, Z.Y., Wang, G.C., Zhou, B.C., Effet of iron growth and lipid aumulation in Chlorella vulgaris. Bioresour. Tehnol. 99, Mansour, M.P., Frampton, D.M.F., Nihols, P.D., Volkman, J.K., Lipid and fatty aid yield of nine stationary-phase miroalgae: Appliation and unusual C24 C28 polyunsaturated fatty aids. J. Appl. Phyol. 17, Meng, X., Yang, J., Xu, X., Zhang, L., Nie, Q., Xiang, M., Biodiesel prodution from oleaginous miroorganisms. Renew. Energy 34, 1 5. Pittman, J.K., Dean, A.P., Osundeko, O., The potential of sustainable algal biofuel prodution using wastewater resoures. Bioresour. Tehnol. 102, Solovheno, A.E., Khozin-Goldberg, I., Didi-Cohen, S., Cohen, Z., Merzlyak, M.N., Effet of light intensity and nitrogen starvation on growth, total fatty aids and arahidoni in the green miroalga Parietohloris inise. J. Appl. Phyol. 20, Sterin, J.R., Handbook of phyologial methods: ulture methods and growth measurements. Cambridge University Press.