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1 Journal of the Taiwan Institute of Chemical Engineers 40 (2009) Contents lists available at ScienceDirect Journal of the Taiwan Institute of Chemical Engineers journal homepage: Study of increasing lipid production from fresh water microalgae Chlorella vulgaris Arief Widjaja a, *, Chao-Chang Chien b, Yi-Hsu Ju b a Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Kampus ITS Keputih Sukolilo, Surabaya, East Java 60111, Indonesia b Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Keelung Road, Section 4, Taipei 10607, Taiwan ARTICLE INFO ABSTRACT Article history: Received 6 March 2008 Received in revised form 30 June 2008 Accepted 1 July 2008 Keywords: Chlorella vulgaris Lipid Nitrogen depletion Harvesting time Lipid productivity Study of increasing lipid production from fresh water microalgae Chlorella vulgaris was conducted by investigating several important factors such as the effect of CO 2 concentration, nitrogen depletion and harvesting time as well as the method of extraction. The drying temperature during lipid extraction from algal biomass was found to affect not only the lipid composition but also lipid content. Drying at very low temperature under vacuum gave the best result but drying at 60 8C still retained the composition of lipid while total lipid content decreased only slightly. Drying at higher temperature decreased the content of triacylglyceride (TG). As long as enough pulverization was applied to dried algal sample, ultrasonication gave no effect whether on lipid content or on extraction time. In addition to the increase of total lipid content in microalgal cells as a result of cultivating in nitrogen depletion media, it was found that changing from normal nutrient to nitrogen depletion media will gradually change the lipid composition from free fatty acid-rich lipid to lipid mostly contained TG. Since higher lipid content was obtained when the growth was very slow due to nitrogen starvation, compromising between lipid content and harvesting time should be taken in order to obtain higher values of both the lipid content and lipid productivity. As the growth was much enhanced by increasing CO 2 concentration, CO 2 concentration played an important role in the increase of lipid productivity. At low until moderate CO 2 concentration, the highest lipid productivity could be obtained during N depletion which could surpassed the productivity during normal nutrition. At high-co 2 concentration, harvesting at the end of linear phase during normal nutrition gave the highest lipid productivity. However, by reducing the incubation time of N depletion, higher lipid content as well as higher lipid productivity may still be achieved under this condition. ß 2008 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. 1. Introduction CO 2 is recognized as the most important of the atmospheric pollutants that contribute to the greenhouse effect. Reducing the build-up of atmospheric CO 2 can be accomplished by utilizing photosynthetic organism which has ability to use CO 2 for the growing. Higher plants, e.g. trees, are capable to do this process. However, these will not be enough to stabilize atmospheric CO 2 levels sufficiently to avoid a future greenhouse world (Benemann, 1997). Microalgae is a photosynthetic microorganism that is able to use the solar energy to combine water with carbon dioxide to create biomass. Because the cells grow in aqueous suspension, they have more efficient access to water, CO 2, and other nutrients. * Corresponding author. Tel.: ; fax: address: arief_w@chem-eng.its.ac.id (A. Widjaja). Microalgae, growing in water, have fewer and more predictable process variables (sunlight, temperature) than higher plant systems, allowing easier extrapolation from one site, even climatic condition, to others. Thus, fewer site-specific studies are required for microalgae than, for example, tree farming. Also, microalgae grow much faster than higher plants and require much less land areas. However, the utilization of microalgae to overcome global warming is not enough without utilizing an algal biomass before degradation. Fatty acid methyl esters originating from vegetable oils and animal fats are known as biodiesel. Biodiesel fuel has received considerable attention in recent years, as it is a biodegradable, renewable and non-toxic fuel. It contributes no net carbon dioxide or sulfur to the atmosphere and emits less gaseous pollutants than normal diesel (Antolin et al., 2002; Lang et al., 2001; Vicente et al., 2004). High dependence on foreign oil, especially transportation sector, gives rise to the importance of producing biodiesel for the sake of national energy security /$ see front matter ß 2008 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi: /j.jtice

2 14 A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) There are four primary ways to make biodiesel, direct use and blending, microemulsions, thermal cracking (pyrolysis) and transesterification (Ma and Hanna, 1999). The most common way is transesterification as the biodiesel from transesterification can be used directly or as blends with diesel fuel in diesel engine (Peterson et al., 1991; Zhang et al., 2003). Biodiesel, primarily rapeseed methyl ester, has been in commercial use as an alternative fuel since 1988 in many European countries (Lang et al., 2001). However, in spite of the favourable impact that its commercialization could provide, the economic aspect of biodiesel production prevents its development and large-scale use, mainly due to the high-feed cost of vegetable oil (Antolin et al., 2002; Lang et al., 2001). Biodiesel usually costs over US $0.5/L, compared to US $0.35/L for normal diesel (Zhang et al., 2003). Exploring ways to reduce the high cost of biodiesel is of much interest in recent biodiesel research, especially for those methods concentrating on minimizing the raw material cost. Microalgae have been suggested as very good candidates for fuel production because of their advantages of higher photosynthetic efficiency, higher biomass production and faster growth compared to other energy crops (Dote et al., 1994; Ginzburg, 1993; Miao and Wu, 2006; Milne et al., 1990; Minowa et al., 1995). Microalgae systems also use far less water than traditional oilseed crops. For these reasons, microalgae are capable of producing more oil per unit area of land, compared to terrestrial oilseed crops. Microalgae are very efficient biomass capable of taking a waste (zero energy) form of carbon (CO 2 ) and converting it into a highdensity liquid form of energy (natural oil). Hundreds of microalgal strains capable of producing high content of lipid have been screened and their lipid production metabolism have been characterized and reported (Sheehan et al., 1998). Most of them are marine microalgae. Recently, Miao and Wu (Miao and Wu, 2004, 2006; Miao et al., 2004; Wu et al., 1992, 1994) reported a heterotrophic growth of Chlorella protothecoides capable of yielding as high as 55% lipid content and converting the lipid to biodiesel. Besides the need of expensive nutrient of thiamine hydrochloride, the need of glucose instead of CO 2 for heterotrophic growth gain less interest in the view of global warming issue. Using CO 2 as carbon source, the strain yielded only about 15% of lipid. Allard and Templier (2000) extracted lipid from a variety of freshwater and marine microalgae and reported that lipid content varied from 1 to 26%. A great deal of attention has been focused on the autotrophic green microalga, Botryococcus braunii, due to its high-hydrocarbon production level (Casadevall et al., 1985; Metzger and Pouet, 1995; Wake and Hillen, 1980). Sawayama et al. (1995) utilized this algae for continuously operated CO 2 fixation and oil production. Despite the high-lipid content of 64%, the growth rate of this strain was reported to be very low (Sheehan et al., 1998). Utilizing marine microalgal strains will give benefit if large pond is used for the system. As these strains can grow in brackish water, that is, water that contains high levels of salt, they will not compete for the land already being used by other biomass-based fuel technologies. Freshwater microalgae, however, still can compete with marine microalgae if, instead of using large pond, closed photobioreactors which require less land area are used. Lower land requirements were also assumed to be possible with the optical fiber devices (Sheehan et al., 1998). These kind of reactors are also able to provide better dark and light photoperiod in the system as evidenced from several experimental works using air lift bioreactor (Novakovic et al., 2005), stirred tank photobioreactor (Huang and Rorrer, 2003) and other types of closed photobioreactor (Rorrer et al., 1996; Rorrer and Mullikin, 1999; Usui and Ikenouchi, 1997; Vernerey et al., 2001). As productivity is measured in terms of biomass produced per day per unit of available surface area, closed photobioreactors will give much higher productivities. Although many reports are available concerning the production of lipid from microalgae, as far as the authors know, there is no report available for a thorough discussion concerning the effect of growth conditions and extraction method on the lipid productivity as well as on its content and composition. These parameters are of vital importance for the application of the system in larger scale. Zhu et al. (2002) extracted fungal lipid of Mortierella alpina and made comparison between wet biomass and biomass dried at 80 8C. Wet biomass was extracted according to the procedure of Bligh and Dyer (1959) and dried biomass was extracted using chloroform:methanol (2:1, v/v ratio). They found that dry extraction was more effective than wet extraction. Solvent extraction was still the main extraction method used by researchers due to its simplicity and relatively inexpensive requiring almost no investment for equipment (Letellier and Budzinski, 1999). Inspite of many extraction methods developed recently including supercritical or subcritical fluid extraction, as well as microwave and ultrasound assisted extraction, the yield of the extraction of lipid from biomass using organic solvents, including chloroform/methanol mixture, was still found superior in comparison to supercritical fluid extraction (Mendes et al., 2006). Comparison between different methods to extract compounds with pharmaceutical importance from microalgae including Chlorella vulgaris was reported (Mendes et al., 2003) but no comparison was made against chloroform/methanol solvent extraction. Much research was focused on the lipid trigger which refers to the observation that under environmental stress, many microalgae produce more lipid. It is generally accepted that the depletion of the nitrogen from the medium induces lipid accumulation (Evans and Ratledge, 1984b; Yoon and Rhee, 1983a). Botham and Ratledge (1979) argued that the glucose conversion into lipids was triggered, when nitrogen was exhausted, due to the high-energy charge (ratio of ATP:AMP) present. The nitrogen source also was reported to alter the amount of lipids (Evans and Ratledge, 1984a,b; Yoon and Rhee, 1983b). Turcotte and Kosaric (1988) studied the biosynthesis of lipids on Rhodosporidium toruloides ATCC under limiting amount of nitrogen required to trigger lipid accumulation. The results showed that lipid accumulation always started sometime after nitrogen reached a level of M and the specific initial lipid productivity, expressed as g/(l h) of storage lipid per g/l of lipid-free cellular material, was constant. Sheehan et al. (1998) reported that the reason for the increase of lipid content was that under nutrient starvation, the rate of production of all cell components is lower, but oil production remains higher, leading to an accumulation of oil in the cells. Environmental stresses like nitrogen depletion lead to inhibition of cell division, without immediately slowing down oil production. They also suggested by controlling the timing of nutrient depletion and cell harvesting, a net increase in total oil productivity might be obtained. The research aimed to produce lipid contained in fresh water microalgae C. vulgaris using a 5-L closed fermentor. The effect of CO 2 concentration, nitrogen concentration, harvesting time, and lipid extraction method on the lipid content, lipid composition and lipid productivity were investigated to obtain best condition under which high-lipid content and high-lipid productivity could be achieved. 2. Materials and methods 2.1. Materials A microalgal strain of C. vulgaris was kindly provided by Prof. Hong-Nong Chou of The Institute of Fisheries Science, National

3 A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) Taiwan University. In addition to its easier growth in a relatively cheap media without a necessity of utilizing very specific compound, this strain was utilized since it gave significant lipid content with good growing rate as evidenced from our preliminary experimental results and in comparison with other freshwater and seawater microalgal strains conducted by Prof. Chou (data not shown). All solvents and reagents were either of HPLC grade or AR grade. All other chemicals used were obtained from commercial sources Medium and cultivation condition The normal nutrition medium for cultivation of C. vulgaris was a modification of Fitzgerald medium (Hughes et al., 1959) made by adding 1 ml of each of IBI (a), IBI (b), IBI (c), IBI (d), and IBI (e) to 1 L distilled water. IBI (a) contained, per 200 ml: NaNO 3, 85.0g; CaCl 2 2H 2 O, 3.70 g. IBI (b) contained, per 200 ml: MgSO 4 7H 2 O, g. IBI (c) contained, per 200 ml: KH 2 PO 4,1.36g;K 2 HPO 4, 8.70 g. IBI (d) contained, per 200 ml: FeSO 4 7H 2 O, g; EDTAtri- Na, g. IBI (e) contained, per 200 ml: H 3 BO 3, g; MnSO 4 H 2 O, g; ZnSO 4 7H 2 O, g; (NH 4 ) 6 Mo 7 O 24 4H 2 O, g; CoCl 2 6H 2 O, g; KBr, g; KI, g; CdCl 2 (5/2) H 2 O, g; Al 2 (SO 4 ) 3 (NH 4 ) 2 SO 4 24H 2 O, g; CuSO 4 5H 2 O, g; 97% H 2 SO 4, 0.56 ml. This normal nutrition medium resulted in a nitrogen content of mg/l medium. The nitrogen depletion mediumwas provided by eliminating the addition of IBI (a) to result in a medium with a nitrogen content of 0.02 mg/l medium Lipid extraction Dry extraction procedure according to Zhu et al. (2002) as a modification of the wet extraction method by Bligh and Dyer (1959) was used to extract the lipid in microalgal cells. Typically, cells were harvested by centrifugation at 8500 rpm for 5 min and washed once with distilled water. After drying the samples using freeze drier, the samples were pulverized in a mortar and extracted using mixture of chloroform:methanol (2:1, v/v). About 50 ml of solvents were used for every gram of dried sample in each extraction step. After stirring the sample using magnetic stirrer bar for 5 h and ultrasonicated for 30 min, the samples were centrifuged at 3000 rpm for 10 min. The solid phase was separated carefully using filter paper (Advantec filter paper, no. 1, Japan) in which two pieces of filter papers were applied twice to provide complete separation. The solvent phase was evaporated in a rotary evaporator under vacuum at 60 8C. The procedure was repeated three times until the entire lipid was extracted. The effect of solvents having different polarities for extracting the lipid, as well as the effect of drying temperature and ultrasonication time were investigated in this study Effect of CO 2 concentration The effect of CO 2 concentration on lipid content, lipid composition and productivity was investigated by varying the CO 2 concentration. At first, the culture was aerated under air flow rate of 6 L/min without additional CO 2. By taking into account the CO 2 content in air of about 0.03%, this condition resulted in about 2 ml/min CO 2 as carbon source. The next batch was conducted under the same air flow rate with the addition of 20, 50, 100, and 200 ml/min pure CO 2 gas, or about 0.33, 0.83, 1.67, and 3.33% CO 2, respectively Effect of nitrogen concentration At first, cells of C. vulgaris was cultivated in 4 L normal nutrition medium and incubated batchwisely at 22 8C. The system was aerated at an air flow rate of 6 L/min with or without the addition of pure CO 2 gas. The fermentor is agitated at 100 rpm. Four pieces of 18 W cool-white fluorescent lamps are arranged vertically, at a 20-cm distance from the surface of fermentor to provide a continuous light to the system. This gave an average light intensity of 30 me/(m 2 s). The optical density of cells was measured at 682 nm every 24 h using UV-530 JASCO Spectrophotometer, Japan. Cells were harvested at the end of linear phase, i.e. at a cell concentration of about cells/ml. To investigate the effect of nitrogen depletion, 1 L of culture from the end of linear phase was diluted by adding 3 L nitrogen depletion medium and the cultivation continued for 7 and 17 d at which time the cells were harvested and the lipid content as well as lipid productivity were measured. Other conditions of incubation such as light intensity, pure CO 2 gas flow rate and temperature were all the same as the corresponding normal nutrition condition Gas chromatography analysis Sample was dissolved in ethyl acetate and 0.5 ml of this was injected into a Shimadzu GC-17A (Kyoto, Japan) equipped with flame ionization detector using DB-5HT (5% phenyl)-methylpolysiloxane non-polar column (15 m 0.32 mm ID); Agilent Tech., Palo Alto, California). Injection and detector temperature both were 370 8C. Initial column temperature was 240 8C, and the temperature was increased to 300 8C at a temperature gradient of 15 8C/min. 3. Results and discussion 3.1. Effect of extraction method on lipid content and composition Dry extraction procedure according to Zhu et al. (2002) and Miao et al. (2004) was the main method employed to be investigated in this study. Several important factors during the extraction such as effect of solvent, drying temperature and ultrasonication was investigated to see which method give the best result in view of total lipid obtained, lipid composition and lipid productivity Effect of solvent on the extraction Instead of chloroform/methanol mixture, Miao and Wu (2006) used hexane to extract lipid from C. protothecoides. Our results using hexane as the solvent in the extraction of lipids resulted in poor yield (data not shown) and chloroform/methanol mixture was employed in this study Effect of drying temperature Fig. 1 shows the effect of drying temperature on the lipid content. Heating at 60 8C resulted in a slight decrease of lipid content but when heating was conducted at 80 8C or higher temperature, the lipid content decreased significantly. Table 1 shows that the content of TG tend to decrease when higher temperature was applied for the drying of algal sample. Oxidation of fatty acid upon exposing to high temperature has been reported (Oehrl et al., 2001). They reported that unsaturated fatty acid, especially polyunsaturated free fatty acid (PUFA), was more susceptible to oxidation than saturated fatty acid. The reasonable explanation of the degradation of TG in Table 1 was the oxidation of TG at high temperature for 12 h. The TG in microalgal cells obtained in the present works should consist of highly unsaturated fatty acids. Bockisch (1998) and Choe and Min (2007) also reported that the degradation of TG by oxidation also resulted in the formation of hydroperoxide group ( OOH) in the chain. The hydroperoxides formed can react further to aldehydes, ketones,

4 16 A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) Table 2 Comparison of lipid composition under different ultrasonication time Major lipid components Composition (%) 0 min 15 3 min 30 3 min 60 3 min C16 FFA C18 FFA DG TG Others Fig. 1. The effect of drying temperature on lipid content. Total lipid content was calculated as w/w ratio of chloroform/methanol soluble fraction to dried algal sample. Drying at 0 8C was provided by freeze drier, while drying at other temperatures was conducted using oven for 12 h until all the water was removed, i.e. final water content of ca. 0%. Data are expressed as mean values deviation (n = 3). Table 1 Comparison of lipid composition under different drying temperature Major lipid components Composition (%) 0 8C 608C 808C 100 8C C16 FFA C18 FFA DG TG Others and fatty acids. The results in Table 1 in which the content of other compounds was increased seemed to confirm the previous observation. The others in Table 1 should therefore contained aldehydes and ketones. The resistance of DG after drying may indicate that they consisted of saturated fatty acids which were not easily broken down in comparison to the highly unsaturated fatty acid that may construct the TG. and it was found that ultrasonication reduce the extraction time without any significant different in yield (data not shown). It can be concluded from this result that most all lipids were accumulated in the extracellular part of the microalgal cells and sufficient pulverization is enough to help all the lipid extracted from the cells without a necessity of applying ultrasonication to obtain higher yield of lipid Effect of CO 2 concentration, nitrogen depletion and harvesting time on microalgal growth, lipid content, lipid composition and productivity Effect of CO 2 concentration on growth Fig. 3 shows the growth of algae under different CO 2 concentration. At 15 d incubation time, the figure shows that increasing CO 2 flow rate until 50 ml/min enhanced the growth tremendously. However, increasing CO 2 flow rate further to 100 and 150 ml/min gave no increase on the growth (data not shown). As shown in the figure, increasing to 200 ml/min CO 2 flow rate seemed to give even worse results by which until 2 d of incubation time the growth rate was lower than that using 50 ml/min CO 2. The data obtained in the experimental results agreed with the data reported by Riebesell et al. (2000) in which they found that increasing CO 2 concentration of up to 1% of air will increase lipid produced by algae. Sorensen et al. (1996) explained the mechanism of converting CO 2 to biomass as follows: Effect of ultrasonication time The effect of ultrasonication during extraction was investigated and the results on total lipid content and lipid composition were shown in Fig. 2 and Table 2, respectively. It can be seen from both Fig. 2 and Table 2 that ultrasonication gave neither effect to the increase of extracted lipid nor to the lipid composition. Extraction without enough pulverization of dried algal sample was conducted Fig. 2. Comparison of total lipid content under different ultrasonication time. Solvent extraction was conducted three times each of which used different ultrasonication time of 0, 15, 30 and 60 min as indicated in the figure. Data are expressed as mean values deviation (n = 3). Using higher concentration of CO 2 may result in decreasing the ph since unutilized CO 2 will be converted to H 2 CO 3. On the other hand, if there is not enough CO 2 gas supply, algae will utilize carbonate to maintain its growth. Since algae use CO 2 (aq) from bicarbonate as a compensation of lacking enough CO 2 from gas supply, this will result in increase of ph. Table 3 shows the ph range under different CO 2 concentration. Higher CO 2 flow rate decreased the ph but during nitrogen starvation, the ph was practically stable at around 7. As can be seen from Fig. 3, atco 2 flow rate of 200 ml/min, the growth was once very slow with ph dropped to about 5. But, after 2 d, the growth increased greatly indicating that the algae recovered from low ph due to exposing at very high-co 2 concentration. At this condition, the ph was monitored to increase from about 5 to 6.4 and constant around this value which was the same ph range as that using lower CO 2 flow rate. As the growth recovered at the same time during the

5 A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) Fig. 3. Comparison of growth curve under normal nutrition at an air flow rate of 6 L/min with the addition of pure CO 2 gas at a flow rate of (*) 0 ml/min, (&) 20 ml/min, (^) 50 ml/min and (~) 200 ml/min. Optical density (OD) was absorbance measured at 682 nm. Table 3 Range of ph measured under different CO 2 concentration [CO 2 ] (ml/min) ph Normal nutrition N depletion Fig. 5. Comparison of lipid productivity during normal nutrition and nitrogen starvation at CO 2 flow rate of 20 ml/min. Incubation time under normal nutrition was conducted for (&) 15 d and (&) 20 d. After normal nutrition, the medium was changed to nitrogen depletion and the growth continued for 7 and 17 d. Oil productivity was calculated as total lipid (mg) obtained per volume (L) of culture per total incubation time (d) since the beginning of normal nutrition culture until harvesting time. Data are expressed as mean values deviation (n = 3). Table 4 Typical information required to calculate lipid productivity Parameters Incubation time 15 d 20 d gradual increase of ph, it was evidenced from this result that the microalgae C. vulgaris could survive under low ph albeit the growth was slow. Iwasaki et al. (1996) reported the similar behavior of a green algae Chlorococcum littorale in which under sudden increase of CO 2, activity of algae decreased temporarily and then recovered after several days. The fact that C. vulgaris can survive at a wide range of ph from 5 to above 8 was beneficial in considering of applying the algae in any conditions such as very low ph under direct flue gas from power plant or higher ph when exposed to not enough CO 2 source Effect of nitrogen depletion and harvesting time Fig. 4 shows the lipid content obtained at the end of linear phase during normal nutrition and the results were compared with lipid content obtained during nitrogen starvation using 20 ml/min CO 2 flow rate. Period of incubation during normal nutrition was also Fig. 4. Comparison of total lipid content during normal nutrition and nitrogen starvation at CO 2 flow rate of 20 ml/min. Incubation time under normal nutrition was conducted for (&) 15 d and (&) 20 d. After normal nutrition, the medium was changed to nitrogen depletion and the growth continued for 7 and 17 d. Total lipid content was calculated as w/w ratio of chloroform/methanol soluble fraction to dried algal sample. Data are expressed as mean values deviation (n = 3). Cell concentration (10 7 cells/ml) Biomass/mL culture (mg/ml) Total lipid content (%) Lipid productivity (mg/(l d)) varied to investigate the difference. Fig. 4 shows that lipid content obtained after 20 d was higher than that obtained after 15 d. This was due to longer incubation time which led to less nitrogen concentration in the medium. Fig. 4 also shows that longer time of nitrogen starvation obviously resulted in higher accumulation of lipid inside the cells. Fig. 5 shows the lipid productivity calculated from the data in Fig. 4. Typical calculation of productivity was given in Table 4. As shown in this table, cell concentration obtained after 20-d incubation was significantly higher than that obtained after 15 d which led to higher amount of dried algal sample for lipid extraction. Higher lipid content and higher biomass obtained resulted in higher productivity after 20-d normal nutrition period. However, Fig. 4 also shows that after exposing to nitrogen starvation condition for 17 d, lipid content were almost the same for both batches. As a consequence, Fig. 5 shows that lipid productivity obtained after 17-d nitrogen depletion was higher since total time required for incubation was shorter. This 15-d period of normal nutrition was employed for further investigation. The results in Figs. 4 and 5 clearly show the two phenomena occurred in this experimental results. The first one was that lipid content was increased by exposing to 7-d nitrogen starvation condition and increased further under longer N starvation of 17 d. The second phenomena was that lipid productivity was once decreased by changing from normal nutrition to 7 d of nitrogen depletion and then increased back again at longer nitrogen starvation time. Figs. 4 and 5 also reveals that higher lipid productivity can be obtained by varying not only the length of nutrient starvation but also the length of normal nutrition.

6 18 A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) Table 5 Composition of major lipid components under different nutrient condition Major lipid components Composition (%) Normal nutrition 7-D nitrogen depletion 17-D nitrogen depletion C16 FFA C18 FFA DG TG Others Lipid composition during normal nutrition and nitrogen starvation was analyzed using gas chromatography and the results were shown in Table 5. It is clear from the table that exposing to longer nitrogen starvation resulted not only in increasing total lipid content but also in increasing the content of TG. The lipid composition gradually changed from free fatty acid-rich lipid to TG-rich microalgal lipid with far less amount of impurities. As far as we know, this is the first report concerning the gradual change of the lipid composition in microalgal cells upon exposing to nitrogen starvation. In the viewpoint of converting the lipid for biodiesel production, this changing is more favorable. Component of others in Table 5 may represent low concentration of other fatty acids, hydrocarbon, and bioactive compound which are usually not valuable for direct production of biodiesel (Allard and Templier, 2000). Isolation of these components, especially important bioactive compound, however, may have significant economical benefits which can support the economical value of the whole biodisel production process Effect of CO 2 concentration on lipid content and productivity The effect of CO 2 on growth as given in Fig. 3 correlates directly to the lipid productivity since growth was enhanced tremendously by increasing the CO 2 concentration. Effect of CO 2 concentration on lipid content and lipid productivity were given in Figs. 6 and 7, respectively. As shown in Fig. 6, under all CO 2 concentrations, the lipid content tend to increase when the algae was exposed to nitrogen starvation condition. Fig. 7 shows that during normal nutrition, lipid productivity increased as CO 2 flow rate was increased. Similar with the results obtained in Fig. 5, exposing at nitrogen starvation condition once resulted in decreasing the lipid productivity although Fig. 6 shows that total lipid content was higher than lipid obtained during normal nutrition (result of 7-d nitrogen depletion). This was caused by the slow growth of algae under nitrogen depletion. However, for 0 and 20 ml/min CO 2, exposing at longer time of nitrogen depletion (17 d) resulted not only in higher lipid content but also in increasing the lipid productivity at about the same or even higher than lipid productivity at the end of normal nutrient. At CO 2 flow rate of 50 ml/min, lipid content after 17-d nutrient starvation reached more than 50% which was the highest value compared to lipid content using lower CO 2 flow rate. Although the lipid productivity under this condition cannot reach the same value as that obtained under normal nutrition, however, this condition may still be better due to its TG-rich lipid composition as mentioned in the previous section. Furthermore, higher productivity than normal nutrition can still be achieved by shortening the incubation time of less than 17 during nitrogen starvation. This study investigated several important parameters responsible for increasing lipid content and lipid productivity from fresh water microalgae C. vulgaris. The biomass and lipid productivity obtained in the present work are not necessarily superior to those reported elsewhere using different strain of microalgae. However, Fig. 6. Effect of CO 2 concentration and nitrogen depletion on total lipid content. After using normal nutrition, the medium was changed to nitrogen depletion and continued the growth for 7 and 17 d. The CO 2 flow rate was ( ) 0 ml/min, (&) 20 ml/min, and (&) 50 ml/min. Total lipid content was calculated in the same way as Fig. 2. Data are expressed as mean values deviation (n = 3). Fig. 7. Effect of CO 2 concentration and nitrogen depletion on lipid productivity. After using normal nutrition, the medium was changed to nitrogen depletion and continued the growth for 7 and 17 d. The CO 2 flow rate was ( ) 0 ml/min, (&) 20 ml/min, and (&) 50 ml/min. Oil productivity was calculated in the same way as Fig. 3. Data are expressed as mean values deviation (n = 3). the simple and inexpensive method conducted in this study which resulted in a significant increase of lipid content while maintaining the productivity at a high value can easily be applied to other strain of microalgae. Improving of nutrient conditions such as the use of spesific but inexpensive compounds which can increase the growth, genetic engineering of DNA responsible for capability of producing high content of lipid, as well as design of photobioreactor giving better performance of the fermentation process are several other parameters that can be optimized. The study in the present report is therefore expected to give one part of significant contributions for the strategy of developing biodiesel production technology Concluding remark Fresh water microalgae C. vulgaris was chosen as the subject of investigating the lipid productivity due to its easy growth and its significant lipid content. Factors responsible for the increase of lipid productivity such as CO 2 concentration, nitrogen depletion, harvesting time and extraction method were investigated.

7 A. Widjaja et al. / Journal of the Taiwan Institute of Chemical Engineers 40 (2009) The drying temperature during lipid extraction from algal biomass was found to affect both the lipid composition and the lipid content. Drying using freeze drier will retain the original composition of microalgal lipid, while higher temperature drying decreased the content of TG. Drying at 60 8C still retained the composition of lipid and only decreases slightly the lipid content. No significant effect was found in the effect of ultrasonication on extraction. Sufficient pulverization is enough to help the entire lipid extracted from the cells. Higher lipid content was obtained by exposing to 7-d nitrogen starvation condition and increased further under 17-d N starvation. Interestingly, the lipid productivity was once decreased under 7-d N depletion condition and then increased back again under 17-d N starvation. Furthermore, it was found that cultivating in nitrogen depletion media will result not only in the accumulation of lipid in microalgal cells but also in gradual changing in the lipid composition from free fatty acid-rich lipid to lipid mostly contained TG. This new finding will be very important in the viewpoint of further application for biodiesel production. Since accumulation of lipid occurs at nitrogen depletion condition under which the growth was much slower or even no growth was encountered, compromising between increasing lipid content and harvesting time was necessary to obtain higher values of both the lipid content and lipid productivity. CO 2 concentration played an important role in the increase of lipid productivity since the growth was much affected by CO 2 concentration. At 0 until 20 ml/min CO 2 flow rate, the lipid productivity during nitrogen depletion could be higher than that obtained at the end of linear phase during normal nutrition. 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