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LIPID PRODUCTION FROM MICROALGAE AS A PROMISING CANDIDATE FOR BIODIESEL PRODUCTION

Arief Widjaja
Department of Chemical Engineering, Institute of Technology Sepuluh November, Surabaya 60111, Indonesia E-mail: [email protected]

Abstract
Recently, several strains of microalgae have been studied as they contain high lipid content capable to be converted to biodiesel. Fresh water microalgae Chlorella vulgaris studied in this research was one of the proof as it contained high triacyl glyceride which made it a potential candidate for biodiesel production. Factors responsible for good growing of microalgae such as CO2 and nitrogen concentration were investigated. It was found that total lipid content was increased after exposing to media with not enough nitrogen concentration. However, under this nitrogen depletion media, the growth rate was very slow leading to lower lipid productivity. The productivity could be increased by increasing CO2 concentration. The lipid content was found to be affected by drying temperature during lipid extraction of algal biomass. Drying at very low temperature under vacuum gave the best result but drying at 60oC slightly decreased the total lipid content. Keywords: biodiesel, lipid, microalgae, nitrogen concentration, productivity

1. Introduction
Microalga 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, CO2, and other nutrients. 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 sitespecific 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. There are several ways to make biodiesel, and the most common way is transesterification as the biodiesel from transesterification can be used directly or as blends with diesel fuel in diesel engine [1-2]. 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 [3-5]. High dependence on foreign oil, especially transportation sector, gives rise to the importance of producing biodiesel for the sake of national energy security. Microalgae have been suggested as very good candidates for fuel production because of their advantages of higher photosynthetic eficiency, higher biomass production and faster growth compared to other Table 1. Several Lipid Producing Microalgae Triolein equivalents (mg x L-1) exponential growth 8 Triolein equivalents (mg x L-1) N deficient growth 1003

Strain

Spesies

NITZS54

ASU3004

FRAGI2

AMPHO27

Nitzschia Bacillariop hyceae Amphora Bacillariop hyceae Fragilaria Bacillariop hyceae Amphora Bacillariop hyceae

593

304

38

235

47

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energy crops [6-7]. 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 (CO2) and converting it into a high density liquid form of energy (natural oil). Table 1 gives several lipid producing microalgae capable to produce biodiesel [8]. The present research aimed to produce lipid contained in fresh water microalgae C. vulgaris in a closed fermentor. The effect of CO2 concentration and nitrogen concentration on lipid content were investigated as well effect of drying temperature during lipid extraction.

using UV-530 JASCO Spectrophotometer, Japan. Cells were harvested at the end of linear phase, i.e. at a cell concentration of about 1.1 x 107 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 days at which time the cells were harvested and the lipid content as well as lipid productivity was measured. Other conditions of incubation such as light intensity, pure CO2 gas flow rate and temperature were all the same as the corresponding normal nutrition condition. Effect of CO2 concentration The effect of CO2 concentration on lipid content, lipid composition and productivity was investigated by varying the CO2 concentration. At first, the culture was aerated under air flow rate of 6 L/min without additional CO2. By taking into account the CO2 content in air of about 0.03%, this condition resulted in about 2 mL/min CO2 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 CO2 gas, or about 0.33, 0.83, 1.67, and 3.33% CO2, respectively. Lipid extraction Dry extraction procedure according to Zhu [9] 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 60oC. The procedure was repeated three times until the entire lipid was extracted. The effect of drying temperature was investigated in this study. Gas chromatography analysis Sample was dissolved in ethyl acetate and 0.5 L 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 x 0.32 mm I.D); Agilent Tech. Palo Alto, California). Injection and detector temperature both were 370oC. Initial column temperature was 240oC, and the temperature was increased to 300oC at a temperature gradient of 15oC/min.

2. Methods
Materials A microalgal strain of C. vulgaris was kindly provided by Prof. Hong-Nong Chou of The Institute of Fisheries Science, National Taiwan University, Taiwan. 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 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: NaNO3, 85.0 g; CaCl2 2H2O, 3.70 g. IBI (b) contained, per 200 mL: MgSO4 7H2O, 24.648 g. IBI (c) contained, per 200 mL: KH2PO4, 1.36 g; K2HPO4, 8.70 g. IBI (d) contained, per 200 mL: FeSO4 7H2O, 1.392 g; EDTA tri Na, 1.864 g. IBI (e) contained , per 200 mL: H3BO3, 0.620 g; MnSO4 H2O, 0.340 g; ZnSO4 7H2O, 0.057 g; (NH4)6Mo7O24 4 H2O, 0.018 g; CoCl2 6H2O, 0.027 g; KBr, 0.024 g; KI, 0.017 g; CdCl2 5/2 H2O, 0.023 g; Al2(SO4)3(NH4)2SO4 24H2O, 0.091 g; CuSO4 5H2O, 0.00004 g; 97% H2SO4, 0.56 ml. This normal nutrition medium resulted in a nitrogen content of 70.02 mg/L medium. The nitrogen depletion medium was provided by eliminating the addition of IBI (a) to result in a medium with a nitrogen content of 0.02 mg/L medium. Effect of nitrogen concentration At first, cells of C. vulgaris were cultivated in 4 L normal nutrition medium and incubated batchwisely at 22oC. The system was aerated at an air flow rate of 6 L/min with or without the addition of pure CO2 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 E/m2s. The optical density of cells was measured at 682 nm every 24 hr

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3. Results and Discussion

Effect of CO2 concentration on growth Sobczuk et al. [10] reported that the yield of biomass increased significantly when the CO2 molar fraction in the injected gas was reduced. They also showed that with less CO2 in the injected gas, the O2 generation rate and the CO2 consumption rate were greater. Riebesell and his co workers [11] studied the effect of varying CO2 concentration on lipid composition. They found that increasing CO2 concentration of up to 1% of air will increase lipid produced by algae. Figure 1 shows the growth of algae under different CO2 concentration. The figure shows that increasing CO2 flow rate until 50 mL/min enhanced the growth tremendously. Further increase of CO2 may result in decreasing the growth rate. Table 2 shows the pH range under different CO2 concentration. Higher CO2 flow rate decreased the pH but during nitrogen starvation, the pH was practically stable at around 7. As can be seen from Figure 1, at CO2 flow rate of 200 mL/min, the growth was once very slow with pH dropped to about 5. But, after two days, the growth increased greatly indicating that the algae recovered from low pH due to exposing at very high CO2 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 CO2 flow rate. As the growth recovered at the same time during the gradual increase of pH, it was evidence from this result that the microalgae C. vulgaris could survive under low pH albeit the growth was slow. Iwasaki et al. [12] reported the similar behavior of green algae Chlorococcum littorale in which under sudden increase of CO2, activity of algae decreased temporarily and then recovered after several days. The fact that C. vulgaris can survive at 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 CO2 source. Effect of nitrogen depletion on lipid content and productivity Figure 2 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. Period of incubation during normal nutrition was also varied to investigate the difference. Figure 2 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. Figure 2 also shows that longer time of nitrogen starvation obviously resulted in higher accumulation of lipid inside the cells. Figure 3 shows the lipid productivity obtained during this period of time. Typical calculation of productivity

was given in Table 3. As shown in this table, cell concentration obtained after 20 days incubation was significantly higher than that obtained after 15 d which led to higher amount of dried algal sample for lipid consequence, lipid productivity obtained after 17 d nitrogen depletion was higher since total time required for incubation was shorter. This 17 d period of normal nutrition was employed for further investigation. Figure 2 and 3 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.
3 2,5 2 OD (Abs) 1,5 1 0,5 0 0 5 10 Time (d) 15 20 25

Figure 1. Growth of Microalgae Under Various CO2 Flow Rrate of ({) 0 mL/min, () 20 mL/min, () 50 mL/min and (U) 200 mL/min, all of which Supplied with an Air Flow Rate of 6 L/min

Table 2. Range of pH Measured Under Different CO2 Concentration

[CO2] mL/min 0 20 50 200

Total lipid content (%) 50 40 30 20 10 0

pH Normal Nutrition N depletion 6.86 8.33 7.49 8.30 6.74 7.15 6.88 7.00 6.16 7.01 6.40 6.90 5.44 6.44 6.01 6.30

normal

7 days N depletion Nutrient condition

17 days N depletion

Figure 2.

Lipid Content in Microalgae at Various N Condition. Incubation Time Under Normal Nutrition was Conducted for () 15 d and () 20 d

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Lipid productivity (mg/L/d)

14 12 10 8 6 4 2 0 normal 7 days N depletion Nutrient condition 17 days N depletion

Effect of drying temperature during lipid extraction Figure 4 shows the effect of drying temperature on the lipid content. Heating at 60oC resulted in a slight decrease of lipid content but when heating was conducted under 80oC or higher temperature, the lipid content decreased significantly. Effect of CO2 concentrantion on lipid productivity The effect of CO2 on growth as given in Figure 1 correlates directly to the lipid productivity since growth was enhanced tremendously by increasing the CO2 concentration. Effect of CO2 concentration on lipid productivity was given in Figure 5. As shown in Figure 5, under all CO2 concentrations, the lipid content tend to increase when the algae was exposed to nitrogen starvation condition. Similar with the results obtained in Figure 3, exposing at nitrogen starvation condition once resulted in decreasing the lipid productivity. This was caused by the slow growth of algae under nitrogen depletion. However, exposing at longer time of nitrogen depletion (17 days) 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.

Figure 3.

Lipid Productivity by Microalgae at Various N Condition. Incubation Time Under Normal Nutrition was Conducted for () 15 d and () 20 d

53.00 Lipid content (%) 52.00 51.00 50.00 49.00 48.00 47.00 0 60 80
o

100

4. Conclusion
Fresh water microalgae C. vulgaris was a good candidate for Biodiesel production due to its lipid content in addition to its easy growth. It was found that cultivating in nitrogen depletion media will result in the accumulation of lipid in microalgal cells. Although lipid productivity was slow under nitrogen starvation due to slow growth rate of algae, its lipid productivity during nitrogen depletion could be higher than that obtained at the end of linear phase during normal nutrition. The drying temperature during lipid extraction from algal biomass was found to affect the lipid content. Drying at 60oC only slightly decrease the lipid content.

Drying temperature ( C)

Figure 4. Lipid Content at Various Drying Temperature Table 3. Typical Information Required to Calculate Lipid Productivity

Incubation time 15 d 20 d Cell concentration 1.1 x 107 cell mL-1 1.3 x 107 cell mL-1 Biomass/mL culture 0.55 mgmL-1 0.86 mgmL-1 Total lipid content 26.71% 29.53% Lipid productivity 9.75 mg L-1d-1 12.77 mgL-1 d-1 Parameters
12 10 8 6 4 2 0 normal 7 days N depletion Nutrient condition 17 days N depletion

Acknowledgement
Lipid productivity (mg/L/d)

The author expresses sincere thanks to Prof. Yi-Hsu Ju from Dept. of Chemical Engineering, NTUST, Taiwan for all the help he provided.

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Figure 5. Lipid Production at Various CO2 Flow Rate of () 0 and () 20 mL/min

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