Department of Chemical and Materials Engineering,. Tim Keener ... Allied chemical, and J. T. Baker, respectively. ... from Fisher Scientific Company. The culture ...
Growth Kinetic Study of Chlorella Vulgaris Jinsoo Kim, Joo-Youp Lee, * Department of Chemical and Materials Engineering, Tim Keener, Department of Civil and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio, 45221, USA Abstract Chlorella Vulgaris is known as one of the fastest growing microalgae, and has been selected for this kinetic study. Its lab-scale growth kinetic data have been obtained in terms of different operating conditions including pH and uptake rates of total inorganic carbon content. As a result, Chlorella vulgaris cultured at initial pH 7, 8, and 9 culture media grew constantly in contrasts to the algae grown at pH 5 and 10 did not grow well due to the harsh condition. Corresponding to the result, the lag time of the algae at pH 5 and 10 is longer than that of other pH conditions. Subsequently, Chlorella vulgaris grown at pH 7 showed the highest maximum cell number. Consequently, it was noted that pH 7 is the best pH condition for Chlorella vulgaris and sodium carbonate can be used as a substantial source of carbon dioxide for the photosynthesis. Introduction Microalgae are getting growing attention as they have a significant amount (e.g., 20−50% dry cell weight [13]) of triacylglycerols for biodiesel production. Chlorella vulgaris is known as one of the fastest growing microalgae and includes a reasonable amount (14-22%) of triacylglycerol [1] for biodiesel production. In this regard, lipid extraction from Chlorella vulgaris was investigated [2], and also several growth kinetic studies of Chlorella vulgaris were explored with respect to different ratios of air-CO2 mixture gas, initial pH [3-5] and phosphorus concentration [6]. Carbon dioxide is one of the critical factors for photosynthesis of plant along with light, water, and nutrients. In this respect, previous researches attempted to blow different ratios of air-CO2 mixture gas (95/5, 90/10 and 80/20 (v/v)) [5]. However, in this case, lots of carbon dioxide was apt to lose in the air because the solubility of carbon dioxide is very low (1.45 g/L at 25 oC, 100 kPa) [7]. When CO2 dissolves in water, three inorganic carbon species are present in the aqueous phase: CO2 (aq), bicarbonate, and carbonate ions as shown in Figure 1. In this study, sodium carbonate is used as a source of inorganic carbon for photosynthesis of Chlorella vulgaris. Sodium carbonate is well dissolved in water due to the high solubility (29.4 g/100 g of H2O (25 oC) in water [8]. This method does not require power consumption for bubbling CO2 gas in the aqueous phase, and can minimize the carbon loss to the atmosphere by saturating the bicarbonate concentrations in an appropriate pH range for algae culture. Bicarbonate ions as well as carbon dioxide are also known to be used for photosynthesis of algae [9-11]. When sodium carbonate is dissolved in water, the pH of a solution also increases and can retain high inorganic carbon content in the aqueous phase.
Figure 1. Schematic representation of water/carbonate equilibrium Experimental 2.1. Preparation of culture medium The preparation of culture medium was following the shuisheng-4 method [12]. Ammonium sulfate, boric acid, cupric sulfate were purchased from EM science, Allied chemical, and J. T. Baker, respectively. Ammonia sulfate, calcium phosphate monobasic monohydrate, magnesium sulfate hetahydrate, sodium bicarbonate, potassium chloride, Iron (III) chloride, potassium phosphate dibasic, manganese chloride, zinc sulfate, sodium molybdate was bought from Fisher Scientific Company. The culture medium was made by mixing with 200 mg/l of ammonia sulfate ((NH4)2SO4), 30 mg/l of calcium phosphate monobasic monohydrate (Ca(H2PO4)2·H2O), 100 mg/l of magnesium sulfate hetahydrate (MgSO4·7H2O3) 80 mg/l, 100 mg/l of sodium bicarbonate (NaHCO3), 25 mg/l of potassium chloride (KCl), 1.5 mg/l of Iron (III) chloride (FeCl3), 10 mg/l of potassium phosphate dibasic (K2HPO4), and 1ml of A5 liquid in distillated water. Subsequently, A5 liquid was prepared by mixing with 2.86 g/l of boric acid (H3BO3), 1.81 g/l of manganese chloride (MnCl2·4H2O), 0.222g/l of zinc sulfate (ZnSO4·7H2O), 0.391 g/l of sodium molybdate (Na2MO4·2H2O), 0.079g/l of cupric sulfate (CuSO4·5H2O) [12]. The culture medium was sterilized at 125 oC for 30 min. 2.2. Controlling pH of each medium and preparing individual sample (S1-S6) The pH of prepared 4.2 liter of culture medium was increased to 10.00 using by 4.5 g of sodium carbonate purchased from Fisher Scientific while mixing with 700 rpm. Then, the resulting medium was transferred to 700 ml of individual six culture solutions (i.e., S1−S6) in 1000 ml Pyrex tall form beakers. The pH of S1 was adjusted to pH 7 by injecting 99.5 % carbon dioxide purchased from Wright Brother, Inc. The pHs of other solutions were adjusted to pH 5 (S2), pH 7 (S3), pH 8 (S4), pH 9 (S5) and pH 10 (S6), respectively, by 36.5−38.0 % of hydrochloric acid and sodium carbonate using Oakton pH 11 Standard Portable meter. Then, every 5 ml of Chlorella vulgaris suspended in the shuisheng-4 culture medium which has 22 (±3)
cell number in a 0.2 mm × 0.2 mm square space (Reichert Bright-Line counting chamber) transfused into respective culture medium. 2.3. Light condition 6,500 K fluorescent lamps were used as the source of light, and the incoming light intensity to beakers was set to 6,000 lux (100.8 µmol⋅m-2⋅s-1) for all of the cultures (i.e., S1−S6) by controlling the distance between the beaker and the lamp. The light intensity was measured using a light intensity meter (HQRP digital lux meter, LX1010BS, Osprey-Talon Company), and a 16-hr light and 8-hr dark cycle was applied to all the cultures. 2.4. Determination of algal cell density Reichert Bright-Line counting chamber (Fisher Scientific, Inc., Pittsburgh, PA) was used for determining the number of algal cells. 0.3 μL of a sample was taken from a culture solution using a 1 μL Hamilton micro syringe (Sigma-Aldrich). Then, the sample was placed on the counting chamber, and the cell number in a 0.2 mm × 0.2 mm square space was counted by using the Nikon Labophot-II microscope. Results and discussion As shown in Figure 2 (black symbol and line), both S1 and S3 continued to grow for 6 days. Sodium carbonate was used as a carbon source for both S1 and S3 samples, but CO2 gas and HCl solution were used to adjust initial pHs for S1 and S3, respectively. Both samples started to grow after 2 days. S1 grow dramatically after 3 days while S3 gradually grow over the same period. It seems that Cl ion does somehow impact on the growth rate. The growth kinetics of Chlorella vulgaris under different initial pHs are shown in Figure 2 (blue symbol and line). S3 through S5 cultured in the initial pHs of 7, 8, and 9 continuously grow for 6 days. In contrast, S2 and S6 cultured in initial pHs 5 and 10 showed longer lag periods as summarized in Table 1 and had smaller maximum cell numbers after 6 days. After a 3-day lag period, S2 cultured with initial pH 5 started to grow, and the cell numbers started to decrease after 5 days. Overall, Chlorella vulgaris grows best in S1 and S3 cultured at initial pH 7. It has been reported that pH increases when algae grow [14]. The pHs of S1, S3, S4, and S5 cultivated with initial pH of 7, 8, and 9 continued to increase and reached up to 9.72, 9.69, 9.60, and 9.66. Although their initial pHs were different, the pHs of the culture solutions reached 9.31, 9.31, 9.31, and 9.28, respectively, after 3 days. It was noted that the growth rapidly increased when pH reached ~9. Meanwhile, the pH of S2, the culture with initial pH 5, reached a maximum pH at 7.38 after 3 days and then started to decrease. In addition, S6, the culture with initial pH 10, did not show significant differences in pH and cell growth.
Figure 2. Growth kinetic and pH change of Chlorella vulgaris dependent upon initial pH variance. (a) Growth kinetic and pH change grown in S1 culture medium (pH 7), (b) growth kinetic and pH changes grown in S2 culture medium (pH 5), (c) growth kinetic and pH change grown in S3 culture medium (pH 7), (d) growth kinetic and pH change grown in S4 culture medium (pH 8), (e)
growth kinetic and pH change in S5 culture medium (pH 9), and (e) growth kinetic and pH change grown in S6 culture medium (pH 10). Table 1. Lag period of Chlorella Vulgaris cultures. Name Carbon source pH Lag phase period (days) S1 Sodium carbonate 7 2 S2 Sodium carbonate 5 3 S3 Sodium carbonate 7 2 S4 Sodium carbonate 8 2 S5 Sodium carbonate 9 2 S6 Sodium carbonate 10 5 Conclusions Chlorella vulgaris has been cultivated under different initial pH conditions with sodium carbonate as a carbon source for photosynthesis. The cultures with initial pH of 7−9 continue to grow after a short 2-day lag period, and the pH of the cultures reached pH=~9.3, where bicarbonate ions are the dominant inorganic carbon species and have been reported to be utilized for their growth. The inorganic carbon equilibrium in the aqueous phase indicates that a maximum amount of bicarbonate ions are present at pH=~8.3. However, a culture with initial pH=8 (S4) did not show the fastest kinetics and the initial pH does not fully account for the growth kinetics. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Becker, E.W. (1994), “10. Microalgae: biotechnology and microbiology,” Cambridge University Press, New York, USA, pp. 177-195. Widjaja, A., Chien, C.C., Ju, Y.H. (2009) “Study of increasing lipid production from fresh water microalgae Chlorella vulgaris,” J. Taiwan. Inst. Chem. Eng. 40, pp. 13-20. Azov, Y. (1982), “Effect of pH on inorganic carbon uptake in algal cultures,” App. Environ. Microbiol. 43(6), pp. 1300-1306. Goldman, J.C., Azov, Y., Riley, C.B. and Dennett, M.R. (1982), “The effect of pH in intensive microalgal cultures. I. Biomass requlation,” J. Exp. Mar. Biol. Ecol. 57, pp.1-13. 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,” Bioresour. Technol. 100, pp. 269-274. Grover, J.P. (1989) “Phosphorus-dependent growth kinetics of 11 species of freshwater algae,” Limnol. Oceanogr., 34(2), pp. 341-348. Wiebe, R., Gaddy, V.L. (1940) “The solubility of carbon dioxide in water at various temperatures from 12 to 40o and at pressures to 500 atmospheres. Critical Phenomena,” J. Am. Chem. Soc. 62, pp. 815-817. Kobe, K.A., Sheehy, T.M (1948) “Thermochemistry of sodium carbonate and its solutions,” Ind. Eng. Chem. 40(1), pp. 99-102. Osterlind, S. (1951) “Inorganic source of gree algae. IV. Photoactivation of some factor necessary for bicarbonate assimilation,” Plant Physiol. 4, pp. 514-527. Arizawa, K. and Miyachi., S. (1986), “Carbonic anhydrase and CO2 concentrating mechanisms in microalgae and cyanobacteria,” FEMS Microbiol. Rev. 39, pp. 215-233.
11. 12. 13. 14.
Moroney, J.V., Somanchi, A. (1999) “How do algae concentrate CO2 to increase the efficiency of photosynthetic productivity of microalgae,” Biotechnol. Bioeng. 69, pp. 693-698. Zhou, Y.X., Zhang, Z.S. (1989) “The toxicity methods in aquatic living,” Agriculture Press, Beijing, China, pp. 36-44. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A. (2008) “Microalgal triacylglycerols as feedstocks as feedstocks for biofuel production: perspectives and advances,” Plant J. 54, pp. 621-639. Anderson, R.A. (2005) “Algal culturing techniques,” Elsevier Inc., San Diego, USA, pp. 35-64.
