- Email: [email protected]
Improved arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation
Bioresource Technology 88 (2003) 265–268
Short Communication
Improved arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation L.J. Yu *, W.M. Qin, W.Z. Lan, P.P. Zhou, M. Zhu School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, China Received 18 October 2001; received in revised form 25 November 2002; accepted 27 November 2002
Abstract The effect of various concentrations of glutamate on arachidonic acid (AA) production from Mortierella alpina in shaker flask culture was studied. Glutamate supplementation promoted Mortierella growth, accelerated substrate metabolism, and increased AA production, and a concentration of 0.8 g/l glutamate resulted in the greatest AA yield (1.41 g/l). In 10 l airlift stirred fermenter culture, AA yield in the cultures exposed to 0.8 g/l glutamate was also greater than that in the control (0.56 g/l). Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Arachidonic acid; Glutamate; Mortierella alpina
1. Introduction
2. Methods
Arachidonic acid (AA), a long chain polyunsaturated fatty acid (PuFA) of the omega-6 class (5,8,11,14-eicosatetraenoic acid), plays important roles in the structure and function of biological membranes (Yamada et al., 1989). In addition, it has recently attracted great interest due to several unique biological activities (Das et al., 1987; Horrobin and Huang, 1987). At the present time, the oil extracted from porcine liver is generally used as the main source of AA. However, the AA content of this oil is so low that it is not a practical source of this fatty acid. Therefore, alternative sources are being sought. The cells of some protozoa, algae and fungi are known to contain AA (Yongmanitchai and Ward, 1989; Shinmen et al., 1989; Gandhi and Weete, 1991). Lower fungi of the class of Zygomycetesm, especially Mortierella alpina, producing substantial quantities of AA (Ward, 1995), are a promising source of AA. The present communication represents studies on the effects of glutamate on AA production in shaker flask and fermenter.
2.1. Microorganism and inoculum preparation
* Corresponding author. Tel.: +86-27-8754-3633; fax: +86-27-87540184. E-mail address: [email protected] (L.J. Yu).
Mortierella alpina M18 was maintained on potato paste–dextrose medium (Totani and Oba, 1988) at )4 °C and subcultured every two months. The inoculum was prepared in 50 ml medium containing (g/l): glucose, 100; yeast extract, 5; MgSO4 , 5. The pH was adjusted to 6.0 before autoclaving at 121 °C for 20 min. In preparation for an experiment, a stock culture was vigorously stirred in 100 ml sterilized water with glass beads for 10 min. Ten milliliters of mycelial suspension was transferred to 250 ml shake flasks containing 50 ml medium and incubated at 24 1 °C with shaking at 130 rpm for three days. 2.2. Experimentation and statistical analysis 2.2.1. Experiment 1: shaker flask cultivation The production medium composition was as follows (g/l): glucose, 100; yeast extract, 5; beef extract, 3; MgSO4 , 1.5. In order to determine the effects of glutamate on the AA production, 0, 0.2, 0.4, 0.6, 0.8 g/l glutamate were respectively added to the production medium. The pH was adjusted to 6.0 before autoclaving at 121 °C for 20 min. The culture was grown at 24 1 °C for seven days with shaking at 130 rpm. Five hundred milliliters conical flasks containing 100 ml production
0960-8524/03/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0960-8524(02)00312-7
266
L.J. Yu et al. / Bioresource Technology 88 (2003) 265–268
medium were inoculated with the 72-h-grown inoculum preparation at a rate of 10% v/v. 2.2.2. Experiment 2: scale-up culture The medium used for scale up studies was the production medium supplemented with 0.8 g/l glutamate. The culture was grown at 24 1 °C for seven days in a 10 l airlift stirred fermenter with a 6 l working volume at a 0.6 m3 /h air flow rate. The scale up experiment was conducted using 6 l of medium inoculated with 1 l of 72h-grown inoculum preparation. 2.2.3. Analytical methods Biomass concentration was determined by centrifugation of the fungal cell suspension, washing with distilled water and drying at 100 °C for 12–16 h. Glucose, nitrogen and phosphate were measured on samples from the supernatant of the cell suspension using 3,5-dinitrosalicyclic acid colorimetry (Miller, 1959), folin phenol reagent (Lowry et al., 1951) and phosphomolybdic acid colorimetry assays, respectively (Eisenreich et al., 1975). The lipids were extracted following the method by Bligh and Dyer (1959). The extracted lipids were dried at 36 °C under a nitrogen atmosphere and methylated (Holub and Skeaff, 1987). AA was determined as follows. A gas chromatograph (GC-9A) equipped with a flame ionization detector (FID) and a glass column (Nishio Kogyo, Tokyo) was used. The oven temperature was 210 °C. Hexane was used as the solvent and 1 ll of solution was injected. The chromatograms were analyzed with CR2AX (Institute of Information, University of Zhejiang, China) software. The corrected area normalization of individual polyunsaturated fatty acid (PuFAs) and the internal standard method (Rounds and Nielsen, 1994) were used for PuFAsÕ quantification. The production of PuFAs was expressed as the weight % fatty acid methyl esters for further yield calculations (g/l).
2.3. Statistical analysis Results obtained were the mean of three or more determinations. Analysis of variance was carried out on all data at p < 0:05 with the help of Microsoftâ Excel (USA). When the ÔF Õ-value was found to be significant, critical difference was calculated by multiplying the standard error of difference (SED) with the corresponding ÔtÕ-values at 5% level of probability. Significant differences among the means were determined by using least significant difference test.
3. Results and discussion 3.1. Experiment 1: shaker flask cultivation Glutamate is involved in nitrogen metabolism and is required as an essential precursor of protein and nucleotide synthesis as well as a substrate for energy metabolism (Wice et al., 1981) in the organism. In addition, glutamate can stimulate aerobic glycolysis (Pellerin and Magistretti, 1994). Therefore, glutamate could potentially have a substantial influence on cell growth. Our results showed that glutamate supply promoted Mortierella growth (Table 1). Cliquet and Jackson (1999) also observed that glutamate supported biomass accumulations of Paecilomyces fumosoroseus. Our results also showed that decreases in glucose, nitrogen and phosphate concentration in the medium were accelerated by glutamate supplementation (Table 1). Recently, glutamate was demonstrated to have a role in activating acetyl-CoA carboxylase (ACC; EC 6.4.1.2). ACC catalyzes the formation of malonyl-CoA, an essential substrate for fatty-acid synthase and for fatty acyl chain elongation systems (Kowluru et al., 2001). Further, glutamate carbon can be utilized for fatty acid synthesis, either directly through the generation of keto
Table 1 Effect of glutamate concentration on AA production in the shaker flask culture on the seventh day Glutamate concentration (g/l) Total biomass (g/l) Total lipids in biomass (% w/w) AA in lipid (% w/w) AA in biomass (% w/w) AA yield (g/l) Glucose (g/l) Nitrogen (g/l) Phosphate (mg/l)
0
0.4
0.6
0.8
1.0
1.2
21.56 0.35a 30.56 1.3 12.74 0.7 3.90 0.13 0.84 0.07 27.80 0.81 1.87 0.15 15.13 0.53
23.27 0.51 35.79 2.1 13.48 1.0 5.24 0.25 1.22 0.23 25.25 0.75 1.69 0.12 13.21 0.6
23.51 0.4 36.63 2.4 14.56 1.5 5.31 0.3 1.25 0.32 25.69 0.6 1.56 0.2 11.56 0.45
24.43 0.45 37.69 2.5 15.29 2.1 5.77 0.42 1.41 0.38 24.01 0.64 1.54 0.1 10.55 0.38
24.47 0.65 34.79 3.1 10.80 1.2 3.76 0.25 0.92 0.25 21.26 0.5 1.45 0.13 9.95 0.4
24.78 0.57 31.78 1.8 10.23 1.6 3.27 0.32 0.81 0.2 20.33 0.63 1.21 0.85 8.34 0.34
AA represents arachidonic acid. Significantly different from the control at p < 0:05. ** Significantly different from the control at f < 0:01. a All values are mean and standard deviation of three replicates. *
L.J. Yu et al. / Bioresource Technology 88 (2003) 265–268
267
Table 2 The effect of glutamate supplementation (0.8 g/l) on day seven for biomass, total lipid in biomass, total lipids, arachidonic acid in lipids and biomass, AA yield in the scale-up culture
Control Glutamate *
Dry biomass (g/l)
Total lipid in biomass (% w/w)
Arachidonic acid In lipids (% w/w)
In biomass (% w/w)
Yield (g/l)
16.25 0.08a 18.54 0.75
28.01 0.85 34.12 1.1
12.36 0.55 14.78 0.97
3.44 0.16 5.02 0.21
0.56 0.43 0.93 0.54
Significantly different from the control at p < 0:05. a All values are mean and standard deviation of three replicates.
acids or acetyl-CoA (Albers et al., 1996). In our experiment it appeared that supplementation of less than 0.8 g/l glutamate could increase total lipids and AA production. However, supplementation of greater than 0.8 g/l glutamate led to a decrease in the total lipids in biomass and AA production compared with the response to 0.8 g/l glutamate. Certik et al. (1999) reported that glutamate was one of the potentially available nitrogen sources for increasing G6PDH activities and enhancing PuFA biosynthesis. However, when glutamate concentration is too high, glutamate will be converted to proline accompanied by NADPH consumption (Andarwulan and Shetty, 1999), which is necessary for AA biosynthesis. A concentration of 0.8 g/l glutamate was most favorable for AA production, therefore, it was selected to further study the AA production in the scaleup culture. 3.2. Experiment 2: scale-up culture The effect of glutamate supplementation (0.8 g/l) on day seven for biomass, total lipid in biomass, total lipids, AA in lipids and biomass, AA yield in the scale-up culture are showed in Table 2. The yield of the scale-up culture (Table 2) was lower than the production of the shaker flask culture (Table 1). The difference might be caused by the potentially different dissolved oxygen concentrations between in the medium and in the fermenter (Higashiyama et al., 1999), as well in the fermenter, it is unavoidable that the shearing strength has damaging effect on cell growth (Enfors et al., 2001). However, AA yield of 0.8 g/l glutamate (0.93 g/l) was significantly greater than the production of the control (0.56 g/l) in 10 l airlift stirred fermenter. This is additional evidence that 0.8 g/l glutamate could improve arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation. 4. Conclusions This report presents that addition of 08 g/l glutamate could promoted Mortierella growth, accelerated substrate metabolism, and increased AA production, and a concentration of 0.8 g/l glutamate resulted in the greatest AA yield (1.41 g/l) in 250 ml shaker flask.
Furthermore, in 10 l airlift stirred fermenter, AA yield in the cultures exposed to 0.8 g/l glutamate was also greater than that in the control. Therefore, the application of the 0.8 g/l glutamate could be useful in developing rational strategies for enhancing the production of AA in large-scale fungal M. alpina cultures.
References Albers, E., Larsson, C., Liden, G.L., Niklasson, C., Gustafsson, L., 1996. Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Appl. Environ. Microbiol. 9, 3187–3195. Andarwulan, N., Shetty, K., 1999. Improvement of pea (Pisum sativum) seed vigour response by fish protein hydrolysates in combination with acetyl salicylic acid. Proc. Biochem. 35, 159–165. Bligh, E.G., Dyer, N.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Certik, M., Megova, J., Horenitzky, R., 1999. Effect of nitrogen sources on the activities of lipogenic enzymes in oleaginous fungus Cunninghamella echinulata. J. Appl. Microbiol. 45, 289– 293. Cliquet, S., Jackson, M.A., 1999. Influence of culture conditions on production and freeze-drying tolerance of Paecilomyces fumosoroseus blastospores. J. Ind. Microbiol. Biotechnol. 23, 97–102. Das, U.N., Begin, M.E., Huang, Y.S., Horrobin, D.F., 1987. Polyunsaturated fatty acids augment free radical generation in tumor cells in vitro. Biochem. Biophys. Res. Commun. 145, 15–24. Eisenreich, S.J., Bannerman, R.T., Armstrong, D.E., 1975. A simplified phosphorus analytical method. Environ. Lett. 9, 45–53. Enfors, S.-O., Jahic, M., Rozkov, A., Xu, B., Hecker, M., J€ urgen, B., Kr€ uger, E., Schweder, T., et al., 2001. Physiological responses to mixing in large scale bioreactors. J. Biotechnol. 85, 175–185. Gandhi, S.R., Weete, J.D., 1991. Production of the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid by the fungus Phythium ultimum. J. Gen. Microbiol. 137, 1825–1830. Higashiyama, K., Murakami, K., Tsujimua, H., Matsumoto, N., Fujikawa, S., 1999. Effects of dissolved oxygen on the morphology of arachidonic acid production by Mortierella alpina 1S-4. Biotech. Bioeng. 63, 442–448. Holub, B.J., Skeaff, C.M., 1987. Nutritional regulation of cellular phosphatidylinositol. Meth. Enzymol. 141, 234–244. Horrobin, D.F., Huang, Y.S., 1987. The role of linoleic acid and its metabolites in the lowering of plasmacholesterol and the prevention of cardiovascular disease. Int. J. Cardiol. 17, 173–180. Kowluru, A., Chen, H.Q., Modrick, L.M., Stefanelli, C., 2001. Activation of acetyl-CoA carboxylase by a glutamate- and magnesium-sensitive protein phosphatase in the islet beta-cell. Diabetes 50, 1580–1587. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Rancall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275.
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L.J. Yu et al. / Bioresource Technology 88 (2003) 265–268
Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428. Pellerin, L., Magistretti, P.J., 1994. Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proceedings of the National Academy of Sciences USA 91,10625–10629. Rounds, M.A., Nielsen, S.S., 1994. Basic principles of chromatography. In: Nielsen, S.S. (Ed.), Introduction to the Chemical Analysis of Foods. Jones and Bartlett, Boston, pp. 403–422. Shinmen, Y., Shimizu, S., Akimoto, K., Yamada, H., 1989. Production of arachidonic acid by Mortierella fungi. Appl. Microbiol. Biotechnol. 31, 11–16.
Totani, N., Oba, K., 1988. A simple method for production of arachidonic acid by Mortierella alpina. Appl. Microbiol. Biotechnol. 28, 135–137. Ward, O.P., 1995. Microbial Production of Fungi and Other Organism. Plenum, New York, pp. 9–48. Wice, B.M., Reitzer, L.J., Kennell, D., 1981. The continuous growth of vertebrate cells in the absence of sugar. J. Biol. Chem. 256, 7812–7819. Yamada, H., Shimizu, S., Shinmen, Y., Kawashima, H., Akimoto, K., 1989. Biotechnological processes for production of polyunsaturated fatty acid. J. Disper. Sci. Technol. 10, 561–579. Yongmanitchai, W., Ward, O.P., 1989. Omega-3 fatty acids: alternative sources of production. Proc. Biochem. 24, 117–125.
Short Communication
Improved arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation L.J. Yu *, W.M. Qin, W.Z. Lan, P.P. Zhou, M. Zhu School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, China Received 18 October 2001; received in revised form 25 November 2002; accepted 27 November 2002
Abstract The effect of various concentrations of glutamate on arachidonic acid (AA) production from Mortierella alpina in shaker flask culture was studied. Glutamate supplementation promoted Mortierella growth, accelerated substrate metabolism, and increased AA production, and a concentration of 0.8 g/l glutamate resulted in the greatest AA yield (1.41 g/l). In 10 l airlift stirred fermenter culture, AA yield in the cultures exposed to 0.8 g/l glutamate was also greater than that in the control (0.56 g/l). Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Arachidonic acid; Glutamate; Mortierella alpina
1. Introduction
2. Methods
Arachidonic acid (AA), a long chain polyunsaturated fatty acid (PuFA) of the omega-6 class (5,8,11,14-eicosatetraenoic acid), plays important roles in the structure and function of biological membranes (Yamada et al., 1989). In addition, it has recently attracted great interest due to several unique biological activities (Das et al., 1987; Horrobin and Huang, 1987). At the present time, the oil extracted from porcine liver is generally used as the main source of AA. However, the AA content of this oil is so low that it is not a practical source of this fatty acid. Therefore, alternative sources are being sought. The cells of some protozoa, algae and fungi are known to contain AA (Yongmanitchai and Ward, 1989; Shinmen et al., 1989; Gandhi and Weete, 1991). Lower fungi of the class of Zygomycetesm, especially Mortierella alpina, producing substantial quantities of AA (Ward, 1995), are a promising source of AA. The present communication represents studies on the effects of glutamate on AA production in shaker flask and fermenter.
2.1. Microorganism and inoculum preparation
* Corresponding author. Tel.: +86-27-8754-3633; fax: +86-27-87540184. E-mail address: [email protected] (L.J. Yu).
Mortierella alpina M18 was maintained on potato paste–dextrose medium (Totani and Oba, 1988) at )4 °C and subcultured every two months. The inoculum was prepared in 50 ml medium containing (g/l): glucose, 100; yeast extract, 5; MgSO4 , 5. The pH was adjusted to 6.0 before autoclaving at 121 °C for 20 min. In preparation for an experiment, a stock culture was vigorously stirred in 100 ml sterilized water with glass beads for 10 min. Ten milliliters of mycelial suspension was transferred to 250 ml shake flasks containing 50 ml medium and incubated at 24 1 °C with shaking at 130 rpm for three days. 2.2. Experimentation and statistical analysis 2.2.1. Experiment 1: shaker flask cultivation The production medium composition was as follows (g/l): glucose, 100; yeast extract, 5; beef extract, 3; MgSO4 , 1.5. In order to determine the effects of glutamate on the AA production, 0, 0.2, 0.4, 0.6, 0.8 g/l glutamate were respectively added to the production medium. The pH was adjusted to 6.0 before autoclaving at 121 °C for 20 min. The culture was grown at 24 1 °C for seven days with shaking at 130 rpm. Five hundred milliliters conical flasks containing 100 ml production
0960-8524/03/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0960-8524(02)00312-7
266
L.J. Yu et al. / Bioresource Technology 88 (2003) 265–268
medium were inoculated with the 72-h-grown inoculum preparation at a rate of 10% v/v. 2.2.2. Experiment 2: scale-up culture The medium used for scale up studies was the production medium supplemented with 0.8 g/l glutamate. The culture was grown at 24 1 °C for seven days in a 10 l airlift stirred fermenter with a 6 l working volume at a 0.6 m3 /h air flow rate. The scale up experiment was conducted using 6 l of medium inoculated with 1 l of 72h-grown inoculum preparation. 2.2.3. Analytical methods Biomass concentration was determined by centrifugation of the fungal cell suspension, washing with distilled water and drying at 100 °C for 12–16 h. Glucose, nitrogen and phosphate were measured on samples from the supernatant of the cell suspension using 3,5-dinitrosalicyclic acid colorimetry (Miller, 1959), folin phenol reagent (Lowry et al., 1951) and phosphomolybdic acid colorimetry assays, respectively (Eisenreich et al., 1975). The lipids were extracted following the method by Bligh and Dyer (1959). The extracted lipids were dried at 36 °C under a nitrogen atmosphere and methylated (Holub and Skeaff, 1987). AA was determined as follows. A gas chromatograph (GC-9A) equipped with a flame ionization detector (FID) and a glass column (Nishio Kogyo, Tokyo) was used. The oven temperature was 210 °C. Hexane was used as the solvent and 1 ll of solution was injected. The chromatograms were analyzed with CR2AX (Institute of Information, University of Zhejiang, China) software. The corrected area normalization of individual polyunsaturated fatty acid (PuFAs) and the internal standard method (Rounds and Nielsen, 1994) were used for PuFAsÕ quantification. The production of PuFAs was expressed as the weight % fatty acid methyl esters for further yield calculations (g/l).
2.3. Statistical analysis Results obtained were the mean of three or more determinations. Analysis of variance was carried out on all data at p < 0:05 with the help of Microsoftâ Excel (USA). When the ÔF Õ-value was found to be significant, critical difference was calculated by multiplying the standard error of difference (SED) with the corresponding ÔtÕ-values at 5% level of probability. Significant differences among the means were determined by using least significant difference test.
3. Results and discussion 3.1. Experiment 1: shaker flask cultivation Glutamate is involved in nitrogen metabolism and is required as an essential precursor of protein and nucleotide synthesis as well as a substrate for energy metabolism (Wice et al., 1981) in the organism. In addition, glutamate can stimulate aerobic glycolysis (Pellerin and Magistretti, 1994). Therefore, glutamate could potentially have a substantial influence on cell growth. Our results showed that glutamate supply promoted Mortierella growth (Table 1). Cliquet and Jackson (1999) also observed that glutamate supported biomass accumulations of Paecilomyces fumosoroseus. Our results also showed that decreases in glucose, nitrogen and phosphate concentration in the medium were accelerated by glutamate supplementation (Table 1). Recently, glutamate was demonstrated to have a role in activating acetyl-CoA carboxylase (ACC; EC 6.4.1.2). ACC catalyzes the formation of malonyl-CoA, an essential substrate for fatty-acid synthase and for fatty acyl chain elongation systems (Kowluru et al., 2001). Further, glutamate carbon can be utilized for fatty acid synthesis, either directly through the generation of keto
Table 1 Effect of glutamate concentration on AA production in the shaker flask culture on the seventh day Glutamate concentration (g/l) Total biomass (g/l) Total lipids in biomass (% w/w) AA in lipid (% w/w) AA in biomass (% w/w) AA yield (g/l) Glucose (g/l) Nitrogen (g/l) Phosphate (mg/l)
0
0.4
0.6
0.8
1.0
1.2
21.56 0.35a 30.56 1.3 12.74 0.7 3.90 0.13 0.84 0.07 27.80 0.81 1.87 0.15 15.13 0.53
23.27 0.51 35.79 2.1 13.48 1.0 5.24 0.25 1.22 0.23 25.25 0.75 1.69 0.12 13.21 0.6
23.51 0.4 36.63 2.4 14.56 1.5 5.31 0.3 1.25 0.32 25.69 0.6 1.56 0.2 11.56 0.45
24.43 0.45 37.69 2.5 15.29 2.1 5.77 0.42 1.41 0.38 24.01 0.64 1.54 0.1 10.55 0.38
24.47 0.65 34.79 3.1 10.80 1.2 3.76 0.25 0.92 0.25 21.26 0.5 1.45 0.13 9.95 0.4
24.78 0.57 31.78 1.8 10.23 1.6 3.27 0.32 0.81 0.2 20.33 0.63 1.21 0.85 8.34 0.34
AA represents arachidonic acid. Significantly different from the control at p < 0:05. ** Significantly different from the control at f < 0:01. a All values are mean and standard deviation of three replicates. *
L.J. Yu et al. / Bioresource Technology 88 (2003) 265–268
267
Table 2 The effect of glutamate supplementation (0.8 g/l) on day seven for biomass, total lipid in biomass, total lipids, arachidonic acid in lipids and biomass, AA yield in the scale-up culture
Control Glutamate *
Dry biomass (g/l)
Total lipid in biomass (% w/w)
Arachidonic acid In lipids (% w/w)
In biomass (% w/w)
Yield (g/l)
16.25 0.08a 18.54 0.75
28.01 0.85 34.12 1.1
12.36 0.55 14.78 0.97
3.44 0.16 5.02 0.21
0.56 0.43 0.93 0.54
Significantly different from the control at p < 0:05. a All values are mean and standard deviation of three replicates.
acids or acetyl-CoA (Albers et al., 1996). In our experiment it appeared that supplementation of less than 0.8 g/l glutamate could increase total lipids and AA production. However, supplementation of greater than 0.8 g/l glutamate led to a decrease in the total lipids in biomass and AA production compared with the response to 0.8 g/l glutamate. Certik et al. (1999) reported that glutamate was one of the potentially available nitrogen sources for increasing G6PDH activities and enhancing PuFA biosynthesis. However, when glutamate concentration is too high, glutamate will be converted to proline accompanied by NADPH consumption (Andarwulan and Shetty, 1999), which is necessary for AA biosynthesis. A concentration of 0.8 g/l glutamate was most favorable for AA production, therefore, it was selected to further study the AA production in the scaleup culture. 3.2. Experiment 2: scale-up culture The effect of glutamate supplementation (0.8 g/l) on day seven for biomass, total lipid in biomass, total lipids, AA in lipids and biomass, AA yield in the scale-up culture are showed in Table 2. The yield of the scale-up culture (Table 2) was lower than the production of the shaker flask culture (Table 1). The difference might be caused by the potentially different dissolved oxygen concentrations between in the medium and in the fermenter (Higashiyama et al., 1999), as well in the fermenter, it is unavoidable that the shearing strength has damaging effect on cell growth (Enfors et al., 2001). However, AA yield of 0.8 g/l glutamate (0.93 g/l) was significantly greater than the production of the control (0.56 g/l) in 10 l airlift stirred fermenter. This is additional evidence that 0.8 g/l glutamate could improve arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation. 4. Conclusions This report presents that addition of 08 g/l glutamate could promoted Mortierella growth, accelerated substrate metabolism, and increased AA production, and a concentration of 0.8 g/l glutamate resulted in the greatest AA yield (1.41 g/l) in 250 ml shaker flask.
Furthermore, in 10 l airlift stirred fermenter, AA yield in the cultures exposed to 0.8 g/l glutamate was also greater than that in the control. Therefore, the application of the 0.8 g/l glutamate could be useful in developing rational strategies for enhancing the production of AA in large-scale fungal M. alpina cultures.
References Albers, E., Larsson, C., Liden, G.L., Niklasson, C., Gustafsson, L., 1996. Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Appl. Environ. Microbiol. 9, 3187–3195. Andarwulan, N., Shetty, K., 1999. Improvement of pea (Pisum sativum) seed vigour response by fish protein hydrolysates in combination with acetyl salicylic acid. Proc. Biochem. 35, 159–165. Bligh, E.G., Dyer, N.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Certik, M., Megova, J., Horenitzky, R., 1999. Effect of nitrogen sources on the activities of lipogenic enzymes in oleaginous fungus Cunninghamella echinulata. J. Appl. Microbiol. 45, 289– 293. Cliquet, S., Jackson, M.A., 1999. Influence of culture conditions on production and freeze-drying tolerance of Paecilomyces fumosoroseus blastospores. J. Ind. Microbiol. Biotechnol. 23, 97–102. Das, U.N., Begin, M.E., Huang, Y.S., Horrobin, D.F., 1987. Polyunsaturated fatty acids augment free radical generation in tumor cells in vitro. Biochem. Biophys. Res. Commun. 145, 15–24. Eisenreich, S.J., Bannerman, R.T., Armstrong, D.E., 1975. A simplified phosphorus analytical method. Environ. Lett. 9, 45–53. Enfors, S.-O., Jahic, M., Rozkov, A., Xu, B., Hecker, M., J€ urgen, B., Kr€ uger, E., Schweder, T., et al., 2001. Physiological responses to mixing in large scale bioreactors. J. Biotechnol. 85, 175–185. Gandhi, S.R., Weete, J.D., 1991. Production of the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid by the fungus Phythium ultimum. J. Gen. Microbiol. 137, 1825–1830. Higashiyama, K., Murakami, K., Tsujimua, H., Matsumoto, N., Fujikawa, S., 1999. Effects of dissolved oxygen on the morphology of arachidonic acid production by Mortierella alpina 1S-4. Biotech. Bioeng. 63, 442–448. Holub, B.J., Skeaff, C.M., 1987. Nutritional regulation of cellular phosphatidylinositol. Meth. Enzymol. 141, 234–244. Horrobin, D.F., Huang, Y.S., 1987. The role of linoleic acid and its metabolites in the lowering of plasmacholesterol and the prevention of cardiovascular disease. Int. J. Cardiol. 17, 173–180. Kowluru, A., Chen, H.Q., Modrick, L.M., Stefanelli, C., 2001. Activation of acetyl-CoA carboxylase by a glutamate- and magnesium-sensitive protein phosphatase in the islet beta-cell. Diabetes 50, 1580–1587. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Rancall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275.
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