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Selection of suitable maintenance medium for the mutant Micrococcus glutamicus [AB.sub.100] as a L-glutamic acid producer.

Introduction

Production of L-amino acids by fermentation method have the advantage of yielding biologically active L-form of amino acids directly [1]. Bacterial isolates produced higher amounts of amino acids directly [2]. But the high amino acid yielding mutant strains show degenerative properties by means of which they again revert bact to their original low amino acid yielding parent strains [3]. Thus, maintenance of pure culture conditions are very much essential to maintain constant productivity [4]. Different reviews shows utilization of different chemical elements for the maintenance of pure bacterial cultures employed for different amino acid production [5-15].

Considering the fact, the present study was intended to select suitable maintenance medium for the auxotrophic mutant Micrococcus glutamicus [AB.sub.100] employed for L-glutamic acid production.

Materials and Methods

Microorganism: Micrococcus glutamicus [AB.sub.100], a biotin requiring auxotrophic mutant, derived from a regulatory mutant Micrococcus glutamicus [AB.sub.1] by induced mutation [16].

Estimation of Dry Cell Weight (DCW): After centrifugation, a few ml of 1.0(M) HCl was poured into the precipitate of the bacterial cells and calcium carbonate to dissolve calcium carbonate. The remaining bacterial cells were washed with water and dried at 100[degrees]C until cell weight remain constant [14].

Medium for L-glutamic acid production: glucose, 10%; urea, 0.8%; K2HPO4, 0.1%; MgSO4 x 7H2O, 0.025%; yeast extract, 0.2%; pH 7.0.

Analysis of Amino acid: Descending paper chromatography was employed for detecting L-glutamic acid in culture medium and was run for 18h on a watman No. 1 chromatographic paper. Solvent system used include, n-butanol: acetic acid: water (2: 1: 1). The spots were visualized by spraying with a solution of 0.2% ninhydrin in acetone by spraying with a solution of 0.2% ninhydrin in acetone and quantitative estimation of L-glutamic acid in the suspension was done using colorimetric estimation method [17,18].

Statistical Analysis: All data were expressed as mean [+ or -] SEM, where n = 6. The data were analyzed by one way ANOVA followed by Dunett's post-hoc multiple comparison test using "prism 4.0" software (Graph pad Ind., USA). A "p" value less than 0.05 was considered significant.

Results and Discussion

The effect of different chemical constituents and condition of the maintenance medium on the maintenance of the auxotrophic mutant Micrococcus glutamicus [AB.sub.100] were depicted in Fig. 1-6.

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Shah et al (2002) reported that maintenance medium for Corynebacterium glutamicum employed beef extract, 1.0%; peptone, 1.0%; peptone, 1.0% NaCl 0.3%. Agar was used as solidifying agent [15]. Yugandhar et al (2006) claimed that maintenance medium contained NaCl, 0.5%; peptone, 1.0%; beef extract, 1.0% for the production of L-glutamic acid by Bervibacterium roseum [14].

Thus, constant rate of production of L-glutamic acid (6.8 mg/ml) was obtained experimentally up to sixteen subculture of Micrococcus glutamicus [AB.sub.100] studied before employed this strain into further optimization study using a maintenance medium composed of: glucose, 1.0%; peptone, 0.5%; beef extract, 0.3%; yeast extract, 0.1% and agar 4.0% (as solidifying agent). pH was maintained at 6.5.

References

[1] Yamada, K., 1972, "The microbial production of Amino Acids", John Wiley and Sons (eds), New York, pp. 264.

[2] Bejwa, M. A., Zahoor, T., Butt, T. M., Atiq, M., and Talib, S., 2010, "Microbial production of L-isoleucine from different substractes using locally isolated bacteria", Int. J. Agric. Biol., 12(5), pp. 668-672.

[3] Nadeem, S., and Ahmad, S. M., 1999, "Amino acid fermentation: a recent perspective", Proc. Pakistan Acad. Sci., 36, pp. 193-206.

[4] Soda, K., Tanaka, H., and Esaki, N., 1983, "Amino acids. In: Biotechnology, Biomass, Microorganisms for special Applications, Microbial products Energy from Renewable Resource", Dellweg, H(ed)., Education Ltd., London, 3, pp. 657-674.

[5] Malumbcrs, M., Meteos, M., and Martin, J. M., 1995, "Microorganisms for amino acid production Escherichia Coli and Corynebacteria. In: Food Becteriology Microorganisms", Hui, Y. H., and Kachatorians, G. G. (eds), VCH Publication, New York, pp. 423-469.

[6] Ikeda, M., 2003, "Amino acid production processes", Adv. Biochem. Eng. Biotechnol., 79, pp. 1-35.

[7] Crueger, W., and Crueger, A., 1990, "Biotechnology: Text book of Industrial Microbiology", Brock, T. D. (ed.), Sinauer associates, Sunderland, Massachusetts, 2nd ed., pp. 56-59.

[8] Caplice, E., and Fitzgerald, G. P., 1999," Food Permentations: role of microorganisms in food production and presentation", Int. J. Food Microbiol, 50, pp. 131-149.

[9] Ahmed, M. S., and Nadeem, S., 1993, Screening of bacterial isolates for amino acid fermentation", Nucleus, 30, pp. 45-49.

[10] Kumagai, H., 2003, "Microbial production of Amino Acids in Japan", In: Advances in Biochemical Engineering / Biotechnology, Fiecheter, A (ed.)., Springer-verlag, Berlin, Heidelberg, 69, pp. 71-85.

[11] Ikeda, M., 2003, " amino Acid production process", In: Advances in Biochemical Engineering / Biotechnology, Thormmel, J., and Faurie, R. (eds.)., Springer-verlag, Berlin, Heidelberg, 79, pp. 1-35.

[12] Hirose, Y. H., Encei, H., and Shibai, 1985, "L-glutamic Acid Fermentation", In: Comprehensive Biotechnology, Moo-Young, M. (ed.)., Pergamon pren, New York, 2, pp. 593-600.

[13] Yugandhar, N. M., Kiran Babu, U., Raju, Ch., A. I., Raju, J., and Rami, DSR., 2006, "Optimization of Glutamic Acid Production by Brevibacterium roseum", Res. J. Microbiol, 1(5) 475-432.

[14] Shah, A. H., Hameed, A., Ahmad, S., and khan, M 2 Gul., 2002, optimization of culture conditions for L-lysine Fermentation by Corynebacterium glutamicum, On Line J. Bio. Sci. 2(3), pp. 151-156.

[15] Tosaka, O., and Takinami, K., 1986, "Biotechnology of amino acid production", In: Progress in industrial microbiology Elsevier, Tokyo, Aida, K., Chibata, I., and Nakayama, K (eds), 24, pp. 151-172.

[16] Ganguly, S., and Banik, A. K., 2010, "Induced mutation and selection of High yielding strain of Micrococcus glutamicus for glutamic acid production", J. Indian Chem. SOC, 87(5), pp. 717-721.

[17] Chinard, F. P., 1952, "Photomeric estimation of proline and Ornithine", J. Biochem, 199, pp. 91-95.

[18] Spies, J. R., 1957, Colorimetric procedure for amino acid, Methods in Enzymol, 3, pp. 468-471.

* S. Ganguly and A.K. Banik

Department of Chemical Engineering Biochemical Engineering Division, Biotechnology Laboratory, University of Calcutta, 92, A.P.C. Road, Kolkata-700 009, West Bengal, India.

* Corresponding Author E-mail: subhadeepgangulyphysiol@rediffmail.com
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Author:Ganguly, S.; Banik, A.K.
Publication:International Journal of Biotechnology & Biochemistry
Date:Sep 1, 2011
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