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