INTRODUCTION:

The industrial demand for L-glutamic acid is rapid expanding. Mono Sodium L-glutamatee (MSG) is the largest product of L-glutamic acid out of all amino acids whose production level is of 1.5 million tons and the market is growing by about 6.0% per year¹. However, research and development carried out mainly in Japan, resulted in the successful and economically valuable production of L-glutamic acid by the microbial fermentation process².

Recent advancement of biotechnology gained the attention of many biotechnologists for the successful application of immobilization technology in this field. Among them entrapment of whole cell in different matrices including calcium alginate beads and agar blocks have been better choices over the other methods for the microbial fermentative production of L-glutamic acid^3-7.

Different reviews claimed that these techniques are simple and their viabilities were estimated over a period of more than 18 months and thus these are considered to be the potential applications for this purpose ⁵.

Various fermentation techniques have been reported for the production of L-glutamic acid using immobilized bacterial cells ^3,4,8-11.

The successful achievement of whole cell immobilization technique for the production of different secondary metabolites including amino acids, prompted us to study the production of L-glutamic acid with immobilized whole cells of the mutant Micrococcus glutamicus AB_100.

The main purpose of this present study was to investigate the potency of the mutant strain immobilized into two different matrices namely calcium alginate beads and agar blocks for L-glutamic acid accumulation using selected suitable synthetic medium.

Materials and Methods:

Organism : Micrococcus glutamicus AB₁₀₀, a biotin requiring Auxotrophic mutant derived from a regulatory mutant Micrococcus glutamicus AB₁ by induced mutation in our laboratory using UV irradiation as physical and ethyleineimine as chemical mutagens were used throughout the study ¹².

Growth medium and growth conditions: The cultures were maintained on agar slants having composition: glucose, 1.0%; peptone, 0.5% beef extract, 0.3%; yeast extract, 0.1% and agar, 4.0%. The pH of the medium was adjusted to 6.5 and incubated at 29^oC for 48h using shake-flask method on a rotary shaker with a shaking speed of 150 rpm ^8,12.

Preparation of inoculum: A full grown slant of 48h old Micrococcus glutamicus AB₁₀₀ were scrapped off and suspended in 10³ sterile water. The cell suspension 4.0% (v/v) of the seed culture (6.0 x 10⁷ cells) of the organism was used as the inoculum ¹².

Preparation of Calcium alginate beads: The cell suspension was slowly added to the sterile solution of sodium alginate (3.0%) and mixed thoroughly with the sterile glass rod. The mixture was continuously extruded into 25 ml Erlenmeyer flask containing 20 ml 0.1 (M) CaCl₂ for 30 minutes. Then the beads were filtered aseptically and washed successively with sterile buffer solution (pH 6.5) and with sterile distilled water^6,7.

Preparation of agar blocks: A defined quantity of agar was dissolved in 18 ml of 0.9% NaCl solution to get final concentration of 4.0% and sterilized by autocleaving. 4.0% (v/v) cell suspension was added to the molten agar maintained at 40^oC shaken well for few seconds (without forming foam), poured into sterile flat bottom 4.0 inch diameter petri plates and allowed to solidify. The solidified agar block was cut into equal size cubes (4.0 mm³), added to sterile 0.1 (M) phosphate buffer (pH 6.5) and kept into the refrigerator (1h) for curing. After curing, phosphate buffer was decanted and cubes were washed with sterile water for 3 to 4 times ¹³.

Composition of selected synthetic medium for the production of L-glutamic acid: The following fermentation medium was used for the production of L-glutamic acid by this mutant strain : glucose, 9.0%; diammonium hydrogen phosphate, 1.4%; dipotassium hydrogen phosphate, 0.15; MgSO₄, 7H₂O, 0.03%; CaCO₃, 0.04%; FeSO₄, 7H₂O, 5.0 µg/ml; ZnSO₄, 7H₂O, 1.0 µg/ml; MnSO₄, 4H₂O, 1.0 µg/ml; and biotin, 0.2 µg/ml. pH was adjusted to 6.5 ¹².

Estimation of L-glutamic acid: Descending paper chromatography was employed for the detection of L-glutamic acid in the culture broth and was run for 16-18h on Whatman 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 and quantitative estimation of L-glutamic acid in the suspension was done using colorimetric estimation method ^14,15.

Estimation of Dry Cell wight (DCW): After centrifugation, a few ml of 1.0 (M) HCl was poured into the precipitate of the bacterial cells and calcium carbonate to dissolved calcium carbonate. The remaining bacterial cells were washed with water and dried at 100^oC until cell weight remains constant ¹⁶.

Statistical analysis: All data were expressed as mean ± SEM, where n = 6. The data were analysed by one way ANOVA followed by Dunett’s post hoc multiple comparison test using “prism 4.0” software (Graph pad Inc., USA). A “p” value less than 0.05 was considered significant and less than 0.01 was considered highly significant.

RESULTS AND DISCUSSION:

Production of L-Glutamic acid by immobilized cells of the mutant Micrococcus glutamicus AB₁₀₀ in Calcium alginate beads:

L-Glutamic acid production conditions for the mutant Micrococcus glutamicus AB₁₀₀ entrapped in calcium alginate beads were investigated considering the following parameters:

Effect of Calcium Chloride in the Synthetic medium : Our synthetic medium contained 0.04% calcium Carbonate. However, in presence of the calcium chloride in the synthetic medium for the calcium alginate bead stability was investigated with 0.1 (M) Calcium Chloride. We have seen that the beads were dissolved into the synthetic medium in absence of calcium chloride, mild L-glutamic acid accumulation was recorded which improved significantly (p<0.01) in presence of 0.1 (M) Calcium Chloride [Fig.1A]. To optimize the calcium chloride concentration in the synthetic medium, different concentrations of calcium chloride [ranging from 0.1 – 0.5 (M)] were studied for L-glutamic acid accumulation. Maximum production was obtained with 0.3 (M) calcium chloride as depicted in Fig. 1B Calcium Chloride is essential for bead stability and pore size of the beads ⁵. The Calcium alginate is unstable in presennce of phosphate and certain cations such as Mg⁺² or K⁺ which are major nutrients for living microorganisms ⁴. For solubilizing effect of calcium alginate beads can also be prevented by supplementing the synthetic medium with calcium chlorider⁵.

Fig. 1A : Effect of CaCl₂ in the synthetic medium on the L-glutamic acid accumulation by the immobilized cells of the mutant Micrococcus glutamicus AB₁₀₀entrapped in calcium alginate beads.

( Where n = 6; values were expressed as Mean ± SEM; ** p < 0.01; 0.0 stands for control )

Fig. 1B : Optimization of CaCl₂ Concentration in the synthetic medium for the production of L-glutamic acid by the immobilized cells of the mutant Micrococcus glutamicus AB₁₀₀ entrapped in calcium alginate beads.

(Where n = 6; values were expressed as Mean ± SEM; *p<0.05, ** p < 0.01; 0.1 (M) Concentration is treated as control)

Effect of Calcium Chloride on Calcium alginate beads formation : Owing to the bead stability, another important aspect of Calcium Chloride was to confer the proper shape and porosity of the calcium alginate beads ¹⁷. Therefore, it is essential to determine the optimum concentration of calcium chloride for the bead formation considering the fact, different concentrations of calcium chloride ranging from 0.05 – 0.4(M) were investigated were 0.2 (M) concentration was proved to be pitimum [Fig.1C].

Fig. 1C : Effect of Calcium Chloride on the calcium alginate bead formation on the L-glutamic acid production by the mutant Micrococcus glutamicus AB₁₀₀.

( Where n = 6; values were expressed as Mean ± SEM; * p < 0.05, ** p < 0.01; 0.1 (M) Concentration is treated as control )

Effect of sodium alginate on the bead formation : Different sodium alginate concentrations ranging from 1.0 – 6.0% with 4.0% (V/V) inoculum volume and 0.2 (M) calcium alginate beads loaded with Micrococcus glutamicus AB₁₀₀ for L-glutamic acid production. At 3.0% sodium alginate concentration, the yield was maximum [Fig.D]. Sodium alginate is very crucial determining factor for the formation of calcium alginate beads. Higher alginate concentration reduces the pore size leading to lower conversion efficiency and lower alginate cencentration increases the pore size leading to leakage of the cells in the medium, thus production falls markedly ¹⁸. Thus, with 3.0% sodium alginate, we got the appropriate bead shape with maximum yield. Therefore, this concentration was used for further study.

Fig. 1D : Effect of Sodium alginate on the L-glutamic acid accumulation by the immobilized cells of the mutant Micrococcus glutamicus AB₁₀₀, entrapped in calcium alginate beads.

( Where n = 6; values were expressed as Mean ± SEM; * p < 0.05, ** p < 0.01; 3.0% sodium alginate concentration was treated as control )

Effect of storage periods of calcium alginate beads : The storage periods of the Calcium alginate beads on the L-glutamic acid production were investigated using different ages of beads [Fig.1E]. 24h aged beads showed maximum production. Bu above this storage period, the production of L-glutamic acid did not affect significantly.

Fig. 1E : Effect of storage periods of Calcium alginate beads entrapping the whole cells of the mutant Micrococcus glutamicus AB₁₀₀.

( Where n = 6; values were expressed as Mean ± SEM; * p < 0.05, ** p < 0.01; 24h aged beads were treated as control )

0 h aged beads showed minimal L-glutamic acid production with deformation of the beads. The cells encapsulated in properly solidified beads had better storage stability than the free cells ^18,19.

The solidification of the beads require a minimum time interval which was 24h in this case.

Effect of cell/alginate ratio : To study the effect of the amount of cell mass entrapped in the gel matrix on L-glutamic acid production, the cell/alginate ratio were varied between 0.33 to 2.66. The beads were prepared using 3.0% sodium alginate and 0.2(M) calcium chloride. The yield of L-glutamic acid was maximum for cell/alginate ratio of 1.33 as shown in Fig.1F. The leakage of cell/alginate ratio of 2.33 and it was maximum will cell/alginate ratio of 1.33. Sunitha et al. (1998) obtained maximum l-glutamic acid with cell alginate ratio of 0.05 ²⁰. Thus, this cell/alginate ratio was considered as optimum and used for further study.

Fig. 1F : Effect of cell/alginate ratio for the fermentation of L-glutamic acid by the mutant Micrococcus glutamicus AB₁₀₀ entrapped in calcium alginate beads.

( Where n = 6; values were expressed as Mean ± SEM; * p < 0.05, ** p < 0.01; cell/alginate ratio of 1.33 was treated as control )

Production of L-Glutamic acid by a mutant Micrococcus glutamicus AB₁₀₀ entrapped in Agar blocks

To increase the production of L-glutamic acid by this mutant entrapped in agar blocks, following parameters were optimized one by one as described below:

Effect of block volume: As the accumulation of L-glutamic acid by bacteria is a function of surface area of the cell or the medium where it is entrapped. Which confer its proper three dimensional configuration²¹. Different agar block sizes (1 – 7 mm³) were studied and maximum production of L-glutamic acid was obtained with 4.0 mm³ blocks [Fig.2A].

Fig. 2A : Effect of Agar block volume on the production of L-glutamic acid by the mutant Micrococcus glutamicus AB₁₀₀ entrapped in the agar blocks.

(Where n = 6; values were expressed as Mean ± SEM; * p < 0.05, ** p < 0.01; 4.00 mmq Agar blocks were considered as control)

Effect of storage periods of agar blocks : Minimum storage period of immobilizing matrices is required for proper entrapment of the organism and solidification of the matrices ²². Thus different storage periods (1.0 – 168h) of the agar blocks were studied for maximum L-glutamic acid accumulation. Maximum yield was obtained with 24h aged blocks above which the production was not altered significantly [Fig.2B].

Fig. 2B : Effect of Storage periods of agar blocks, trapping with the mutant Micrococcus glutamicus AB₁₀₀ for the production of L-glutamic acid.

( Where n = 6; values were expressed as Mean ± SEM; * p < 0.05; one hour aged blocks were treated as control )

Effect of cell/agar ratio : To determine the maximum amount of cell mass entrapment with maximum yield of L-glutamic acid different cell/agar ratio (0.25 – 2.0) were taken under consideration. Blocks were prepared with 4.0% agar and got maximum production with a cell/agar ratio of 1.0 as shown in Fig.2 C.

Fig. 2C : Effect of cell/agar ratio on the production of L-glutamic acid by the mutant Micrococcus glutamicus AB₁₀₀ entrapped in agar blocks. ( Where n = 6; values were expressed as Mean ± SEM; * p < 0.05, ** p < 0.01; cell/agar ratio of 1.0 was considered as control)

Comparative study between free and immobilized cells of Micrococcus glutamicus AB₁₀₀ for the fermentation of L-glutamic acid

A comparative study was made on the fermentation of L-glutamic acid between the free and immobilized cells of Micrococcus glutamicus AB₁₀₀. For immobilization of two different matrics (Calcium alginate and agar) were used. The result of fermentation of L-glutamic acid is shown in Table 1.

From the result it is very clear that the production was significantly (p<0.05) increased with Calcium alginate beads and decreased significantly (p<0.05) with agar blocks compared to the production by free cells of the mutant, considered as control. Hence, the advantage of the immobilization was attributed to the immobilized cells of Micrococcus glutamicus AB₁₀₀ entrapped in calcium alginate beads with high retention of the cellular activities.

ACKNOWLEDGEMENT:

We express our sincere gratitude to the librarian of Bose Institute for his kind Co-operation in finding necessary information regarding this work without which we could not finish the work.

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Received on 10.04.2011 Modified on 08.05.2011

Asian J. Research Chem. 4(6): June, 2011; Page 1014-1018

Conditions of the cells	L-Glutamic acid (mg/ml)	Dry cell weight (mg/ml)
Free cells of Micrococcus glutamicus AB₁₀₀	23.6 ± 1.28	9.2
Immobilized whole cells of Micrococcus glutamicus AB₁₀₀ entrapped in calcium alginate beads	24.8 * ± 2.41	Equivalent to 0.61
Immobilized whole cells of Micrococcus glutamicus AB ₁₀₀ entrapped in agar blocks	21.8 * ± 1.13	Equivalent to 0.61