Abstract: Experimental studies were carried out to produces L-glutamic acid with Brevibacterium roseum free cells, immobilized B. roseum and co-immobilized B. roseum and Escherichia intermedia type 1, production conditions were optimized with B. roseum free cells. Incubation time 96 h, pH 6.0, temperature 30°C, 150 rpm agitation and biotin 1.0 mg mL-1 were optimum for the maximum yield of glutamic acid. When the nutrient ingredients were optimized the glutamic acid yield increased to 40.5 mg mL-1. Immobilized B.roseum produced 37.2 mg mL-1 glutamic acid. Immobilized conditions like alginate concentration, calcium ion concentration, storage period of beads and initial cell loading were optimized. Co-immobilized B. roseum and Escherichia intermedia type-1 produced 39.6 mg mL-1 glutamic acid.
INTRODUCTION
The demand for amino acids as food supplements like fortification of vegetable protein and in pharmaceutical industries is fast expanding. Kinoshita et al. (1957) isolated L-glutamic acid producing microorganisms and subsequent research brought about the economic production of L-glutamic acid by fermentation process. This opened the way to fermentative production of various amino acids. Mono Sodium L-glutamate (MSG) is the largest product out of all amino acids and the recent survey indicates that the annual production level is of 1.5 million tons and the market is growing by about 6% per year. Fermentative production of L-glutamic acid has been employed during last few decades and the production yield has been improved considerably over the years (Tauro et al., 1963; Kim and Ryu, 1982). In the last few years different strategies were employed to optimize the glutamate fermentation. These include cell recycling (Izhizaki et al., 1993), culturing the microorganisms in the solid substrates (Nampoothiri and Pandey, 1996b), nutrient formulation (Nampoothiri and Pandey, 1995) and the use of different raw materials (Das et al., 1995; Nampoothiri and Pandey, 1996b). The practical importance of immobilized cells/enzymes has been successfully applied to large scale production of various fermentation products with reduced process cost. The applications of immobilized process have been highlighted from the view point of long term utilization of bio catalysts and continuous operation of stabilized systems (Fukui and Tanaka, 1982; Nampoothiri and Pandey, 1998). A number of amino acids have been produced using this methodology. Amin et al. (1993) made an attempt to study the formation of by-product during glucose conversion to glutamic acid using Corynebacterium glutamicum immobilized in polyurethane foam. Entrapment of protoplast of Brevibacterium flavum in matrices of agar-acetyl cellulose filtering to produce L-glutamic acid (Karube et al., 1985). Binding of the defective enzyme from an external source to free or immobilized microorganisms or immobilization of mixed culture capable of carrying out two or multi step conversions into single step conversions leads to co-immobilized cells can open up new possibilities of synergetic action and result in more yield/conversion which cannot be obtained to the same extent by separately immobilized cells (Jagannadha Rao, 1992).
The present study is aimed to determine the feasibility of glutamic acid production using free, immobilized and co immobilized cells of Brevibacterium and E. intermedia type-1 entrapped in calcium alginate beads. Glutamic acid production conditions were optimized which include fermentation, pH, temperature, gel concentration, bead size and initial biomass.
MATERIALS AND METHODS
Microorganisms
Brevibacterium roseum NCIM 2270 and Escherichia intermedia
type-1 NCIM 2490, obtained from National Chemical Laboratory, Pune, India were
used for this study.
Growth Medium and Growth Conditions
Cultures were maintained on agar slants having composition (g L-1)
peptone-10, Beef extract-10, Sodium chloride-5 and Agar-20. The pH of the medium
adjusted to 7.0 and incubated at 37°C for 24 h. For the seed culture development,
the following inoculation medium was used containing (g L-1) glucose-25,
yeast extract-10, urea-8, K2HPO4-1, MgSO4.7H2O-2.5,
the pH of the medium was adjusted to 7.0 before inoculation.
Inoculum Preparation and Cell Harvest
A fully grown slant of 24 h old Brevibacterium roseum and Escherichia
intermedia type 1 were scrapped off and suspended in 0.01 M citrate buffer
(pH-7.0). The cell suspension was shaken thoroughly to break up the aggregates.
The cell count was determined by plating 1 mL of above cell suspension, after
making serial dilution. The cell count is adjusted in the range of 10-7
to 108 cells mL-1. The cells were grown for 24 h at 30°C
in 250 mL Erlenmeyer flasks containing 50 mL of inoculation medium on a rotary
shaker at 180 rpm. Cells were separated from the inoculation medium by centrifugation
and washed thoroughly with 0.01 M citrate buffer (pH 7.0).
Preparation of Calcium Alginate Beads
The cell suspension is slowly added to the ether sterilized sodium alginate
(3% w/v) and mixed thoroughly with sterile glass rod. The mixture was extruded
as drops into a solution of CaCl2 (0.5 M). Bead size was controlled
by gauge number of the hypodermic needle used during extrusion. The beads were
cured in the same solution at room temperature for an hour and stored in a freshly
prepared 0.1 M CaCl2 solution at 4°C.
Production Medium and Conditions
Production medium contained (g L-1): glucose-5.0, Urea 5, K2HPO4
1.2, CaCO3 16, biotin 0.05 and (mL L-1) mineral
solution, 10 FeSO4.7H2O; MnSO4.4H2O;
MgSO4.7H2O; ZnSO4.6H2O and NaCl
each 10 mg. Glucose and mineral solution were separately autoclaved and mixed
aseptically. CaCO3 was sterilized by dry heat sterilization and added
to the production medium. Urea and biotin were filter sterilized. The pH of
the medium was adjusted to 7.0. All fermentations were carried out in 250 mL
Erlenmeyer flasks containing 100 mL of production medium at 30°C on a rotary
shaker.
Analytical Methods
Reducing sugars were determined by Miller (1959). Thin layer chromatography
(Silica gel G, solvent moisture: n-butanol/glacial acetic acid/water 4:1:1 v/v)
was used for the qualitative determination of L-glutamic acid (Brenner and Neiderwiser,
1967) and it was estimated quantitatively by a nin-hydrin color reaction (Spies,
1957).
The project was done at the Center for Biotechnology, Department of Chemical Engineering, Andhra University, INDIA in the year 2006.
RESULTS AND DISCUSSION
Glutamic acid production conditions like fermentation time, pH, temperature, agitation and biotin concentration were optimized.
The influence of fermentation time on glutamic acid was shown in Table 1. The fermentation time from 36 to 108 h with 12 h increments were studied. The maximum glutamic yield is 27.6 mg mL-1 at 96 h of incubation and the lowest being 15.9 mg mL-1 at 36 h. The effect of pH on glutamic acid was determined. The pH range between 4.5 to 6.5 is used for the study. Table 2 indicates that the maximum glutamic acid (27.8 mg mL-1) was recorded at pH 6.0.
The production medium was adjusted to pH 6.0 and incubated at different temperatures (26-34°C). The maximum yield of glutamic acid was observed at 30°C temperature (Table 3). The production medium with fermentation pH 6.0, 96 h and 30°C temperature were incubated at different agitation ranging from 100 to 300 rpm (Table 4). The change of agitation did not affect the glutamic acid content but a marginal increase (28.1 mg mL-1) was observed with 150 rpm.
| Table 1: | Effect of fermentation time on glutamic acid production |
| Table 2: | Effect of pH on glutamic acid production |
| Table 3: | Effect of temperature on glutamic acid production |
| Table 4: | Effect of agitation on glutamic acid production |
Different biotin concentrations were employed in the production medium (0.8, 1.0, 2.0, 3.0 and 4.0 mg mL-1) to determine its effect on glutamic acid. Biotin concentration of 1.0 mg mL-1 was found to be optimum with 32.7 mg mL-1 of glutamic acid. When the medium ingredients were optimized, the yield of glutamic acid increased from 37.2 to 40.5 mg mL-1 (data not shown).
Immobilization of Brevibacterium roseum
Employing the same production medium and optimum parameters, experiments
were carried out with immobilization of Brevibacterium roseum and co-immobilization
of Brevibacterium roseum and Escherichia intermedia type-1.
Immobilization of Brevibacterium roseum
Production of glutamic acid with immobilized B. roseum was carried
out and optimized the parameters which include effect of alginate concentration,
CaCl2 concentration, storage periods of beads and initial cell loading
on glutamic acid.
Effect of Alginate Concentration
Sodium alginate concentrations 2, 3 and 5% (W/V) were used for the immobilized
beads. The maximum concentration of glutamic acid was obtained with 3% calcium
alginate beads after 96 h of fermentation (Table 5). Sodium
alginate concentration had an influence on density of the beads, higher alginate
concentration showed the lower conversion efficiency, which might be due to
reduced pore size of the bead. The lower alginate concentration effect the leakage
of biomass from the beads, which could be due to increase in pore size of the
beads. Shimmyo et al. (1982), Nasri et al. (1989), Nampoothiri
and Pandey (1998) and Sunitha et al. (1998) also reported the similar
findings.
Effect of Calcium Ions
At 0.03 M concentration, the yield of glutamic acid was more (37.0 mg mL-1)
than at the concentration of CaCl2 higher or lower (Table
6). The concentration of CaCl2 is important for the stability
and pore size of bead. The calcium alginate gel is unstable in the presence
of phosphates and certain cations such as Mg++ or K+,
which are major nutrients for living microorganisms (Lu and Chen, 1998). However,
the solubilizing effect of these agents can be overcome by supplementing the
growth medium with CaCl2 (Nampoothiri and Pandey, 1988).
Influence of Storage Period of Beads
The calcium alginate beads were stored for 4 and 24 h. Table
7 indicates that with 4 h storage beads only 32.0 mg mL-1 glutamic
acid was obtained after 96 h of fermentation. The prolonged storage of beads
(24 h) influenced the glutamic acid yield. After 96 h fermentation the maximum
yield of glutamic was 37.1 mg mL-1. The cells encapsulated in properly
solidified beads had better storage stability than the free cells (Lu and Chen,
1988).
| Table 5: | Effect of alginate concentration on glutamic acid production |
| Table 6: | Effect of calcium ion on glutamic acid production |
| Table 7: | Effect of storage period of beads on glutamic acid production |
| Table 8: | Effect of initial biomass on glutamic acid production |
| Table 9: | Production of glutamic with free/immobilized/co immobilized cells |
Effect of Initial Cell Loading
The cell concentration ranged between 0.25-2.0 g per 20 mL gel was used
(Table 8). It was evident that the glutamic acid production
was influenced by the initial biomass. After 96 h fermentation with 0.75 cell
concentration the glutamic acid was more. The increase of cell in gels, oxygen
was consumed faster then it could diffuse into beads (Gosmann and Rehm, 1986).
The mixed culture of B. rosem and E. intermedia type-1 were immobilized together in calcium alginate gel matrix were used to find out feasibility of co-immobilization for the glutamic acid production in single process. The same medium and predetermined optimum conditions which were employed for free cell and immobilization cells were used for this study. The co immobilized B. rosem and E. intermedia type1 produced 39.6 mg mL-1 of glutamic acid.
The results of microbial fermentation carried out using the selected production medium under similar experimental conditions revealed that the glutamic acid (40.5 mg mL-1) obtained with Brevibacterium roseum is substantially greater than the yield of glutamic acid (37.2 mg mL-1) obtained with immobilized Brevibacterium roseum. It is further observed that the co immobilized cells of B. roseum and E. intermedia type-1 produced 39.6 mg mL-1 glutamic acid which was higher than immobilized Brevibacterium roseum but slightly lower than the glutamic acid yield with B. roseum free cells (Table 9). The study shows that the mixed culture has greater potential for glutamic acid production instead of single organism because the conversion of glucose to α-Ketoglutarate by B. roseum in the first step and the subsequent conversion of α-Ketoglutarate to glutamic acid in the next step by Escherichia intermedia type 1.
CONCLUSIONS
Production of glutamic acid by Brevibacterium roseum was enhanced by the manipulation of media ingredient concentrations. Further the results obtained with co-immobilization of B. roseum and E. intermedia type 1, are quite promising. The increased production of glutamic acid by co-immobilized whole cells might be due to the presence of mixed culture with gel matrix. Hence the advantage of immobilized cells can be attributed to co-immobilized whole cells than the free cell fermentation and the immobilized cells can be recycled for the continuous production of glutamic acid.
ACKNOWLEDGMENTS
The project was financed by University Grants Commission (SAP phase-III), New Delhi and the Center for Biotechnology, Department of Chemical Engineering, Andhra University for providing the necessary chemicals and laboratory facilities.