Improved Production of L-Lysine by Immobilized Biotin Auxotropic Mutant Micrococcus glutamicus AB₂₀₀

 

A. Gupta*, S. K. Das and A. K. Banik

Department of Chemical Engineering, University of Calcutta, Kolkata – 700 009

*Corresponding Author E-mail: amritagupta1982@gmail.com

Experimental studies were carried out to examine the efficiency of whole cell immobilization in calcium alginate beads and agar blocks for the production of L-lysine using a biotin autotrophic mutant Micrococcus glutamicus AB₂₀₀ developed in laboratory by induced mutation from a regulatory mutant Micrococcus glutamicus AB₅. Under the same physico- chemical environment, using selected suitable synthetic medium, different parameters for both calcium alginate beads and agar blocks were optimized one by one to maximize the production of L-lysine. Production was increased significantly with cells entrapped in calcium alginate beads compared to free cells with the presence of 0.2(M) CaCl₂ in synthetic medium, 0.2 (M) CaCl₂ for bead formation, 4% sodium alginate, 24 h storage period of beads and 1.5 cell/alginate ratio but the accumulation of was decreased significantly with the whole cell entrapped in agar blocks, compared to free cells.

 

 

INTRODUCTION:

Lysine is an important amino acid in human as well as in animal nutrition. It is used as a feed additive to enhance their nutritional value [1]. Out of 20 naturally occurring amino acids, L-lysine is one of the nine essential amino acids. It is used as feed additives to enhance their nutritional value. Fermentation technology has played crucial roles over a period of time and currently the amino acid produced by fermentation represent chief products of biotechnology in both volume and value [2]. The application of alginate for the purpose of whole cell immobilization was first reported in 1975 [3].Immobilization of cells for fermentation has been developed to eliminate inhibition caused by high concentration of substrate and product, also to enhance the productivity [4, 5, 6].

 

After successful achievement of whole cell immobilization technique for the production of different secondary metabolites including amino acids, this study is intended to produce L-lysine with immobilized whole cell of the mutant Micrococcus glutamicus AB_200.

 

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

 

MATERIALS AND METHODS:

Organism: 

Micrococcus glutamicus AB₂₀₀, a biotin requiring auxotropic mutant derived from a regulatory mutant Micrococcus glutamicus AB₅ by induced mutation in the laboratory using UV irradiation as physical and methyl methane sulphonate as chemical mutagens were used throughout the study[7].

 

Growth medium and growth condition: 

The cultures were maintained on agar slants having composition: Glucose: 1%, Urea: 0.5%, K₂HPO₄:0.1%, MgSO₄.7H₂O:0.02%, Biotin: 0.1 µg/mL, Agar: 4%, pH: 7.5, incubation temperature: 30 ^o C, incubation time: 48h.

 

Preparation of calcium alginate beads:

The cell suspension is slowly added to the sterile solution of sodium alginate (3%) and mixed thoroughly with the sterile glass rod. The mixture was continuously extruded into 100mL Erlenmeyer flask containing 20mL 0.1 (M) CaCl₂ for 30 minutes. Then the beads were filtered aseptically and washed successively with sterile buffer solution pH 7.5 and with sterile distilled water [8].

 

Preparation of agar block:

A defined quantity of agar was dissolved in 18 mL of 0.9% NaCl solution to get final concentration of 4.0% (v/v) cell suspension was added to the molten agar maintained at 40 ^o C 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 7.5 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 [9, 10].

 

Estimation of L-lysine:

Descending paper chromatography was employed for detecting L-Lysine in culture medium and was run for 16 to 18 h on Watman No: 1 chromatographic paper. Solvent system: n-butanol:acetic acid: water 2:1:1. The spots were visualized by spraying with a solution of 0.2% ninhydrine in acetone and quantitative estimation of L-lysine in the suspension was done using colorimetric estimation method.

 

Recovery and identification of L-lysine:

For the identification of L-lysine the fermentation broth after separation of cells was adjusted to pH = 5 with HCl and filtered. The filtrate was then treated with active charcoal to remove the colored impurity. After filtration the clear filtrate was finally passed through a Dowex 50(H⁺ type) column in which the amino acid was absorbed. After washing with water and elution with 0.24(N) HCl, the elute was further passed through an amberlite (H⁺ type) for elimination of chloride ion. The elute was concentrated by evaporation in vacuum and the product was obtained in a crystalline form in the cold. The pure material is proved to be L-Lysine both by chemical test, optical rotation and by paper chromatographic method. Insoluble picrate derivative of L-Lysine was prepared and melting point of the derivative 266^oC. [11].

 

Elemental analysis of the pure material gave the following values: C – 49.31%, H – 9.63%, N – 19.17%, O – 21.88%. Whereas the molecular calculation shows the following values: C – 49.29%, H – 9.65%, N – 19.16%, O – 21.89%. Optical rotation was estimated as (L)²⁵_{D =20.1}  (C=2, 5N HCl). These data agreed with those of L-lysine.

 

Estimation of dry cell weight:

After centrifugation, a few ml of 1.0 (M) HCl was poured into the precipitate of the bacterial cells to dissolve calcium carbonate. The residual bacterial cells were washed with water and dried at 100^o C until cell weight remains constant [11]

 

Statistical Analysis:

Data were expressed as mean ± SEM, where n=6. The data was analyzed by student’t test, by using MS Excel. Two measurements were considered significant if the corresponding p value<0.05 and considered highly significant if p<0.01.

 

RESULTS AND DISCUSSIONS:

  I.     Production of L-lysine by immobilized cells of the mutant Micrococcus glutamicus AB₂₀₀ in calcium alginate beads:

L-lysine production conditions for the mutant Micrococcus glutamicus AB₂₀₀ entrapped in calcium alginate beads were investigated considering the following parameters:

 

   i.     Effect of calcium chloride in the synthetic medium: The finally selected synthetic medium contained 0.05% calcium carbonate. However, calcium alginate bead stability was investigated with different concentration of calcium chloride [ranging from 0 to 0.25 (M)] in the synthetic medium for L-lysine accumulation. Maximum production was obtained with 0.2 (M) calcium chloride as depicted in Fig 1. Calcium chloride is essential for bead stability and pore size of the beads [9]. The calcium alginate is unstable in presence of phosphate and certain cation such as Mg²⁺ or K⁺ which are major nutrients for living microorganisms [12]. Solubilizing effect of calcium alginate beads can also be prevented by supplementing the synthetic medium with calcium chloride [9].

 

 ii.     Effect of calcium chloride on calcium alginate bead formation: Owing to bead stability, another important aspect of calcium chloride was to confer the proper shape and porosity of the calcium alginate beads [13]. Different concentrations of calcium chloride ranging from 0 to 0.25 (M) were investigated where 0.2 (M) concentration was proved to be optimum.[Fig 2]

 

iii.     Effect of sodium alginate concentration on bead formation: When alginate gel containing the organism were added drop by drop to 0.2 (M) calcium chloride solution calcium alginate beads were formed with the organisms encapsulated in the beads. So there should be an optimum sodium alginate concentration for maximum stability of the bead. 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 AB₂₀₀ for L-lysine production. At 4% sodium alginate concentrations, the yields were maximum [Fig 3]. 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 concentration increases the pore size leading to  leakage of the cells in the medium, thus production falls markedly [13]. thus with 4% sodium alginate, we got the appropriate bead  shape with maximum yield. Therefore, this concentration was used for further study.

 

iv.     Effect of storage periods of calcium alginate beads: The storage periods of calcium alginate beads on the L-lysine production were investigated using different ages of beads ranging from 18 hours to 144 hours [Fig 4]. 24 h aged beads showed maximum production. But above this storage period, the production of L-lysine was not affected significantly. 0 h beads showed minimal L-lysine production with deformation of the beads. The cells encapsulated in properly solidified beads had better storage stability than the free cells [14]. The solidification of the beads require a minimum time interval which was 24 h in this case.

 

 v.     Effect of cell/alginate ratio: To study the effect of the amount of cell mass entrapped in the gel matrix on L-lysine production, the cell/alginate ratio was varied from 0.5 to 2.0. The beads were prepared using 4% sodium alginate and 2.0 (M) calcium chloride. The yield of L-lysine was maximum for cell/alginate ratio of 1.5 as shown in Fig 5. With higher values of cell alginate ratio, leakage of cells occurs. Thus cell/alginate 1.5 was considered as optimum and used for further study [Fig 5]

 

II.     Production of L-lysine by immobilized cells of the mutant Micrococcus glutamicus AB₂₀₀ entrapped in agar block:

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

 

   i.     Effect of block volume: The accumulation of L-lysine by bacteria is a function of surface area of the cell or the medium where it is entrapped [15]. Different agar block sizes (1-6 mm³) were studied and maximum production of L-lysine was obtained with 3.0 mm³ of agar block [Fig.6].

 

 ii.     Effect of storage period of agar block: Minimum storage period of immobilized matrices is required for proper entrapment of the organism and solidification of the matrices [15]. Thus different storage periods (18-144 h) of the agar blocks were studied for maximum L-lysine accumulation. Maximum yield was obtained with 48 h aged agar blocks above which the production was not altered significantly [Fig.7].

 

iii.     Effect of cell/agar ratio: to determine the maximum amount of cell mass entrapped with maximum yield of L-lysine different cell/agar ratio (0.5 to 2.0) were taken under consideration. Blocks were prepared with 4.0% agar and got maximum production with a cell/agar ratio of 1.25 as shown in Fig 8.

 

Studies on stability & reusability of immobilized Micrococcus glutamicus AB₂₀₀ :

An experimental study was carried out to examine the stability of immobilized cell of the mutant and number of times the cell can be reused safely. It was observed that immobilized Micrococcus glutamicus AB₂₀₀ can be used safely at least 12 times without any significant fall in lysine production.

 

So the bead can be used several times without any deformation of the bead and degeneration of the microorganism.

 

 

Fig 1-5: Production of L-lysine by a mutant Micrococcus glutamicus AB₂₀₀  entrapped in calcium alginate bead . Values expressed as mean ± SEM. Where n=6.

 

Fig 6-8: Production of L-lysine by a mutant Micrococcus glutamicus AB₂₀₀  entraped in agar blocks . Values expressed as mean ± SEM. Where n=6.

 

 

Table 1

 

 

 

Fig 9

 

Fig: 10

Photographic representation of immobilized Micrococcus glutamicus AB₂₀₀ entrapped in agar block [Fig 9] and calcium alginate bead [Fig 10]

 

Comparison of L-lysine accumulation by free and immobilized cells of the mutant Micrococcus glutamicus AB₂₀₀

Table 2

 

CONCLUSION:

From the present study it is clear that the production of L-lysine was increased with calcium alginate beads and decreased significantly 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 AB₂₀₀ entrapped in calcium alginate beads with high retention of the cellular activities.

 

ACKNOWLEDGEMENT:

We express our sincere thanks to Council of scientific and industrial research (CSIR) for providing us the financial support for this research work and to the librarians of Bose Institute, Kolkata.

 

REFERENCES:

1.       Sarva Rao, B., Muralidhararao  and Swamy, AV.N., 2011 “Studies on Continuous production Kinetics of L-Lysine by a Immobilized Corynebacterium glutamicum” 13032, Middle-East Journal of Scientific Research,  7 (2): 235-240.

2.       Ikeda M., Amino acid production process. Adv. Biochem. Eng/Biotechnol., 2003; 79; 1-35

3.       Hackel U., Klein J., Megnet R., Wagner F. 1975 Immobilization of microbial cells in polymeric matrices. Eur. J. APPL. Microbiol., 1, 291-293.

4.       Groboilot A., Boadi DK., Poncelet D. and Neufeld RJ, 1994, Immobilization of cells for application in the food industry. Crit. Rev. Biotechnol., 14: 75-107.

5.       Prasad B. and Mishra IM., 1995, On the kinetics and effectiveness of immobilized whole cell batch culture. Biores. Technol., 53: 269-275.

6.       Ganguly S., Banik A.K., Production of L-glutamic acid by Immobilized Micrococcus glutamicus AB₁₀₀ Asian J. Research Chem. 4(6): June, 2011.

7.        Gupta A,  Das S.K and  Banik A.K,  Induced mutation and selection of a high yielding strain of Micrococcus glutamicus  for L-lysine production. International Journal of Pharma and Bio Science. 3(2): 2012; B35-B42.

8.       Beshay U., Production of alkaline protease by Terredinobacter turniae cells immobilized in calcium alginate beads. African Journal of biotechnology, 2, 2003, 60-65.

9.       Nampoothiri K. M. and Panday A., Immobilization of Brevibacterium cells  for the production of L-glutamic acid. Boi resource technology, 63,1998, 101-106.

10.     Ganguly S. and Banik A.K. Production of L-glutamic acid by Immobilized Micrococcus Glutamicus AB_{100. ­}Asian journal of research in chemistry.

11.       Shah A.H., Hameed A, Ahmad S. and Khan GM. Optimization of culture condition for L-lysine fermentation by Corynebacterium glutamicum. Online Journal of Biological Science. 2(3); 2002: 151-156  

12.     Lu W.M. and Chem W.C., Production of L-glutamate using entrapped living cells of Bevibacterium ammoniagenes with calcium alginate gel, In proceedings of the national Science Council, Taipail, Taiwan, 6, 1988, 400-406.

13.     Shimmyo A., Kimura H. and Okada H., Physiology of L-amylase production by immobilized Bacillus amyloliquefaciens. European Journal of Applied Microbiology Biotechnology.

14.     Gosmann B. and Rehm HJ. Oxygen uptake of microorganisms entrapped in calcium alginate. Applied Microbiology and Biotechnokogy, 23, 1986, 163-167.

15.     Ramkrishna S.V. and Prakasham R.S. Microbial fermentation with immobilized cells. Current Science, 87, 1999, 87-100.

 

 

Received on 06.05.2013       Modified on 30.05.2013

Accepted on 16.06.2013      © AJRC All right reserved