Effect of various immobilization matrices on Lactobacillus delbrucekii cells for optically pure L (+) lactic acid production

Ch. Subba Rao¹, R.S. Prakasham¹*, Ch. Sri Lakshmi² and A. Bhaskar Rao²

^1-Bioengineering and environmental center, ^2- Organic chemistry-1,

Indian Institute of Chemical Technology,Hyderabad – 500 007

Corresponding author: Dr. R. S.Prakasham

Telefax: +91-40-27193159 ; Email: prakasam@iict.res.in

Current Trends in Biotechnology and Pharmacy , Volume 3 (3) July - 2009

Abstract

The present study reveals the effect of Lactobacillus delbrucekiicells immobilized in various matrices for the production of optically pure L (+) lactic acid. Functionalized alginate matrices were effective and suitable for higher L (+) lactic acid yields compared to other matrices. Repeated batch fermentation showed productivity of 1.74, 1.44, 1.48 and 1.52Yp/s with functionalized alginate, Ca-alginate, k-Carrageenan, and glass beads, respectively. L. delbrucekii cells were immobilized in natural and functionalized alginate beads. The scanning electron microscopic studies showed increase in entrapped microbial cell biomass in modified immobilized beads compared to other matrices. These modified alginate beads showed enhanced stability and selectivity towards L (+) lactic acid production in higher yields with an enantiomeric selectivity of 99% and low by-product production.

Key words: Alginate, Enantiomeric selectivity, Fermentation, Immobilization, L(+) lactic acid, Lactobacillus delbrucekii

Introduction

Optically pure lactic acid is gaining importance in the current biotechnological era as it has vast application potential especially as feedstock for biodegradable polymers, oxygenated chemicals, plant growth regulators, synthesis of environmental friendly green solvents and specially chemical intermediates (1 ).  Hence, it became a large volume (one lakh tones per annum) chemical commodity with an annual growth rate of 15 % in global market with an estimate of 3.9 million tones in the year 2008 (2).Though lactic acid could be synthesized either by chemical means or by fermentation methods using specialized microbial strain, the fermentation methodology is becoming prominent (50 % of the world supply is by conventional free cell fermentation) due to the production potential of optically pure lactic acid (3).  In fact, the efficiency and economics of microbial product production has always been a concern with respect to fermentation medium development (4, 5, 6), microbial strain selection (7), substrate economics (8), biomass development and downstream processing (9,10).  In order to improve the lactic acid productivity, several microbial strains were isolated and studied in detail to understand their potential in industrial scale. However, it achieved a limited success mainly due to low productivity associated with a change of fermentation environment (increased acidity of the fermentation medium) which resulted ing in reduced growth of microbial strain and productivity. Efforts have been made to enhance the biomass concentration, online removal of product, reduction of by-product formation and improve the metabolism and most of the work has been focused in by using starch, beet molasses, whey and cane sugar as the fermentation media including glucose containing other wastes (11).  In this context, immobilization technology has shown ed promising role as most of above problems such as cell-retaining capacity, reduced susceptibility to contamination, and reuse of the biocatalyst with higher product conversion capability over free-cell fermentations could be solved without much alteration in the fermentation conditions (12-14).  Variety of immobilization matrices including natural (alginate, carrageenan, agar-agar, glass beads, etc) and synthetic (polyvinyl alcohol, poly acrylamide, etc) containing sodium alginate, calcium pectate gels, chemically modified chitosan and alginate beads were evaluated ( 15-18). Among all immobilization matrices studied, sodium alginate attracted scientific attention due to its eco-friendly nature, cost-effectiveness, the mild conditions required for immobilization, its simplicity and non-toxic nature (17).  Studies with Lactobacillus delbrucekii strain entrapped in the alginate bead matrix indicated that the mass transfer of substrates and products diffuse in and out easily. However, stability of the beads was important to maintain high conversion of substrate to product as this matrix has certain demerits like losing its stability under extreme pH and certain ionic concentration (14, 20, 21), which was influenced by the concentration of sodium alginate and bead diameter (18, 19, 22). Studies made by different scientific communities using different immobilization matrices, fermentation media, different microbial strains under different fermentation environments caused ing difficulty in comparison of the results and their subsequent use at process evaluation steps.  Hence, in the present investigation, an effort has been made to compare the growth pattern of L. delbrucekii under different immobilization environments (natural and functionalized alginate, carrageenan and glass bead), also evaluate its substrate utilization and obtain optically pure lactic acid production pattern under prescribed fermentation conditions along with its by-product formation.  

Materials and Methods

Organism and Medium

The organism, L.delbrucekii (NCIM 2365) was used in this study. The culture was maintained on deMan–Rogosa–Sharpe (MRS) agar (Hi-media) slabs stabs and sub cultured twice in a month. Sodium alginate (A2158; viscosity approximately 250 cps at 25 °C) was procured fromSigma-Aldrich,USA.Spherical porous sintered glass beads having SIKUG04 matrix type consisting of 0.4-1.0 mm dimension, 55-60 % pore volume, 120 µm pore diameter and 600 g/l density were purchased from SIRAN (Schott Glassware, MainZ, Germany) and was used for immobilization. All chemicals and solvents used in this study were of analytical grade and procured from standard firms.

Derivatization of Alginic Acid and Analysis

Alginate succinylation and palmitoylation was performed according to modified method of Phillips et al. (21) and Le-Tein et al. (24),respectively. The degree of succinylation was determined by the titration method as described byWurzburg (25), while the degree of palmitoylation of alginate was determined by ninhydrin method (detecting free amino groups) as described by Phillips et al. (21).

Cell Immobilization

L. delbrucekii cells were immobilized in κ-carrageenan, sodium alginate and succinylated alginate beads using 2 % (v/v) inoculum (10⁵ CFU/ml) under sterile conditions. To 4% sodium alginate/succinylated alginate solution, an equal volume of 24 h grown L. delbrucekii cell culture was added and mixed thoroughly. The resultant cell suspension was dropped as droplets into 2% calcium chloride solution to get the calcium alginate/ succinylated/ palmitoylated alginate-immobilized beads. In κ-carrageenan immobilization, 4% solution was prepared at 70°C, cooled to room temperature, and equal volumes of bacterial suspension and κ-carrageenan solution were mixed and immobilized by dropping into 2% KCl solution at 10°C. The resultant immobilized beads were washed with sterile distilled water, and the resultant immobilized cell beads were then incubated at constant shaking condition at 150 rpm at 37°C. The stability of immobilized beads was measured in terms of time taken for dissolution of five beads from each matrix in 3 M phosphate buffer (pH 5.0).

Fermentations

Lactic acid production with both the free and immobilized cell fermentations was performed at 37 °C using the production medium (glucose, 100 g/l; corn steep liquor, 68 ml/l; trace mineral solution, 1 ml/l; and CaCO₃, 100 g/l adjusted to pH 6.4)and 24-h grown inoculum (5%, v/v) having an optical density as 1.0 at 600 nm. The cell-free samples were then collected at predetermined time intervals and were analyzed using high-performance liquid chromatography (HPLC). Repeated batch fermentations were carried out regularly in a fresh medium after every 144 h, using the immobilized cell beads. The process was repeated till the lactic acid production was continued by viable cells present in the immobilized matrix. The data presented in this study was were the average values of three repeated experiments.

Cell mass estimation 

Cell growth was determined spectrophotometrically at 600 nm and converted to cell count using a conversion factor (one unit at 600 nm was equivalent to 10⁵ CFU/ml,which corresponded to 32 mg protein that was calculated by plotting standard graphs). In case of all immobilized conditions except glass bead immobilization, 5 beads were dissolved in 2ml of phosphate buffer (3M; pH 5.0).  and Cells were collected by centrifugation at 5000 rpm at room temperature and used for cell mass measurement,by serial dilution plate count method, using agar-based growth medium.  The total number of released cells was determined by standard plate count method using agar plates after incubating at 37 °C for 24 h. At the end of each batch, the cell densities in the beads were enumerated using similar method to study the total cell loss upon repeated use. Viable cell counts were performed in duplicates and expressed in CFU/ml of immobilized beads. While, cells immobilized on glass beads were measured indirectly by determination of total protein content with a bicinchoninic acid (BCA) assay (26, 27), carrier samples were washed with 1 ml phosphate buffer (17 mM; pH 7.6), followed by addition of 1 ml lyses-buffer (0.1 M TRICIN (N-Tris (Hydroxymethyl)-methyl) glycine, 0.2% Triton X-100, pH 7.6).  The resultant solution was subjected to strong overtaxing for 15 min. The BCA-assay was performed with the supernatant of the lyses solution. For this, 50 ml sample was mixed with 1 ml standard working reagent and then incubated at 60 ^oC for 30 min. After incubation, the samples were cooled and its absorbance was measured at 562 nm. This was converted to CFU according to a standard curve, which was constructed before, by applying the BCA-assay with cell suspension of known cell density. 

Scanning Electron Microscopy

For microscopic studies,immobilized beads containing L.delbrucekii cells were transferred to vials and fixed in 3.5% gluteraldehyde in 0.05 M phosphate buffer (pH 7.2) for 24 h at 4 °C. These cells were then post fixed by incubating in 2 % aqueous osmium tetroxide in the same buffer for 2 h. The samples were then dehydrated by gradient alcohol series and dried to the critical point by incubating in an Electron Microscopy Science CPD unit.The dried samples were then mounted over the stubs with double-sided conductivity tape. Finally, a thin layer of platinum metal was applied over the sample using an automated sputter coater (JEOL JFC 1600) for about 90 sec. The samples were then scanned under scanning electron microscope (model: JOEL-JSM 5600) at various magnifications using 5 kV (accelerating voltage) at RUSKA Lab,c College of Veterinary Sciences,ANGRAU, Hyderabad, India.

HPLC Analysis

Concentrations of glucose and organic acids (lactic, formic, propionic and acetic acids) present in filtered fermentation culture broth were determined by HPLC using GROM Resin ZH column (250×8 mm) using mobile phase 5 mM H₂SO₄ and absorbance at 210 nm. The optical purity of the lactic acid was analyzed using a chiral column [chiral pak MA (+) obtained from Daicel Chemical] using 2 mM CuSO₄ as an eluent and absorbance at 250 nm. All the experiments were carried out in three replicates and the results given were the mean values.

Results and Discussion

Lactic acid production pattern was studied under submerged fermentation environment using production medium with different immobilized Lactobacillus delbrucekii beads.  The acid production pattern differed with the type of immobilization matrix used for L. delbreckii cell indicating the selected matrix material’s impact on metabolism related to the production of lactic acid, in this L. delbrucekii, with similar fermentation environment.  Maximum lactic acid production was noticed with derivatized alginate immobilized beads and minimum production was observed with free cell fermentation.  A 57 % improvement in lactic acid production was noticed with immobilized beads compared to free cell fermentation.  Lactic acid production was observed to be 51.2 and 80.5 g/l with free and immobilized cells, respectively.  Among immobilized cell fermentations, natural polymer immobilized cells (alginate - 63.5 g/l and carrageenan - 68.9 g/l)showed less lactic acid productivity compared with glass bead (72.5 g/l),succinylated (76.2 g/l) and palmitoylated (80.5 g/l) alginate immobilized.  A 30 % of productivity was improved with palmitoylated alginate immobilized cells compared to alginate immobilized L.delbrucekii cells suggesting that the chemical variation of matrix material had influenced the metabolism of L. delbrucekiicells.  This enhanced lactic acid production under immobilized conditions may be attributed to improved buffering activity of fermentation medium. Such increased production profile as well as the associated metabolic affects on cell growth and subsequent product formation was well documented in literature (18,19, 28).  Li Ten et al., (24) observed an improved pH stability and better survival of succinylated alginate immobilized L.delbrucekii cells during intestinal passage.  Further analysis of fermentation broth was performed to investigate the optical purity of produced lactic acid under different immobilized environments.  It was observed that the optical purity of L (+) lactic acid was 99% in both functionalized (both the succinylated and palmitoylated) alginate beads, compared to other immobilization matrices where it varied from 88 to 92 % (results were not shown).

Immobilization dependent improved product productivity in different microbial strains waswere attributed for metabolic shift towards product production rather than microbial strain growth,in addition to improved biomass density in immobilized bead environment and low cell wash off as compared to free cell fermentation (14, 17, 29).  Hence, the cell morphology under different immobilized conditions were was studied by analyzing the scanning electron micrographs of surface and cross sections of different matrices immobilized cell beads and were reported in (Fig 1-5).  The cell density was differed in different immobilized beads (with respect to matrix material), through the same cell concentration was used for all immobilizations, indicating the impact of chemical nature of immobilization matrix on support of microbial cell growth and its retention during fermentation conditions.  Maximum cell density was observed with succinylated and palmitoylated alginate immobilized beads with pocketed distribution on /or in the bead (Fig 3 and 4).  In case of glass beads, the cells were mostly located in pores along with calcium carbonate crystals (Fig 2).  A more or less uniform distribution was observed in case of carrageenan and alginate immobilization indicating, functionalization has an  impact on cell distribution under immobilized environment (Fig 1 and 5). 

Comparative evaluation of lactic acid production values with respect to cell biomass under immobilized conditions reveals that properties of immobilization matrix hasvean influence on L. delbrucekiimetabolism associated lactic acid production. This was concluded, based on observation, that there was variation of lactic acid production and an observed difference in cell densities y under different immobilized environments by L. delbrucekii cells, under similar fermentation conditions (Fig 1 and 2).  These results were in accordance with observed metabolite production values by same microbial strain with immobilization matrix variation (17).

In most of the lactic producing microbial strains, lactic acid production is associated with other by-products such as ethanol, acetic and propionic acid production (30, 31). These by-products have major impact on production of biodegradable polymer production during polymerization (3).  Production of these lactic acid associated by-products depends on culture conditions, fermentation medium composition and microbial genetic nature.  Analysis of by-product profile in the cell free fermentation broth of different immobilized L. delbrucekii cells indicated that ethanol, acetic and propionic acid profile varies with the chemical nature of the matrix chemical nature (Fig 6).  Among all tested immobilization matrices, palmitoylated alginate showed less impurity profile followed by succinylated and glass bead immobilization. Natural alginate immobilized cells produced more amount of by-products compared to carrageenan immobilized cells.  Ethanol impurity was not observed in derivatized alginate as well as in the  and glass bead immobilized cells. 

Stability studies indicated that palmitoylated alginate immobilized beads were more stable as compared to natural alginate, succinylated and carrageenan immobilized during fermentation.  Our fermentation studies also revealed that palmitoylated alginate beads could be used more than eight fermentation cycles compared to six cycles in case of alginate.  Though glass bead immobilized cells revealed higher fermentation cycles than palmitoylated, the productivity values decreased drastically after its  6^th fermentation cycle (results not shown).

Conclusion

Lactic acid along with its by-products production were studied with L. delbrucekii cells using immobilized natural polymers (alginate and carrageenan), functionalized polymers (succinylated and pamitoylted) and glass beads, and these were compared with free cell fermentation results.  Palmitoylated alginate immobilized cell beads showed improved L (+) lactic acid production with less by-product formation, higher stability and more fermentation cycles compared to other fermentation studies.  Improved cell density was also observed in derivatized alginate immobilized beads compared to alginate.  Overall, effective optically pure L (+) lactic acid and less by-product (ethanol, acetic and Propionic acid) production were observed with palmitoylated alginate as an immobilization matrix using L. delbrucekii cells, indicating its commercial significance.

Acknowledgement:

The authors acknowledge Dr. Sanjay Nene, Scientist, NCL, Pune for providing L. delbrucekii culture and Dr. Y Yogeshwara Rao and Dr. Meenakshi Singh of CSIR,New Delhi for financial support under NMITLI programme, and to CSIR for providing SRF to Mr.Ch. Subba Rao.

References

1.Tong, W.Y., Fu, X.Y., Lee, S.M., Yu, J., Lui, J.W. & Wei, D.Z. (2004). Purification of l-lactic acid from fermentation broth with paper sludge as a cellulosic feedstock using weak anion exchanger Amberlite IRA-92. Biochemical Engineering Journal, 18: 89–96.

2.Ueno, T. (2003). Lactic acid production using two food processing wastes, canned pineapple syrup and grape invertase, as substrate and enzyme. Biotechnology Letters, 25: 573–577.

3.Datta, R. & Tsai, S.P. (1995). Technological and economic potential of poly(lactic aid)and lactic acid derivates. Journal of FEMS Microbiological Reviews, 16:221–231.

4.Prakasham, R. S., Subba Rao, Ch., Sreenivas Rao, R. & Sarma, P.N. (2005). Alkaline protease production by an isolated Bacillus circulans under solid state fermentation using agroindustrial waste: Process parameters optimization. Biotechnology Progress, 21: 1380-1388.

5.Prakasham, R.S., Subba Rao, Ch. & Sarma, P.N. (2006).  Green gram husk-an inexpensive substrate for alkaline protease production by Bacillus sp. in solid state fermentation.  Bioresource Technology, 97: 1449-1454.

6.Prakasham, R.S., Subba Rao, Ch, Sreenivas Rao, R., Suvarna Lakshmi, G. & Sarma, P.N. (2007). L-asparaginase production by isolated Staphylococcus sp.  – 6A. Design of experiment considering interaction effect for process parameter optimization.  Journal of Applied Microbiology, 102: 1382-1391.

7.Sreenivas Rao, R., Prakasham, R.S.,Krishna Prasad, K., Rajesham, S., Sharma, P.N. & Venkateshwara Rao, L. (2004). Xylitol production by Candida sp. : Parameter optimization using Taguchi approach.  Process Biochemistry, 39: 951-956.

8.Sreenivas Rao R, Pavana Jyothi Ch, Prakasham R S, Subba Rao Ch Sarma P N and Venkateswar Rao L. Strain improvement of Candida tropicalis for the production of xylitol: Biochemical and physiological characterization of wild and mutant strain CT-OMV5. The J Microbiol. 44, 113-120, 2006.

9.Subba Rao,Ch., Sathish, T., Mahalaxmi, M., Suvarna Laxmi, G., Sreenivas Rao, R. & Prakasham, R.S. (2008).  Modelling and optimization of fermentation factors for enhancement of alkaline protease production by isolated Bacillus circulans using feed-forward neural network and genetic algorithm.  Journal of Applied Microbiology, 104: 889-898.

10. Mahalaxmi, Y.,Subba Rao,Ch., Suvarnalaxmi, G., Sathish, T., Sudhakar, P. & Prakasham, R. S. (2008). Rifamycin B production pattern in Nocardia RSP-3 strain and influence of barbital on antibiotic production.  Current Trends in Biotechnology and Pharmacy, 2: 173-181.

11.Idris, A. & Suzana, W. (2006). Effect of sodium alginate concentration, bead diameter, initial pH and temperature on lactic acid production from pineapple waste using immobilized Lactobacillus delbrucekii Process Biochemistry,41: 1117-1123.

12.Lamboley, L., Lacroix, C., Artignan, J.M., Champagne, C.P. & Vuillemard, J.C. (1999). Long-term mechanical and biological stability of an immobilized cell reactor for continuous mixed-strain mesophilic lactic starter production in whey permeate. Biotechnology Progress, 15: 646-654.

13.Lamboley, L., Lacroix, C., Sodini, I., Lemay, M.J. & Champagne, C.P. (2001). Effect of inoculum composition and low KCL supplementation on the biological and rheological stability of an immobilization-cellsystem for mixed mesophilic lactic starter production. Biotechnology Progress, 17: 1071-1078.

14.Srinivasulu, B., Prakasham, R.S.,Annapurna, J., Srinivas, S., Ellaiah, P. & Ramakrishna, S.V. (2002). Neomycin Production with free and immobilized cells of Streptomycin marinensis in Air-lift Reactor. Process Biochemistry, 38: 593-598.

15.Kosseva, M., Beschkov, V., Kennedy, J.F. & Lloyd, L.L. (1998). Malolactic fermentation in chardonnay wine by immobilized Lactobacillus casei cells. Process Biochemistry, 33: 793–797.

16.Long, Z., Huang, Y., Cai, Y., Cong, W. & Ouyang, F. (2004).  Immobilization of Acidithiobacillus ferrooxidans by a PVA–boric acid method for ferrous sulphate oxidation. Process Biochemistry, 39: 2129–2133.

17.Ramkrishna, S.V. & Prakasham, R.S. (1999). Microbial fermentations with immobilized cells. Current Science, 7: 87–100.

18.Subba Rao,Ch., Prakasham, R.S., Bhaskar Rao, A. & Yadav, J.S. (2008). Functionlized alginate as Immobilization Matrix in Enantioselctive L (+) Lactic acid production by Lactobacillus delbrucekii. Applied Biochemistry and Biotechnology, 149: 219-228.

19.Subba Rao,Ch., Prakasham, R. S., Bhaskara Rao, A. & Yadav, J.S. (2008).  Production of L(+) lactic acid by Lactobacillus delbrueckii immobilized in functionalized alginate matrices.  World Journal of Microbiology and Biotechnology, 24: 1411-1415.

20.Shapiro, L. & Cohen, S. (1997). Novel alginate sponges for cell culture and transplantation. Biomaterials,18: 583–590.

21.Phillips, D.L., Xing, J., Chong, C.K., Liu, H. & Corke, H. (2000). Determination of degree of succinylation in drives modified starches by raman spectroscopy. Journal of Agricultural Food Chemistry, 48: 5105–5108.

22.Mohamed, A., Abdel-Naby, M. & Reyad, A-F. F. (2000). Biosynthesis of cyclodextrin glucosyltransferase by immobilized Bacillus amyloliquefaciens in batch and continuous cultures. Journal of Biochemical Engineering,5:1–9.

23.Subba Rao,Ch., Madhavendra, S.S., Sreenivas Rao, R. & Prakasham, R.S. (2008).  Studies on improving the immobilized bead reusability and alkaline protease production by isolated immobilized Bacillus circulans (MTCC 6811) using overall evaluation criteria. Applied Biochemistry and Biotechnology, 150: 65-83.

24.Le-Tien, C., Millette, M., Mateescu, M.A. & Lacroix, M. (2004). Modified alginate and chitoson alginate for lactic acid bacteria immobilization. Biotechnology and Applied Biochemistry, 39: 347-354.

25.Wurzburg, O.B. (1964). In: Methods in carbohydrate chemistry, Alhistler, R.L., (Eds). Academic Press,New York, pp.286-288.

26.Smith, P.K., Krohn, R. & Hermanson, E.K. (1985). Measurement of protein using bicinchoninic acid. Anal Biochemistry, 150: 76-85.

27.Beshay, U. & Moreira, A. (2003). Reapted batch production of alkaline protease using porous sintered glass as carrier. Process Biochemistry, 38: 1463-1469.

28.Boyaval, P. & Goulet, J. (1988). Optimal conditions for production of lactic acid from cheese whey permeate by Ca-alginate entraped Lactobacillus helveticules. Enzyme Microbial Technology,10: 725-728.

29.Prakasham, R.S., Kuriakose, B. & Ramakrishna, S.V. (1999). The Influence of Inert Solids on Ethanol Production by Saccharomyces cerevisiae.  Applied Biochemistry and Biotechnology, 82: 127-134

30.Lee, Ho-J., Xie, Y., Koo, Y.-M. & Linda Wang, N–H.  (2004). Separation of Lactic Acid from Acetic Acid Using a Four-Zone SMB. Biotechnology Progress 20: 179-192.

31.Patel, M.A., Ou, M.S., Ingram, L.O. & Shanmugam, K.T. (2005). Simultaneous saccharification and co-fermentation of cystalline cellulose and sugarcane begassae hemicellulose hydrolysate to lactate by a thermotolerant acidophilic Bacillus sp.  Biotechnology Progress, 21: 1453-1460.

Fig 1: Scanning electron micrographs of L. delbrucekii cells immobilized using carrageenan A) surface B) section

Fig2: Scanning electron micrographs of L. delbrucekii cells immobilized using porous glass beads A) surface B) section

Fig 3: Scanning electron micrographs of L. delbrucekii cells immobilized using palmitoylated alginate A) surface B) section

Fig 4: Scanning electron micrographs of L. delbrucekii cells immobilized using Succinylated alginate A) surface B) section

Fig 5: Scanning electron micrographs of L. delbrucekii cells immobilized using alginate A) surface B) section

Fig 6: lactic acid and other impurities production profile during fermentation by free cells and different immobilized cells of L. delbrucekii

F.C.–free cells, A-alginate S.A- Succinylated alginate, P.A –palmitoylated alginate, C-carrageenan, G.B- glass beads