Poly-L-hydroxybutyrate production by lactic acid bacteria

Transcription

1 FEMS Microbiology Letters 159 (1998) 293^297 Poly-L-hydroxybutyrate production by lactic acid bacteria Belma Asl m a; *, Fikret C al șkan a, Yavuz Beyatl a, Ufuk Guënduëz b a Gazi University, Faculty of Arts and Science, Department of Biology, Ankara, Turkey b Middle East Technical University, Faculty of Arts and Sciences, Department of Biology, Ankara, Turkey Received 8 September 1997; revised 24 November 1997; accepted 10 December 1997 Abstract Poly-L-hydroxybutyrate was determined in lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, Pediococcus and Streptococcus. Lactobacilli were grown in MRS broth, the others were grown in Elliker broth medium. Cell biomass was obtained by centrifugation. The cell walls were lysed with sodium hypochlorite. Poly-L-hydroxybutyrate was extracted using chloroform in a Soxhlet system. Then it was converted to crotonic acid using sulfuric acid and the amount of crotonic acid was measured spectrophotometrically. The yield of poly-l-hydroxybutyrate (% of cell dry weight) of Lactobacillus species was 6.6^35.8%. The values for Lactococcus, Pediococcus and Streptococcus species were 9.0^20.9, 1.1^8.0 and 6.8^17.2, respectively. It was observed that one of the Lactobacillus species did not produce poly-l-hydroxybutyrate. Generally, Lactobacillus species produced more poly-l-hydroxybutyrate than the other tested bacteria and no significant correlation was observed between poly-l-hydroxybutyrate production and cell density of the cultures. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords: Lactobacillus; Lactococcus; Pediococcus; Streptococcus; Poly-L-hydroxybutyrate production 1. Introduction Poly-L-hydroxybutyrate (PHB) is a thermoplastic polyester. It is biocompatible and biodegradable, and therefore, of industrial interest [1]. In the cell, PHB is an intracellular storage material synthesized during unbalanced growth conditions. All bacteria which are capable of PHB synthesis accumulate PHB during the stationary phase of growth when the cells become limited for an essential nutrient but have an excess for carbon sources [2^4]. Species from more than 50 genera are known to be capable of synthesizing PHB [5]. Despite the fact that PHB was also detected in actinomycetes [6] * Corresponding author. and yeasts [7], it is most often accumulated by bacteria of various morphological and physiological groups. Only the monomer was detected in the mycelium of micromycetes [8]. The amount of PHB production by the genera of Lactobacillus, Streptococcus and Pediococcus is not well studied and documented. The purpose of this study was to determine PHB production by di erent genera of lactic acid bacteria. 2. Materials and methods 2.1. Bacterial strains and culture media The species of Lactobacillus, Streptococcus and / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S (97)

2 294 B. Asl m et al. / FEMS Microbiology Letters 159 (1998) 293^297 Pediococcus used in this study were isolated and identi ed from di erent dairy and food products produced in Turkey. The strains used, their sources and optimal growth temperature [9] are listed in Table 1. Mesophilic and thermophilic Lactobacillus strains were grown twice in MRS broth, Lactococcus, Pediococcus and Streptococcus strains were grown twice in Elliker broth medium at the appropriate temperature (Table 1) for 24 h [10,11]. Lactic acid cultures were maintained in glycerol^ skim milk at 310³C. Prior to use they were subcultured twice in MRS or Elliker broth medium Analytical procedures Lactic cultures grown were inoculated at 2% (v/v) in sterile MRS or Elliker broth medium. Cultures were incubated for 48 h at optimal growth temperature (Table 1). Bacterial cells were centrifuged at 6000 rpm for 15 min. Pellets were dried at 40 þ 1³C for 24 h. The dry weight of the pellets was determined. Bacterial cell walls were lysed by adding sodium hypochloride, mixing and incubation at 60³C for 1 h. Supernatant was obtained by centrifugation and transferred to a Soxhlet system. Cell lipids and other molecules (except PHB) were extracted by adding and evaporating 5 ml 96% ethanol and acetoin. PHB was extracted by hot chloroform (adding 10 ml chloroform in a water bath). Then, chloroform was evaporated to obtain crystals of PHB. By adding 10 ml 98% sulfuric acid at 60³C for 1 h, PHB crystals were converted into crotonic acid. The absorbance of the solution was measured at 235 nm in a UV spectrophotometer against a sulfuric acid blank. The amount of PHB per gram dry weight of bacterial cells was determined using a standard curve of PHB [12,13]. All experiments were repeated three or sometimes four times, the average values of this procedure are given in the tables Statistical analysis The correlation between bacterial cell dry weight Table 1 Bacterial strains and their sources Strain Identi cation no. Source Optimal growth temperature (³C) 1. Lactobacillus plantarum A-C1 Lab. Biotechnol. a 30 þ 1 2. Lactobacillus plantarum B-C2 Lab. Biotechnol. a 30 þ 1 3. Lactobacillus brevis C3 Lab. Biotechnol. a 30 þ 1 4. Lactobacillus acidophilus N (1-4)-C4 Dept. Microbiol. b 37 þ 1 5. Lactobacillus casei N (B-1)-C5 Dept. Microbiol. b 37 þ 1 6. Lactobacillus bi dus N (3-4)-C6 Dept. Microbiol. b 37 þ 1 7. Lactobacillus fermentum N (3-2)-C7 Dept. Microbiol. b 40 þ 1 8. Lactobacillus bulgaricus B121,L-C8 Lab. Biotechnol. a 40 þ 1 9. Lactobacillus bulgaricus B79,L-C9 Lab. Biotechnol. a 40 þ Lactococcus lactis TO (1-1)-A1 R.S.H.I. c 30 þ Lactococcus lactis subsp. diacetylactis P-90-A2 R.S.H.I. c 30 þ Lactococcus cremoris TO(2-2)(19-12)-A3 R.S.H.I. c 30 þ Pediococcus halophilus C4S-B1 Lab. Biotechnol. a 37 þ Pediococcus halophilus C19S-B2 Lab. Biotechnol. a 37 þ Pediococcus halophilus B20S-B3 Lab. Biotechnol. a 37 þ Pediococcus halophilus B15S-B4 Lab. Biotechnol. a 37 þ Pediococcus halophilus A10S-B5 Lab. Biotechnol. a 37 þ Pediococcus halophilus A15S^B6 Lab. Biotechnol. a 37 þ Streptococcus thermophilus B35S-E1 Lab. Biotechnol. a 40 þ Streptococcus thermophilus B22S-E2 Lab. Biotechnol. a 40 þ Streptococcus thermophilus B34S-E3 Lab. Biotechnol. a 40 þ Streptococcus thermophilus 52S-E4 Lab. Biotechnol. a 40 þ 1 a Gazi University, Faculty of Sciences and Arts, Department of Biology, Section of Biotechnology, Ankara, Turkey. b Prof. Dr. Nezihe Tunail, Ankara University Faculty of Agriculture, Department of Microbiology, Ankara, Turkey. c Turkish Health Ministry, Re k Saydam Health Institute, Ankara, Turkey.

3 B. Asl m et al. / FEMS Microbiology Letters 159 (1998) 293^ Fig. 1. Amount of cell dry weight and PHB produced by all Lactobacillus strains in Table 2. In some of the strains, a correlation was observed between PHB production and dry cell weight (strain C5). (g l 31 ) and PHB production (g l 31 ) of the bacteria was determined according to Spearman's b correlation coe cient test. The b value was estimated with the formula b ˆ 16 X xi3yi 2 n n 2 31 and explained using Conver's table [14]. 3. Results and discussion The amount of cell dry weight and the yield of PHB produced by Lactobacillus species are shown in Table 2 and Fig. 1. Lactobacillus strains were grown in MRS broth medium for 48 h. The biomasses of the cultures were di erent (Table 2, Fig. 1). The yields of PHB (%) accumulated in the cells according to dry weight were also di erent: 13.8% for L. plantarum A, 7.2% for L. plantarum B, 29.4% for L. brevis, 17.1% for L. acidophilus, 29.0% for L. casei, 35.8% for L. bulgaricus, 19.1% for L. bi dus, and 6.6% for L. fermentum. L. bulgaricus 121L showed higher PHB production than the other Lactobacillus species. L. bulgaricus C9 did not seem to produce any PHB. The absence of PHB in this strain may be explained by the absence of active PHB genes. In one of the studies conducted by Lee et al. [15], it was reported that when the concerned plasmid DNA was transferred from Alcaligenes eutrophus into Escherichia coli, the recipient bacteria produced a high concentration of PHB. Manchak and William [16] mentioned that the accumulation of PHB in Azotobacter vinelandii UWD was related to enzyme Table 2 PHB production by some Lactobacillus species Bacteria Cell dry weight (g l 31 ) PHB a (g l 31 ) Yield of PHB b (%) L. plantarum A,C þ þ L. plantarum B,C þ þ L. brevis C þ þ L. acidophilus C þ þ L. casei C þ þ L. bi dius C þ þ L. fermentum C þ þ L. bulgaricus C þ þ L. bulgaricus C þ 0.43 ^c ^c a Determined at cell dry weight. According to cell dry weight. No PHB production.

4 296 B. Asl m et al. / FEMS Microbiology Letters 159 (1998) 293^297 Fig. 2. Cell dry weights and the amount of PHB produced by Lactococcus, Pediococcus and Streptococcus strains. It was observed that there was a correlation between PHB production and dry cell weight in some strains (A1, A2, A3). activities of 3-ketothiolase, acetoacetyl-coa and L- hydroxybutyrate dehydrogenase. Speci cally, there was no relation between high cell density and PHB content of Lactobacillus cultures. L. bulgaricus B121L, L. casei and L. brevis produced PHB more than other Lactobacillus strains. The b value was calculated to be b = This value was compared with the 0.05 level of the critical table value b = [14]. The results show that there was no signi cant correlation between cell dry weight (g l 31 ) and PHB amount (g l 31 ). The amounts of PHB produced by Lactococcus, Pediococcus and Streptococcus are reported in Table 3 and Fig. 2. The highest yield of PHB accumulation according to dry weight was obtained in L. lactis A1 (20.9%). Statistical analysis showed that there was no correlation between cell dry weight (g l 31 ) and PHB (g l 31 ) content of the cultures. In spite of this observation, a correlation was found in some strains used such as L. casei C5 (Fig. 1), L. lactis A1,. L. lactis subsp. diacetylactis A2 and L. cremoris A3 (Figs. 1 and 2). In general, the amount of PHB produced by some Lactobacillus species was higher than the amount of PHB produced by Lactococcus, Pediococcus and Streptococcus strains (Tables 2 and 3). On the other Table 3 PHB production by Lactococcus, Pediococcus and Streptococcus species Bacteria Cell dry weight (g l 31 ) PHB a (g l 31 ) Yield of PHB b (%) L. lactis A þ þ L. lactis subsp.diacetylactis A þ þ L. cremoris A þ þ P. halophilus B þ þ P. halophilus B þ þ P. halophilus B þ þ P. halophilus B þ þ P. halophilus B þ þ P. halophilus B þ þ S. thermophilus E þ þ S. thermophilus E þ þ S. thermophilus E þ þ S. thermophilus E þ þ a Determined at cell dry weight. b According to cell dry weight.

5 B. Asl m et al. / FEMS Microbiology Letters 159 (1998) 293^ hand, Lactococcus lactis produced more PHB than the Pediococcus and Streptococcus strains. The b value between dry cell weight and PHB content of all bacteria listed in Table 3 was calculated as b = When this value was compared with the 0.05 level of the critical value, s [14], the results showed that at the 0.05 level there is a statistically signi cant but low correlation between cell dry weight and PHB contents of the bacteria. Comparing the values reported in the literature, the amount of PHB estimated in lactic acid bacteria was generally lower than the amount of PHB produced by the soil bacteria Alcaligenes latus [17], Alcaligenes eutrophus [18], Azospirillum brasilense [19] and Rhizobium species [20]. References [1] Byrom, D. (1987) Polymer synthesis by microorganisms; technology and economics. Trend Biotechnol. 5, 246^250. [2] Anderson, A.J. and Dawes, E.A. (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial poly hydroxy alkanolates. Microbiol. Rev. 54, 450^472. [3] Page, W.J. (1989) Production of poly-l-hydroxybutyrate by Azotobacter vinelandii strain UWD during growth on molasses and other complex carbon sources. Appl. Microbiol. Biotechnol. 31,329^333. [4] Kim, B.C., Lee, S.C., Lee, S.Y., Chang, H.N., Chang, Y.K. and Woo, S.I. (1994) Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control. Biotechnol. Bioeng. 43, 892^898. [5] Doi, Y. (1990) Microbial Polyesters. VCH Publishers, New York. [6] Kannan, L.V. and Rehacek, Z. (1970) Formation of poly-lhydroxybutyrate by actinomycetes. Ind. J. Biochem. 7, 126. [7] Nuti, M.P. and Lepidi, A.A. (1974) Poly-L-hydroxybutyrate occurrence in Saccharomyces cerevisiae and its signi cance in the fermentation process. In: Proc. Fourth Int. Symp. of Yeasts, Part 1, Vienna, p [8] Nuti, M.P., Brooks, J.B. and Lepidi, A.A. (1975) Occurrence of K-, L-, and Q-hydroxybutyrates in some soil microfungi. Trans. Br. Mycol. Soc. 64, 79. [9] Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T. and Williams, S.T. (1994) Bergey's Manual of Determinative Bacteriology, 9th edn. Williams and Wilkins, Baltimore, MD. [10] Harrigan, W.F. and McCance, M.E. (1966) Laboratory Methods in Microbiology. Academic Press, New York. [11] Klaenhammer, T.R., McKay, L.L. and Baldwin, K.A. (1978) Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid. Appl. Environ. Microbiol. 35, 592^600. [12] Kuniko, M., Nakamura, Y. and Doi, Y. (1988) New bacterial copolyesters produced in Alcaligenes eutrophus from organic acids. Polymer Commun. 29, 174^176. [13] Bowker, R.R. (1981) Manual of Methods for General Bacteriology. American Society for Microbiology, Washington, DC. [14] Conver, W. J. (1971) Practical Nonparametric Statics, pp. 244^248. John Wiley and Sons, New York. [15] Lee, S.Y., Yim, K.S., Chang, H.N. and Chang, Y.K. (1994) Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly (3-hydroxybutyric acid) by recombinant Escherichia coli. J. Biotechnol. 32, 203^211. [16] Manchak, J. and Page, W. J. (1994) Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953^963. [17] [18] Doi, Y., Segawa, A., Kawaguchi, Y. and Kunioka, M. (1990) Cyclic nature of poly (3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus. FEMS Microbiol. Lett. 67, 165^170. [19] Okon, Y. and Itzigsohn, R. (1992) Poly-L-hydroxybutyrate metabolism in Azospirillum brasilense and the ecological role of PHB in the rhizosphere. FEMS Microbiol. Rev. 103, 131^ 140. [20] Tombolini, R. and Nuti, M.D. (1989) Poly (L-hydroxyalkanolates) biyosynthesis and accumulation by di erent Rhizobium species. FEMS Microbiol. Lett. 60, 299^304.