Zehra Nur YÜKSEKDAĞ, BELMA ASLIM, Yavuz BEYATLI

Determination of poly-beta-hydroxybutyrate (PHB) production by some mesophilic and thermophilic lactic acid bacteria

Bu çalışmada, Lactobacillus, Lactococcus ve Streptococcus cinslerine dahil olan bazı laktik asit bakterilerinin PHB üretimleri tespit edilmiştir. Laktobasiller MRS besiyerinde ve diğerleri Elliker Broth besiyerinde geliştirilmiştir. Hücre süspansiyonu santrifüj ile elde edilmiş ve hücre huvarları sodyum hipoklorit ile parçalanmıştır. Poli-ß-Hidroksibütirat sokslet sisteminde kloroform kullanılarak ekstrakte edilmiştir. PHB, sülfürik asitle krotonik asite dönüştürülerek krotonik asit miktarı spektrofotometrik olarak (235 nm) ölçülmüştür. Bakteri türlerinin PHB verimi (hücre kuru ağırlığına göre) Lactobacillus türlerinde % 0.52-25.55, Lactococcus ve Streptococcus türlerinde % 0.61-14.81 ve % 1 .20-13.69 olarak saptanmıştır. Streptococcus 'un bir, Lactococcus'un altı suşunun PHB üretmediği gözlenmiştir. Genellikle, Lactobacillus türlerinin diğer bakteri türlerine kıyasla daha fazla PHB ürettikleri gözlenmiştir. PHB üretimi ve kültürlerin hücre yoğunluğu arasında önemli bir ilişki gözlenmemiştir. Ayrıca, PHB üretimine göre mesofilik ve termofilik laktik asit bakterileri arasında önemli bir fark belirlenmemiştir.

Bazı mesofilik ve termofilik laktik asit bakterilerinin poli-beta-hidroksibütirat (PHB) üretim yeteneklerinin incelenmesi

Accumulated poly-ß-hydroxybutyrate (PHB) was determined in lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus and Streptococcus. Lactobacilli were grown in MRS broth and the others were grown in Elliker broth medium. Cell biomass was obtained by centrifugation. The cell walls were lysed with sodium hypochlorite. Poly-ß-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-ß-hydroxybutyrate % of cell dry weight of Lactobacillus species was 0.52-25.55%. The values for Lactococcus and Streptococcus species were 0.61 -14.81 and 1.20-13.69%, respectively. It was observed that one of the Streptococci and six of Lactococcus species did not produce poly-ß-hydroxybutyrate. Generally, Lactobacillus species produced more poly-ß-hydroxybutyrate than the other tested bacteria did and no significant correlation was observed between poly-ß-hydroxybutyrate production and cell density of the cultures. Additionally, no significant difference was observed between mesophilic and thermophilic lactic acid bacteria according to PHB yield.

PDF

___

1. Suziki, T., Yamane, T. and Shimuzu, S., Kinetics and effect of nitrogen source feeding on production of poly-p-hydroxybutyric acid by fed-batch culture. Appl. Microbiol. Lett., 24: 366-369(1986).
2. Anderson, AJ. and Dawes, A.E., Occurrence, Metabolism,Metabolic Role and Industrial Uses of Bacterial Polyhydroxyalkanoates. Microbiol. Rev., 54: 450-472 (1990).
3. Lee, S.Y., Yim, K.S., Chang, H.N. and Chang, Y.K., Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly (3-hydroxybutyric acid) by recombinant Escherichia coll J. Biotechnol. 32: 203-211 (1994).
4. Doi, Y., Microbial Polyesters. VCH Publishers, New York. (1990).
5. Miller, N.N. and Williams, D.F., On the biodegradation of poly-p-hydroxybutyrate (PHB) homopolymer and poly-p-hydroxybutyrate-hydroxyvalerate copolymers. Biomaterials. 8: 129-137 (1987).
6. Yan, Y., Wu, 0. and Zhang, R., Dynamic accumulation and degradation of poly (3-hydroxyalkanoate)s in living cells of Azotobacter vinelandii UWD characterized by 13C NMR. FEMS Microbiol. Lett. 193: 269-273 (2000).
7 .Holt, J. C, Krieg, N.R., Sneathe, P.H.A., Staley, J.T. and Williams,ST., Bergey's Manual of Determinative Bacteriology, 9th ed.Williams and Wilkins. Baltimore, MD. (1994).
8. Harrigan, W.F. and" McCance, M.E., Laboratory Methods in Microbiology. Academic Press. New York (1966).
9. Klaenhammer, T.R., McKay, L.L. and Baldwin, K.A., Improved lysis of group N Streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid. Appl. Environ. Microbiol. 35: 592-600(1978).
10.Kuniko, M., Nakamura, Y. and Doi, Y., New bacterial copolyesters produced in Alcaligenes eutrophus from organic acids. Polymer Commun. 29. 174-176(1988).
11.Bowker, R.R., Manual of Methods for General Bacteriology. American Society for Microbiology, Washington, DC. (1981).
12.Conver, W.J., Practical Nonparametric Statistics. Pp. 244-248. John Wiley and Sons, New York. (1971).
13.Aslim, B., Çalışkan, F., Beyatli, Y. and Gunduz, U., (1998). Poly-p-hydroxybutyrate production by lactic acid bacteria. F'EMS Microbiol. Lett. 159, 293-297.
14.Madder, L.A., Anderson, A.J., Asrar, J., Berger, P. and Garrett, P., Production and characterization of poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-4- hydroxybutyrate) synthesized by Ralstonia eutropha in fed-batch cultures. Polymer. 41: 3499-3505 (2000).
15.Sardesai, N. and Babu C.R., Poly-p-hydroxybutyrate metabolism is affected by changes in respiratory enzymatic activities due to cold stress in two psychrotrophic strains of Rhizobium. Curr. Microbiol., 42: 53-58(2001).
16.Labuzek, S. and Radecka, I., Biosynthesis of PHB tercopolymer by Bacillus cereus UW85. J. Appl. Microbiol., 90: 353-357 (2001).