Optimization of Polyhydroxyalkanoates (PHAs) Production from Bacillus cereus Strain PE3
Abstract
Polyhydroxyalkanoates (PHAs) are biodegradable, environmentally friendly and biocompatible thermoplastics. Various bacterial species accumulate PHAs granules as a carbon and energy reserve inside their cells. The present study aimed to optimize culture conditions for the production of polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from Bacillus cereus strain PE3 previously isolated from soil sample in Thailand. Effect of medium, incubation time, carbon sources, nitrogen sources and initial pH on PHAs production were investigated. Strain PE3 showed maximum PHB concentration of 3.1 g/L and PHAs content of 79.5% (3.9 g/L of cell dry weight) when cultured in Nitrogen deficient medium with 3% (w/v) glucose as a carbon source and 0.25% (w/v) peptone and 0.25% (w/v) yeast extract as nitrogen sources at pH 7, 37°C and 48 hours cultivation. Effect of carbon source and concentration of PHBV production were obtained with 5% (v/v) molasses and 0.5% (w/v) propionic acid. PHB and PHBV were analyzed using fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). The accumulate PHB granule was detected by transmission electron microscopy (TEM) showed 2-7 granules in cell. Keywords : Bacillus cereus, polyhydroxybutyrate (PHB), polyhydroxybutyrate-co-hydroxyvalerates (PHBV), polyhydroxyalkanoates (PHAsReferences
Aarthi, N. & Ramana, K.V. (2011). Identification and characterization of polyhydroxybutyrate producing Bacillus cereus and Bacillus mycoides strains. International Journal of Environmental Sciences, 1, 744-756.
Aldor, S.I. & Keaslingy, J.D. (2003). Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates. Current Opinion in Biotechnology, 14, 475-483.
Anderson, A.J. & Dawes, E.A. (1990). Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiological Reviews, 54, 450-472.
Babruwad, P.R., Prabhu, S.U., Upadhyaya, K.P. & Hungund, B.S. (2015). Production and characterization of thermostable polyhydroxybutyrate from Bacillus cereus PW3A. Journal of Biochemical Technology, 6, 990-995.
Collins, S. (1987). Choice of substrate in polyhydroxybutyrate synthesis. Special Publications of the Society for General Microbiology, 21, 161-168.
Gouda, M.K., Swellam, A.E. & Omar, S.H. (2001). Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources. Microbiological Research, 156, 201-207.
Gowda, V. & Shivakumar, S. (2013). Poly(3)-hydroxybutyrate (PHB) production in Bacillus thuringiensis IAM 12077 under varied nutrient limiting conditions and molecular detection of class IV PHA synthase gene by PCR. International Journal of Pharma and Bio Sciences, 4, 794-802.
Grothe, E. & Chisti, Y. (2000). Poly(-hydroxybutyric acid) thermoplastic production by Alcaligenes latus: behavior of fed-batch cultures. Bioprocess Engineering, 22, 441-449.
Hahn, S.K., Chang, Y.K., Kim, B.S. & Chang, H.N. (1994). Optimization of microbial poly(3-hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform. Biotechnology and Bioengineering, 44, 256-261.
Hourniel, K.L., Slater, S., Broyles, D., Casagrande, L., Colburn, S., Gonzalez, K., Mitsky, T.A., Reiser, S.E., Shah, D., Taylor, N.B., Tran, M., Valentin, H.E. & Gruys, K.J. (1999). Poly(-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus. Planta, 209, 547-550.
Joraleerut, P., Somyoonsap, P., Samosorn, S., Chansiri, K. & Sriyapai, T. (2014). Isolation and production by polyhydroxybutyrate (PHB) producing bacterial from soil. In the 26th Annual Meeting of the Thai Society for Biotechnology and International Conference. (pp. 229-236). Mae Fah Luang University.
Keshavarz, T. & Roy, I. (2010). Polyhydroxyalkanoates: bioplastics with a green agenda. Current Opinion in Microbiology, 13, 321-326.
Khanna, S. & Srivastava, A.K. (2005). Statistical media optimization studies for growth and PHB production by Ralstonia eutropha. Process Biochemistry, 40, 2173-2182.
Kim, B.S., Lee, S.C., Lee, S.Y., Chang, H.N., Chang, Y.K. & Woo, S.L. (1994). Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control. Biotechnology and Bioengineering, 43, 892-898.
Kulpreecha, S., Boonruangthavorn, A., Meksiriporn, B. & Thongchul, N. (2009). Inexpensive fed-batch cultivation for high poly(3-hydroxybutyrate) production by a new isolate of Bacillus megaterium. Journal of Bioscience and Bioengineering, 107, 240-245.
Lamoigne, M. (1926). Products of dehydration and polymerization of -hydroxybutyric acid. Bulletin de la Société de chimie biologique, 8, 770–782.
Lee, S.Y. & Choi, J. (1999). Production and degradation of polyhydroxyalkanoates in waste environment. Waste Management, 19, 133-139.
Linko, S., Vahrte, H. & Seepällä, J. (1993). Production of poly--hydroxybutyrates by Alcaligenes eutrophus on different carbon sources. Applied Microbiology and Biotechnology, 39, 11-15.
Matsusaki, H., Manji, S., Taguchi, K., Kato, Kato, M., Fukui, T. & Doi, Y. (1998). Cloning and molecular analysis of the poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3. Journal of Bacteriology, 180, 6459-6467.
Mittendorf, V., Robertson, E.J., Leech, R.M., KrÜger, N., SteinbÜchel, A. & Poirier, Y. (1998). Synthesis of medium-chain-length polyhydroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid -oxidation. Proceedings of the National Academy of Sciences of the United States of America, 95, 13397-13402.
Nurbas, M. & Kutsal, T. (2004). Production of PHB and P(HB-co-HV) biopolymers by using Alcaligenes eutrophus. Iranian Polymer Journal, 13, 45-51.
Ojumu, T.V., Yu, J. & Solomon, B.O. (2004). Production of polyhydroxyalkanoates, a bacterial biodegradable polymer. African Journal of Biotechnology, 43, 18-24.
Page, W.J. (1992). Production of polyhydroxyalkanoates by Azotobacter vinelandii UWD in beet molasses culture. FEMS Microbiology Reviews, 103, 149-158.
Ramsay, B.A., Lomaliza, K., Chavarie, C., Dubé, B., Bataille, P. & Ramsay, J.A. (1990). Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids. Applied and Environmental Microbiology, 56, 2093-2098.
Rohini, D., Phadnis, S. & Rawal, S.K. (2006). Synthesis and characterization of poly--hydroxybutyrate from Bacillus thuringiensis R1. Indian Journal of Biotechnology, 5, 276-283.
Singh, P. & Parmar, N. (2011). Isolation and characterization of two novel polyhydroxybutyrate (PHB)-producing bacteria. African Journal of Biotechnology, 10, 4907-4919.
SteinbÜchel, A. & Schlegel, H.C. (1991). Physiology and molecular genetics of poly(-hydroxy-alkanoic acid) synthesis in Alcaligenes eutrophus. Molecular Microbiology, 5, 535-542.
Suriyamongkol, P., Weselake, R., Narine, S., Moloney, M. & Shah, S. (2007). Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants. Biotechnology Advances, 25, 148- 175.
Tabanden, F. & Vasheghani-Farahani, E. (2002). Biosynthesis of poly--hydroxybutyrate as a biodegradable polymer. Iranian Polymer Journal, 12, 37-42.
Valappil, S.P., Rai, R., Bucke, C. & Roy, I. (2008). Polyhydroxyalkanoate biosynthesis in Bacillus cerus SPV under
varied limiting conditions and an insight into the biosynthetic genes involved. Journal of Applied Microbiology, 104, 1624-1635.
Wang, J.G. & Bakken, L.R. (1998). Screening of soil bacteria for poly--hydroxybutyric acid production and its role in the survival of starvation. Microbial Ecology, 35, 94-101.
Aldor, S.I. & Keaslingy, J.D. (2003). Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates. Current Opinion in Biotechnology, 14, 475-483.
Anderson, A.J. & Dawes, E.A. (1990). Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiological Reviews, 54, 450-472.
Babruwad, P.R., Prabhu, S.U., Upadhyaya, K.P. & Hungund, B.S. (2015). Production and characterization of thermostable polyhydroxybutyrate from Bacillus cereus PW3A. Journal of Biochemical Technology, 6, 990-995.
Collins, S. (1987). Choice of substrate in polyhydroxybutyrate synthesis. Special Publications of the Society for General Microbiology, 21, 161-168.
Gouda, M.K., Swellam, A.E. & Omar, S.H. (2001). Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources. Microbiological Research, 156, 201-207.
Gowda, V. & Shivakumar, S. (2013). Poly(3)-hydroxybutyrate (PHB) production in Bacillus thuringiensis IAM 12077 under varied nutrient limiting conditions and molecular detection of class IV PHA synthase gene by PCR. International Journal of Pharma and Bio Sciences, 4, 794-802.
Grothe, E. & Chisti, Y. (2000). Poly(-hydroxybutyric acid) thermoplastic production by Alcaligenes latus: behavior of fed-batch cultures. Bioprocess Engineering, 22, 441-449.
Hahn, S.K., Chang, Y.K., Kim, B.S. & Chang, H.N. (1994). Optimization of microbial poly(3-hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform. Biotechnology and Bioengineering, 44, 256-261.
Hourniel, K.L., Slater, S., Broyles, D., Casagrande, L., Colburn, S., Gonzalez, K., Mitsky, T.A., Reiser, S.E., Shah, D., Taylor, N.B., Tran, M., Valentin, H.E. & Gruys, K.J. (1999). Poly(-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus. Planta, 209, 547-550.
Joraleerut, P., Somyoonsap, P., Samosorn, S., Chansiri, K. & Sriyapai, T. (2014). Isolation and production by polyhydroxybutyrate (PHB) producing bacterial from soil. In the 26th Annual Meeting of the Thai Society for Biotechnology and International Conference. (pp. 229-236). Mae Fah Luang University.
Keshavarz, T. & Roy, I. (2010). Polyhydroxyalkanoates: bioplastics with a green agenda. Current Opinion in Microbiology, 13, 321-326.
Khanna, S. & Srivastava, A.K. (2005). Statistical media optimization studies for growth and PHB production by Ralstonia eutropha. Process Biochemistry, 40, 2173-2182.
Kim, B.S., Lee, S.C., Lee, S.Y., Chang, H.N., Chang, Y.K. & Woo, S.L. (1994). Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control. Biotechnology and Bioengineering, 43, 892-898.
Kulpreecha, S., Boonruangthavorn, A., Meksiriporn, B. & Thongchul, N. (2009). Inexpensive fed-batch cultivation for high poly(3-hydroxybutyrate) production by a new isolate of Bacillus megaterium. Journal of Bioscience and Bioengineering, 107, 240-245.
Lamoigne, M. (1926). Products of dehydration and polymerization of -hydroxybutyric acid. Bulletin de la Société de chimie biologique, 8, 770–782.
Lee, S.Y. & Choi, J. (1999). Production and degradation of polyhydroxyalkanoates in waste environment. Waste Management, 19, 133-139.
Linko, S., Vahrte, H. & Seepällä, J. (1993). Production of poly--hydroxybutyrates by Alcaligenes eutrophus on different carbon sources. Applied Microbiology and Biotechnology, 39, 11-15.
Matsusaki, H., Manji, S., Taguchi, K., Kato, Kato, M., Fukui, T. & Doi, Y. (1998). Cloning and molecular analysis of the poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3. Journal of Bacteriology, 180, 6459-6467.
Mittendorf, V., Robertson, E.J., Leech, R.M., KrÜger, N., SteinbÜchel, A. & Poirier, Y. (1998). Synthesis of medium-chain-length polyhydroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid -oxidation. Proceedings of the National Academy of Sciences of the United States of America, 95, 13397-13402.
Nurbas, M. & Kutsal, T. (2004). Production of PHB and P(HB-co-HV) biopolymers by using Alcaligenes eutrophus. Iranian Polymer Journal, 13, 45-51.
Ojumu, T.V., Yu, J. & Solomon, B.O. (2004). Production of polyhydroxyalkanoates, a bacterial biodegradable polymer. African Journal of Biotechnology, 43, 18-24.
Page, W.J. (1992). Production of polyhydroxyalkanoates by Azotobacter vinelandii UWD in beet molasses culture. FEMS Microbiology Reviews, 103, 149-158.
Ramsay, B.A., Lomaliza, K., Chavarie, C., Dubé, B., Bataille, P. & Ramsay, J.A. (1990). Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids. Applied and Environmental Microbiology, 56, 2093-2098.
Rohini, D., Phadnis, S. & Rawal, S.K. (2006). Synthesis and characterization of poly--hydroxybutyrate from Bacillus thuringiensis R1. Indian Journal of Biotechnology, 5, 276-283.
Singh, P. & Parmar, N. (2011). Isolation and characterization of two novel polyhydroxybutyrate (PHB)-producing bacteria. African Journal of Biotechnology, 10, 4907-4919.
SteinbÜchel, A. & Schlegel, H.C. (1991). Physiology and molecular genetics of poly(-hydroxy-alkanoic acid) synthesis in Alcaligenes eutrophus. Molecular Microbiology, 5, 535-542.
Suriyamongkol, P., Weselake, R., Narine, S., Moloney, M. & Shah, S. (2007). Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants. Biotechnology Advances, 25, 148- 175.
Tabanden, F. & Vasheghani-Farahani, E. (2002). Biosynthesis of poly--hydroxybutyrate as a biodegradable polymer. Iranian Polymer Journal, 12, 37-42.
Valappil, S.P., Rai, R., Bucke, C. & Roy, I. (2008). Polyhydroxyalkanoate biosynthesis in Bacillus cerus SPV under
varied limiting conditions and an insight into the biosynthetic genes involved. Journal of Applied Microbiology, 104, 1624-1635.
Wang, J.G. & Bakken, L.R. (1998). Screening of soil bacteria for poly--hydroxybutyric acid production and its role in the survival of starvation. Microbial Ecology, 35, 94-101.
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2016-11-18
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