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Biosynthesis of PHB from a new isolated Bacillus megaterium strain: Outlook on future developments with endospore forming bacteria

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Abstract

Diverse Bacillus strains are known as producers of polyhydroxyalkanoates (PHAs) under nutrient-limiting conditions. However, these limiting conditions have the same nutritional characteristics that stimulate spore generation in Gram-positive microorganisms. In the present work, a new isolated Bacillus megaterium strain was characterized based on 16S rRNA gene sequences (1,411 bp) and studied in terms of its ability for producing polyhydroxybutyrate (PHB) by implementing different fermentation configurations on formulated media. The isolated strain was able to produce PHB up to 59 and 60% of its dry cell weight during bioreactor experiments employing glucose and glycerol as carbon source, respectively. The produced biopolymer was characterized and identified by using carbon-13 nuclear magnetic resonance (13C-NMR) and Fourier transform infrared (FTIR) techniques. In spite of the sporulation phenomenon existing in Bacillus strains, obtained results demonstrate that the new isolated strain has the potential of accumulating high levels of intracellular PHB. Supported by these experimental results and by those reported by other authors, the last section of this paper gives an outlook of future research topics on PHB and polyhydroxyalkanoate (PHA) copolymers production by Gram-positive bacteria. The importance of combining bioprocessing/biorefinering concepts with bioreactor optimization approaches is stressed and analyzed based on current PHAs research trends.

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References

  1. Koslowski, H. J. (2007) Global market trends for synthetic fiber polymers. Chem. Fibers Int. 57: 287–291.

    Google Scholar 

  2. Lee, S. Y. (1996) Plastic bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria. Trends Biotechnol. 14: 431–438.

    Article  CAS  Google Scholar 

  3. Steinbüchel, A. and B. Füchtenbusch (1998) Bacterial and other biological systems for polyester production. Trends Biotechnol. 16: 419–427.

    Article  Google Scholar 

  4. Chen, G. Q. and Q. Wu (2005) The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials. 26: 6565–6578.

    Article  CAS  Google Scholar 

  5. Lee, S. Y., J. I. Choi, and H. H. Wong (1999) Recent advances in polyhydroxyalkanoate production by bacterial fermentation: Mini-review. Int. J. Biol. Macromol. 25: 31–36.

    Article  CAS  Google Scholar 

  6. Valappil, S. P., S. K. Misra, A. R. Boccaccini, T. Keshavarz, C. Bucke, and I. Roy (2007) Large-scale production and efficient recovery of PHB with desirable material properties, from the newly characterised Bacillus cereus SPV. J. Biotechnol. 132: 251–258.

    Article  CAS  Google Scholar 

  7. Wu, Q., H. Huang, G. Hu, J. Chen, K. P. Ho, and G. -Q. Chen (2001) Production of poly-3-hydroxybutyrate by Bacillus sp.JMa5 cultivated in molasses media. Antonie Van Leeuwenhoek. 80: 111–118.

    Article  CAS  Google Scholar 

  8. Solaiman, D. K. Y., R. D. Ashby, T. A. Foglia, and W. N. Marmer (2006) Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates). Appl. Microbiol. Biotechnol. 71: 783–789.

    Article  CAS  Google Scholar 

  9. Posada, J. A., J. M. Naranjo, J. A. López, J. C. Higuita, and C. A. Cardona (2011) Design and analysis of poly-3-hydroxybutyrate production processes from crude glycerol. Process Biochem. 46: 310–317.

    Article  CAS  Google Scholar 

  10. Pochon, J. and L. Tardieu (1962) Techniques d’analyse en microbiologie du sol. De la Tourelle, Paris.

  11. Buddingh, G. J. (1975) Bergey’s Manual of Determinative Bacteriology: 8th edition, edited by R. E. Buchanan and N. E. Gibbons. xxvii + 1,246 pages, illustrated. The Williams and Wilkins Company, Baltimore, Maryland.

    Google Scholar 

  12. Kitamura, S. and Y. Doi (1994) Staining method of poly(3-hydroxyalkanoic acids) producing bacteria by Nile blue. Biotechnol. Tech. 8: 345–350.

    Article  CAS  Google Scholar 

  13. Takagi, Y. and T. Yamane (1997) Replica technique for screening poly(3-hydroxyalkanoic acid)-producing bacteria by Nile blue staining. J. Ferment. Bioeng. 83: 121–123.

    Article  CAS  Google Scholar 

  14. Gorenflo, V., A. Steinbüchel, S. Marose, M. Rieseberg, and T. Scheper (1999) Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining. Appl. Microbiol. Biotechnol. 51: 765–772.

    Article  CAS  Google Scholar 

  15. DeLong, E. F. (1992) Archaea in coastal marine environments. P. Natl. Acad. Sci. USA. 89: 5685–5689.

    Article  CAS  Google Scholar 

  16. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman (1990) Basic local alignment search tool. J. Mol. Biol. 215: 403–410.

    CAS  Google Scholar 

  17. Chun, J., J. H. Lee, Y. Jung, M. Kim, S. Kim, B. K. Kim, and Y. W. Lim (2007) EzTaxon: A web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int. J. Syst. Evol. Microbiol. 57: 2259–2261.

    Article  CAS  Google Scholar 

  18. Braunegg, G., B. Sonnleitner, and R. M. Lafferty (1978) A rapid gas chromatographic method for the determination of poly β hydroxybutyric acid in microbial biomass. Eur. J. Appl. Microbiol. 6: 29–37.

    Article  CAS  Google Scholar 

  19. Manna, A., R. Banerjee, and A. K. Paul (1999) Accumulation of poly (3-hydroxybutyric acid) by some soil Streptomyces. Curr. Microbiol. 39: 153–158.

    Article  CAS  Google Scholar 

  20. Kulpreecha, S., A. Boonruangthavorn, B. Meksiriporn, and N. Thongchul (2009) Inexpensive fed-batch cultivation for high poly(3-hydroxybutyrate) production by a new isolate of Bacillus megaterium. J. Biosci. Bioeng. 107: 240–245.

    Article  CAS  Google Scholar 

  21. Sabra, W. and D. M. Aboud-Zeid (2008) Improving feeding strategies for maximizing polyhydroxybutyrate yield by Bacillus megaterium. Res. J. Microbiol. 3: 308–318.

    Article  CAS  Google Scholar 

  22. Pandian, S. R., V. Deepak, K. Kalishwaralal, N. Rameshkumar, M. Jeyaraj, and S. Gurunathan (2010) Optimization of fed-batch production of PHB utilizing dairy waste and sea water as nutrient sources by Bacillus megaterium SRKP-3. Bioresour. Technol. 101: 705–711.

    Article  Google Scholar 

  23. Yilmaz, M. and Y. Beyatli (2005) Poly-β-hydroxybutyrate (PHB) production by a Bacillus cereus M5 strain in sugarbeet molasses. Zuckerindustrie. 130: 109–112.

    CAS  Google Scholar 

  24. Reddy, S. V., M. Thirumala, and S. K. Mahmood (2009) production of PHB and P (3HB-co-3HV) biopolymers by Bacillus megaterium strain OU303A isolated from municipal sewage sludge. World J. Microbiol. Biotechnol. 25: 391–397.

    Article  Google Scholar 

  25. Reddy, S. V., M. Thirumala, and S. K. Mahmood (2009) A novel Bacillus sp. accumulating poly (3-hydroxybutyrate-co-3-hydroxyvalerate) from a single carbon substrate. J. Ind. Microbiol. Biotechnol. 36: 837–843.

    Article  CAS  Google Scholar 

  26. Thirumala, M., S. V. Reddy, and S. K. Mahmood (2010) Production and characterization of PHB from two novel strains of Bacillus spp. isolated from soil and activated sludge. J. Ind. Microbiol. Biotechnol. 37: 271–278.

    Article  CAS  Google Scholar 

  27. Tajima, K., T. Igari, D. Nishimura, M. Nakamura, Y. Satoh, and M. Munekata (2003) Isolation and characterization of Bacillus sp.INT005 accumulating polyhydroxyalkanoate (PHA) from gas field soil. J. Biosci. Bioeng. 95: 77–81.

    CAS  Google Scholar 

  28. Braunegg, G., R. Bona, and M. Koller (2004) Sustainable poly mer production. Polym. Plast. Technol. Eng. 43: 1779–1793.

    Article  CAS  Google Scholar 

  29. Demirbas, A. (2009) Progress and recent trends in biodiesel fuels. Energy Convers. Manage. 50: 14–34.

    Article  CAS  Google Scholar 

  30. Thomsen, M. H. (2005) Complex media from processing of agricultural crops for microbial fermentation. Appl. Microbiol. Biotechnol. 68: 598–606.

    Article  CAS  Google Scholar 

  31. Bayari, S. and F. Severcan (2005) FTIR study of biodegradable biopolymers: P(3HB), P(3HB-co-4HB) and P(3HB-co-3HV). J. Mol. Struct. 744–747: 529–534.

    Article  Google Scholar 

  32. Hong, K., S. Sun, W. Tian, G. Q. Chen, and W. Huang (1999) A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy. Appl. Microbiol. Biotechnol. 51: 523–526.

    Article  CAS  Google Scholar 

  33. Doi, Y., M. Kunioka, Y. Nakamura, and K. Soga (1986) 1H and 13C NMR analysis of poly(-hydroxybutyrate) isolated from Bacillus megaterium. Macromolecules. 19: 1274–1276.

    Article  CAS  Google Scholar 

  34. Koutinas, A. A., Y. Xu, R. Wang, and C. Webb (2007) Polyhydroxybutyrate production from a novel feedstock derived from a wheat-based biorefinery. Enzyme Microb. Technol. 40: 1035–1044.

    Article  CAS  Google Scholar 

  35. Xu, Y., R. H. Wang, A. A. Koutinas, and C. Webb (2010) Microbial biodegradable plastic production from a wheat-based biorefining strategy. Process Biochem. 45: 153–163.

    Article  CAS  Google Scholar 

  36. Koutinas, A., N. Arifeen, R. Wang, and C. Webb (2007) Cerealbased biorefinery development: integrated enzyme production for cereal flour hydrolysis. Biotechnol. Bioeng. 97: 61–72.

    Article  CAS  Google Scholar 

  37. Koutinas, A. A., R. Wang, and C. Webb (2003) Modelling Studies of a Process to Produce a Generic Bioconversion Feedstock from Wheat. Food Bioprod. Process. 81: 239–249.

    Article  CAS  Google Scholar 

  38. Koutinas, A. A., R. H. Wang, and C. Webb (2005) Development of a process for the production of nutrient supplements for fermentations based on fungal autolysis. Enzyme Microb. Technol. 36: 629–638.

    Article  CAS  Google Scholar 

  39. Wang, R., S. M. Shaarani, L. C. Godoy, M. Melikoglu, C. S. Vergara, A. Koutinas, and C. Webb (2010) Bioconversion of rapeseed meal for the production of a generic microbial feedstock. Enzyme Microb. Technol. 47: 77–83.

    Article  CAS  Google Scholar 

  40. López, J. A., V. Bucalá, and M. A. Villar (2010) Application of dynamic optimization techniques for poly(β-hydroxybutyrate) production in a fed-batch bioreactor. Ind. Eng. Chem. Res. 49: 1762–1769.

    Article  Google Scholar 

  41. Valappil, S. P., A. R. Boccaccini, C. Bucke, and I. Roy (2007) Polyhydroxyalkanoates in Gram-positive bacteria: insights from genera Bacillus and Streptomyces. Antonie Van Leeuwenhoek. 91: 1–17.

    Article  CAS  Google Scholar 

  42. Bauer, H. and A. J. Owen (1988) Some structural and mechanical properties of bacterially produced poly-B-hydroxybutyrate-copoly-B-hydroxyvalerate. Colloid Pol. Sci. 266: 241–247.

    Article  CAS  Google Scholar 

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Correspondence to Jimmy A. López.

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López, J.A., Naranjo, J.M., Higuita, J.C. et al. Biosynthesis of PHB from a new isolated Bacillus megaterium strain: Outlook on future developments with endospore forming bacteria. Biotechnol Bioproc E 17, 250–258 (2012). https://doi.org/10.1007/s12257-011-0448-1

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