Biosynthesis and Characterization of Poly3-hydroxyalkanote (PHA) from Newly Isolated Bacterium Bacillus sp. AZR-1

Research Paper

Abstract

Bacterial poly3-hydroxyalkanotes are the polyesters that are accumulated in conditions of un-balanced nutrients. Soil samples were collected from Hilly areas of Muzaffarabad, and oil processing area of Lahore, Pakistan to isolate PHA producing bacteria. The bacteria were screened on Sudan black B and Nile blue A staining methodologies. An efficient PHA producing strain AZR-1 was isolated and characterized morphologically and biochemically. The strain was identified as Bacillus sp. AZR-1 on the genetic basis of 16S rRNA gene sequence. Bacillus sp. AZR-1 produced 40% PHA contents when grown on N-limited mineral salt medium supplemented with glucose in shake flasks. Starch and sodium gluconate were found to be other putative carbon sources for PHA production as the bacterium produced 22 and 17% PHA on these sources. FTIR confirmed the presence of scl-PHA monomers in the polymer extracted from the bacterium. The PHA synthase gene (phaC) was partially amplified and sequenced which showed maximum similarity with the PHA synthase of Bacillus weihenstephanensis KBAB4.

Keywords

Biopolymers scl-PHA Bacillus phaC gene FTIR 

Notes

Acknowledgements

Authors are thankful to the University of the Punjab, Pakistan and Higher Education Commission of Pakistan for providing the funds to conduct this research work. Funding was provided by University of the Punjab and Higher Education Commission, Pakistan.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

References

  1. Ali I, Jamil N (2014) Enhanced biosynthesis of poly (3-hydroxybutyrate) from potato starch by Bacillus cereus strain 64-INS in a laboratory-scale fermenter. Prep Biochem Biotechnol 44:822–833CrossRefGoogle Scholar
  2. Ali I, Jamil N (2016) Polyhydroxyalkanoates: current applications in the medical field. Front Bio 11:19–27CrossRefGoogle Scholar
  3. Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472Google Scholar
  4. Ataei SA, Vasheghani-Farahani E, Shojaosadati SA, Tehrani HA (2008) Isolation of PHA–producing bacteria from date syrup waste. Macromol Symp 269:11–16CrossRefGoogle Scholar
  5. Ausubel FM (2002) Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology, vol 2. Wiley, New York, USAGoogle Scholar
  6. Cappuccino JG, Sherman N (2008) Microbiology: a laboratory manual. Pearson/Benjamin Cummings, San Francisco, USAGoogle Scholar
  7. Chaudhry WN, Jamil N, Ali I, Ayaz MH, Hasnain S (2011) Screening for polyhydroxyalkanoate (PHA)-producing bacterial strains and comparison of PHA production from various inexpensive carbon sources. Ann Microbiol 61:623–629CrossRefGoogle Scholar
  8. Ciesielski S, Gorniak D, Mozejko J, Swiątecki A, Grzesiak J, Zdanowski M (2014) The diversity of bacteria isolated from Antarctic freshwater reservoirs possessing the ability to produce polyhydroxyalkanoates. Curr Microbiol 69:594–603CrossRefGoogle Scholar
  9. Dalal J, Sarma PM, Lavania M, Mandal AK, Lal B (2010) Evaluation of bacterial strains isolated from oil-contaminated soil for production of polyhydroxyalkanoic acids (PHA). Pedobiologia 54:25–30CrossRefGoogle Scholar
  10. Full T, Jung D, Madigan M (2006) Production of poly-β-hydroxyalkanoates from soy molasses oligosaccharides by new, rapidly growing Bacillus species. Lett Appl Microbiol 43:377–384CrossRefGoogle Scholar
  11. Hahn SK, Chang YK, Kim BS, Chang HN (1994) Optimization of microbial poly (3-hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform. Biotechnol Bioeng 44:256–261CrossRefGoogle Scholar
  12. Halami PM (2008) Production of polyhydroxyalkanoate from starch by the native isolate Bacillus cereus CFR06. World J Microbiol Biotechnol 24:805–812CrossRefGoogle Scholar
  13. Kansiz M, Billman-Jacobe H, McNaughton D (2000) Quantitative determination of the biodegradable polymer poly (β-hydroxybutyrate) in a recombinant Escherichia coli strain by use of mid-infrared spectroscopy and multivariative statistics. Appl Environ Microbiol 66:3415–3420CrossRefGoogle Scholar
  14. Lee W-H, Loo C-Y, Nomura CT, Sudesh K (2008) Biosynthesis of polyhydroxyalkanoate copolymers from mixtures of plant oils and 3-hydroxyvalerate precursors. Bioresour Technol 99:6844–6851CrossRefGoogle Scholar
  15. Madison LL, Huisman GW (1999) Metabolic engineering of poly (3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Bio Rev 63:21–53Google Scholar
  16. Mizuno K, Ohta A, Hyakutake M, Ichinomiya Y, Tsuge T (2010) Isolation of polyhydroxyalkanoate-producing bacteria from a polluted soil and characterization of the isolated strain Bacillus cereus YB-4. Polym Degrad Stab 95:1335–1339CrossRefGoogle Scholar
  17. Mohapatra S, Mohanta P, Sarkar B, Daware A, Kumar C, Samantaray D (2015) Production of polyhydroxyalkanoates (PHAs) by Bacillus strain isolated from waste water and its biochemical characterization. Proc Nat Acad Sci India B Biol Sci 1–8. doi: 10.1007/s40011-015-0626-6
  18. Naheed N, Jamil N, Hasnain S, Abbas G (2012) Biosynthesis of polyhydroxybutyrate in Enterobacter sp. SEL2 and Enterobacteriaceae bacterium sp. PFW1 using sugar cane molasses as media. Afr J Biotechnol 11:3321–3332CrossRefGoogle Scholar
  19. Obruca S, Marova I, Melusova S, Mravcova L (2011) Production of polyhydroxyalkanoates from cheese whey employing Bacillus megaterium CCM 2037. Ann Microbiol 61:947–953CrossRefGoogle Scholar
  20. Park SJ, Park JP, Lee SY (2002) Production of poly (3-hydroxybutyrate) from whey by fed-batch culture of recombinant Escherichia coli in a pilot-scale fermenter. Biotechnol Lett 24:185–189CrossRefGoogle Scholar
  21. Phukon P, Saikia JP, Konwar BK (2011) Enhancing the stability of colloidal silver nanoparticles using polyhydroxyalkanoates (PHA) from Bacillus circulans (MTCC 8167) isolated from crude oil contaminated soil. Colloids Surf B Biointerfaces 86:314–318CrossRefGoogle Scholar
  22. Rehm B (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376:15–33CrossRefGoogle Scholar
  23. Saadoun I (2002) Isolation and characterization of bacteria from crude petroleum oil contaminated soil and their potential to degrade diesel fuel. J B Microbiol 42:420–428CrossRefGoogle Scholar
  24. Shamala T, Chandrashekar A, Vijayendra S, Kshama L (2003) Identification of polyhydroxyalkanoate (PHA) producing Bacillus spp. using the polymerase chain reaction (PCR). J Appl Microbiol 94:369–374CrossRefGoogle Scholar
  25. Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Steinbüchel A (1999) A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171:73–80CrossRefGoogle Scholar
  26. Steinbuchel A, Lütke-Eversloh T (2003) Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochem Eng J 16:81–96CrossRefGoogle Scholar
  27. Tajima K, Igari T, Nishimura D, Nakamura M, Satoh Y, Munekata M (2003) Isolation and characterization of Bacillus sp. INT005 accumulating polyhydroxyalkanoate (PHA) from gas field soil. J Biosci Bioeng 95:77–81CrossRefGoogle Scholar
  28. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Bio Evol 28:2731–2739CrossRefGoogle Scholar
  29. Verlinden RA, Hill DJ, Kenward M, Williams CD, Radecka I (2007) Bacterial synthesis of biodegradable polyhydroxyalkanoates. J Appl Microbiol 102:1437–1449CrossRefGoogle Scholar
  30. Xu K, Xu P (2014) Efficient production of L-lactic acid using co-feeding strategy based on cane molasses/glucose carbon sources. Bioresour Technol 153:23–29CrossRefGoogle Scholar
  31. Younas T, Ali I, Jamil N (2015) Polyhydroxyalkanoates production using canola oil by bacteria isolated from paper pulp industry. Kuwait J Sci 42:236–249Google Scholar
  32. Yu J (2001) Production of PHA from starchy wastewater via organic acids. J Biotechnol 86:105–112CrossRefGoogle Scholar

Copyright information

© Shiraz University 2016

Authors and Affiliations

  1. 1.Microbiology and Molecular GeneticsUniversity of the PunjabLahorePakistan

Personalised recommendations