Journal of Polymers and the Environment

, Volume 26, Issue 11, pp 4176–4187 | Cite as

Biosynthesis of Poly(3-hydroxybutyrate) from Cheese Whey by Bacillus megaterium NCIM 5472

  • Srija Das
  • Amrita Majumder
  • Vasu Shukla
  • Priya Suhazsini
  • P. RadhaEmail author
Original Paper


Microbial polyhydroxyalkonate such as homopolyester of poly(3-hydroxybutyrate) (PHB) was produced from cheese whey by Bacillus megaterium NCIM 5472. Due to their numerous potential industrial applications, the focus was given to competently enhance the amount of PHB produced. The amount of PHB produced from whole cheese whey, and ultrafiltered cheese whey was first compared, and after observing a rise in PHB production by using ultrafiltered cheese whey, cheese whey permeate was chosen for further analysis. The presence of PHB was then confirmed by GCMS. Since the main aim of the study was to increase the amount of PHB produced through batch fermentation, various process parameters like time, pH, C/N ratio, etc. were optimized. After optimization, it was found that B. megaterium NCIM 5472 was capable of accumulating 75.5% of PHB of its dry weight and a PHB yield of 8.29 g/L. The chemical structure of the polymer was further analyzed by using FTIR and NMR spectroscopy methods. Also, the physical and thermal properties were studied by using Differential scanning calorimetry and Thermogravimetric analysis. It was found that the polymer produced had excellent thermal stability, thus allowing the possibility to exploit its properties for industrial purposes such as adhesives, packaging materials, etc.


Biopolymer Poly(3-hydroxybutyrate) Bacillus megaterium Cheese whey permeate 



The authors wish to express their sincere thanks to the management of SRM Institute of Science and Technology and Department of Biotechnology for the research facilities and their constant support throughout this research study. We would also like to thank Dr. M. Venkatesh Prabhu, Assistant Professor (S.G) for his technical help during this research.


  1. 1.
    Povolo S, Casella S (2003) Bacterial production of PHA from lactose and cheese whey permeate. Macromol Symp 197:1–10CrossRefGoogle Scholar
  2. 2.
    Nawrath C, Poirier Y, Somerville C (1995) Plant polymers for biodegradable plastics: cellulose, starch and polydroxyalkanoates. Mol Breed 1:105–122CrossRefGoogle Scholar
  3. 3.
    Wang B, Shivappa RRS, Olson JW, Khan SA (2013) Production of polyhydroxybutyrate (PHB) by Alcaligenes latus using sugarbeet juice. Ind Crops Prod 43:802–811CrossRefGoogle Scholar
  4. 4.
    Ojumu TV, Yu J, Solomon BO (2004) Production of polyhydroxyalkanoates, a bacterial biodegradable polymer. Afr J Biotechnol 3(1):18–24CrossRefGoogle Scholar
  5. 5.
    Chanprateep S, Katakura Y, Visetkoop S, Shimizu H, Kulpreecha S, Shioya S (2008) Characterization of new isolated Ralstonia eutropha strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production. J Ind Microbiol Biotechnol 35(11):1205–1215CrossRefGoogle Scholar
  6. 6.
    Zhu C, Nomura CT, Perrotta J, Stipanovic AJ, Nakas JP (2010) Production and characterization of poly-3-hydroxybutyrate from biodiesel-glycerol by Burkholderia cepacia ATCC 17759. Biotechnol Prog 26:424–430PubMedGoogle Scholar
  7. 7.
    Koller M, Bona R, Chiellini E, Fernandes EG, Horvat P, Kutschera C, Hesse P, Braunegg G (2008) Polyhydroxyalkanoate production from whey by Pseudomonas hydrogenovora. Bioresour Technol 99(11):4854–4863CrossRefGoogle Scholar
  8. 8.
    Yellore V, Desai A (1998) Production of poly-3-hydroxybutyrate from lactose and whey by Methylobacterium sp. ZP24. Lett Appl Microbiol 26:391–394CrossRefGoogle Scholar
  9. 9.
    Baldasso C, Barros TC, Tessaro IC (2011) Concentration and purification of whey proteins by ultrafiltration. Desalination 278:381–386CrossRefGoogle Scholar
  10. 10.
    Barile D, Tao N, Lebrilla CB, Coisson JD, Arlorio M, German JB (2009) Permeate from cheese whey ultrafiltration is a source of milk oligosaccharides. Int Dairy J 19(9):524–530CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Chee JY, Tan Y, Samian MR, Sudesh K (2010) Expression of Aeromonas caviae polyhydroxyalkanoate synthase gene in Burkholderia sp. USM (JCM15050) enables the biosynthesis of SCL-MCL PHA from palm oil products. J Polym Environ 18:584–592CrossRefGoogle Scholar
  12. 12.
    Bunk B, Schulz A, Stammen S, Münch R, Warren MJ, Rohde M, Biedendieck R (2010) A short story about a big magic bug. Bioeng Bugs 1(2):85–91CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Shamala TR, Chandrashekar A, Vijayendra SVN, Kshama L (2003) Identification of polyhydroxyalkanoate (PHA)-producing Bacillus spp. using the polymerase chain reaction (PCR). J Appl Microbiol 94:369–374CrossRefGoogle Scholar
  14. 14.
    Obruca S, Marova I, Melusova S, Mravcova L (2011) Production of polyhydroxyalkanoates from cheese whey employing Bacillus megaterium CCM 2037. Ann Microbiol 61(4):947–953CrossRefGoogle Scholar
  15. 15.
    Kanjanachumpol P, Kulpreecha S, Tolieng V, Thongchul N (2013) Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019. Bioprocess Biosyst Eng 36:1463–1474CrossRefGoogle Scholar
  16. 16.
    Phasakanon J, Chookietwattana K, Dararat S (2014) Polyhydroxyalkanoate production from sequencing batch reactor system treating domestic water mixed with glycerol waste. APCBEE Procedia 8:161–166CrossRefGoogle Scholar
  17. 17.
    Kim YB, Lenz RW (2001) Polyesters from microorganisms. Adv Biochem Eng/Biotechnol 71:51–79CrossRefGoogle Scholar
  18. 18.
    Helm J, Wendlandt KD, Jechorek M, Stottmeister U (2008) Potassium deficiency results in accumulation of ultra-high molecular weight poly-beta-hydroxybutyrate in a methane-utilizing mixed culture. J Appl Microbiol 105(4):1054–1061CrossRefGoogle Scholar
  19. 19.
    Koller M, Hesse P, Salerno A, Reitner A, Braunegg G (2011) A viable antibiotic strategy against microbial contamination in biotechnological production of polyhydroxyalkanoates from surplus whey. Biomass Bioenergy 35(1):748–753CrossRefGoogle Scholar
  20. 20.
    Bugnicourt E, Cinelli P, Lazzeri A, Alvarez V (2014) Polyhydroxyalkanoate (PHA): review of synthesis, characteristics, processing and potential applications in packaging. Express Polym Lett 8(11):791–808CrossRefGoogle Scholar
  21. 21.
    Keshavarz T, Roy I (2001) Polyhydroxyalkanoates: bioplastics with a green agenda. Curr Opin Microbiol 13:321–326CrossRefGoogle Scholar
  22. 22.
    Alkotaini B, Sathiyamoorthi E, Kim BS (2015) Potential of Bacillus megaterium for production of polyhydroxyalkanoates using the red algae Gelidium amansii. Int J Biol Macromol 20:856–860Google Scholar
  23. 23.
    Irsath H, Santhosh S, Hemalatha V, Vikramathithan M, Dhanasekar R, Dhandapani R (2015) Production and optimization of polyhydroxybutyrate using Bacillus subtilis BP1 isolated from sewage sample. Int J Pure Appl Biosci 3(1):158–166Google Scholar
  24. 24.
    Ray S, Prajapati V, Patel K, Trivedi U (2016) Optimization and characterization of PHA from isolate Pannonibacter phragmitetus ERC8 using glycerol waste. Int J Biol Macromol 86:741–749CrossRefGoogle Scholar
  25. 25.
    Koller M, Hesse P, Bona R, Kutschera C, Atlicʹ A, Braunegg G (2007) Biosynthesis of high quality polyhydroxyalkanoate co-and terpolyesters for potential medical application by the archaeon Haloferax mediterranei. Macromol Symp 253(1):33–39CrossRefGoogle Scholar
  26. 26.
    Chaudhry W, 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(3):623–629CrossRefGoogle Scholar
  27. 27.
    Sathiyanarayanan G, Saibaba G, Kiran GS, Selvin J (2013) Optimization of polyhydroxybutyrate production by marine Bacillus megaterium MSBN04 under solid state culture. Int J Biol Macromol 59:170–177CrossRefGoogle Scholar
  28. 28.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedPubMedCentralGoogle Scholar
  29. 29.
    Mishra SK, Suh WI, Farooq W, Moon MS, Shrivastav A, Park MS, Yang JW (2014) Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method. Bioresour Technol 155:330–333CrossRefGoogle Scholar
  30. 30.
    Greenberg AE, Clesceri LS, Eaton AD (1995) Standard methods for the examination of water and wastewater, 19th ed. American Public Health Association, Washington, DCGoogle Scholar
  31. 31.
    Yang YH, Brigham CJ, Budde CF, Boccazzi P, Willis LB, Hassan MA, Sinskey AJ (2010) Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha. Appl Microbiol Biotechnol 87:2037–2045CrossRefGoogle Scholar
  32. 32.
    Bradstreet RB (1954) Kjeldahl method for organic nitrogen. Anal Chem 26(1):185–187CrossRefGoogle Scholar
  33. 33.
    Jacquel N, Lo CW, Wei YH, Wu HS, Wang SS (2008) Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochem Eng J 39(1):15–27CrossRefGoogle Scholar
  34. 34.
    Pandian SR, Deepak V, Kalishwaralal K, Rameshkumar N, Jeyraj M, Gurunathan S (2010) Optimization and fed-batch production of PHB utilizing dairy waste and sea water as nutrient sources by Bacillus megaterium SRKP-3. Bioresour Technol 101:705–711CrossRefGoogle Scholar
  35. 35.
    Rodriguez-Contreras A, Koller M, Miranda de Sousa Dias M, Calafell-Monfort M, Braunegg G, Marques-Calvo MS (2013) High production of poly (3-hydroxybutyrate) from a wild Bacillus megaterium Bolivian strain. J Appl Microbiol 114(5):1378–1138CrossRefGoogle Scholar
  36. 36.
    Law JH, Slepecky RA (1961) Assay of poly-β-hydroxybutyric acid. J Bacteriol 82:33–36PubMedPubMedCentralGoogle Scholar
  37. 37.
    Mohapatra S, Samantaray DP, Samantaray SM, Mishra BB, Das S, Majumdar S, Pradhan SK, Rath SN, Rath CC, Akthar J, Achary KG (2013) Structural and thermal characterization of PHAs produced by Lysinibacillus sp. through submerged fermentation process. Int J Biol Macromol 93(Pt A):1161–1167Google Scholar
  38. 38.
    Li A, Ha Y, Wang F, Li W, Li Q (2012) Determination of thermally induced trans-fatty acids in soybean oil by attenuated total reflectance fourier transform infrared spectroscopy and gas chromatography analysis. J Agric Food Chem 60(42):10709–10713CrossRefGoogle Scholar
  39. 39.
    Gumel AM, Annuar MSM, Heidelberg T (2012) Biosynthesis and characterization of polyhydroxyalkanoates copolymers produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent. PLoS ONE 7(9):1227–1231CrossRefGoogle Scholar
  40. 40.
    Shah KR (2012) FTIR analysis of polyhydroxyalkanoates by a locally isolated novel Bacillus sp. AS 3-2 from soil of Kadi region, North Gujarat, India. J Biochem Technol 3(4):380–383Google Scholar
  41. 41.
    Bhattacharyya A, Pramanik A, Maji SK, Haldar S, Mukhopadhyay UK, Mukherjee J (2012) Utilization of vinasse for production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei. AMB Express 2(1):34CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Mitra T, Sailakshmi G, Gnanamani A, Raja STK, Thiruselvi T, Gowri VM, Selvaraj NV, Ramesh G, Mandal AB (2011) Preparation and characterization of a thermostable and biodegradable biopolymers using natural cross-linker. Int J Biol Macromol 48(2):276–285CrossRefGoogle Scholar
  43. 43.
    Babruwad PR, Prabhu SU, Upadhyaya KP, Hungund BS (2015) Production and characterization of thermostable polyhydroxybutyrate from Bacillus cereus PW3A. J Biochem Technol 6(3):990–995Google Scholar
  44. 44.
    Dhangdhariya JH, Dubey S, Trivedi HB, Pancha I, Bhatt JK, Dave BP, Mishra S (2015) Polyhydroxyalkanoate from marine Bacillus megaterium using CSMCRI’s dry sea mix as a novel growth medium. Int J Biol Macromol 76:254–261CrossRefGoogle Scholar
  45. 45.
    Sharma P, Bajaj BK (2015) Production and characterization of poly-3-hydroxybutyrate from Bacillus cereus PS 10. Int J Biol Macromol 81:241–248CrossRefGoogle Scholar
  46. 46.
    Singh G, Kumari A, Mittal A, Goel V, Yadav A, Aggarwal NK (2013) Cost effective production of poly-β-hydroxybutyrate by Bacillus subtilis NG05 using sugar industry waste water. J Polym Environ 21:441–449CrossRefGoogle Scholar
  47. 47.
    Obruca S, Marova I, Snajdar O, Svoboda Z (2010) Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator from waste rapeseed oil using propanol as a precursor of 3-hydroxyvalerate. Biotechnol Lett 32:1925–1932CrossRefGoogle Scholar
  48. 48.
    Anjum A, Zuber M, Zia KM, Noreen A, Anjum MN, Tabasum S (2016) Microbial production of polyhydroxyalkanoates (PHAs) and its copolymers: a review of recent advancements. Int J Biol Macromol 89:161–174CrossRefGoogle Scholar
  49. 49.
    Masood F, Hassan F, Ahmed S, Chen P, Hameed A (2012) Biosynthesis and characterization of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) from Bacillus cereus S10. J Polym Environ 20:865–871CrossRefGoogle Scholar
  50. 50.
    Lathwal P, Nehra K, Singh M, Rana JS (2015) Optimization of culture parameters for maximum polyhydroxybutyrate production by selected bacterial strains isolated from rhizospheric soils. Pol J Microbiol 64(3):227–239CrossRefGoogle Scholar
  51. 51.
    Randria S, Renard E, Guerin P, Langlois V (2003) Fourier transform infrared spectroscopy for screening and quantifying production of PHAs by Pseudomonas grown on sodium octanoate. Biomacromolecules 4:1092–1097CrossRefGoogle Scholar
  52. 52.
    Lopez NI, Pettinari MJ, Stackebrandt E, Tribelli PM, Potter M, Steinbuchel A, Mendelez BS (2009) Pseudomonas extremaustralis sp. nov., a poly(3-hydroxybutyrate) producer isolated from an Antarctic environment. Curr Microbiol 59(5):14–519CrossRefGoogle Scholar
  53. 53.
    Biradar GG, Shivasharana CT, Kaliwal BB (2017) Characterization of polyhydroxybutyrate (PHB) produced by novel bacterium Lysinibacillus sphaericus bbkgbs6 isolated from soil. J Polym Environ 25:1–17CrossRefGoogle Scholar
  54. 54.
    Arcos-Hernández M, Laycock B, Donose BC, Pratt S, Halley P, Al-Luaibi S, Werker A, Lant PA (2013) Value-added bioplastics from services of wastewater treatment. Eur Polym J 49:904–913CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Srija Das
    • 1
  • Amrita Majumder
    • 1
  • Vasu Shukla
    • 1
  • Priya Suhazsini
    • 1
  • P. Radha
    • 1
    Email author
  1. 1.Bioprocess and Bioseperation Laboratory, Department of Biotechnology, School of BioengineeringSRM Institute of Science and TechnologyChennaiIndia

Personalised recommendations