Applied Microbiology and Biotechnology

, Volume 87, Issue 6, pp 2037–2045 | Cite as

Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha

  • Yung-Hun Yang
  • Christopher J. Brigham
  • Charles F. Budde
  • Paolo Boccazzi
  • Laura B. Willis
  • Mohd Ali Hassan
  • Zainal Abidin Mohd Yusof
  • ChoKyun Rha
  • Anthony J. SinskeyEmail author
Biotechnological Products and Process Engineering


We employed systematic mixture analysis to determine optimal levels of acetate, propionate, and butyrate for cell growth and polyhydroxyalkanoate (PHA) production by Ralstonia eutropha H16. Butyrate was the preferred acid for robust cell growth and high PHA production. The 3-hydroxyvalerate content in the resulting PHA depended on the proportion of propionate initially present in the growth medium. The proportion of acetate dramatically affected the final pH of the growth medium. A model was constructed using our data that predicts the effects of these acids, individually and in combination, on cell dry weight (CDW), PHA content (%CDW), PHA production, 3HV in the polymer, and final culture pH. Cell growth and PHA production improved approximately 1.5-fold over initial conditions when the proportion of butyrate was increased. Optimization of the phosphate buffer content in medium containing higher amounts of butyrate improved cell growth and PHA production more than 4-fold. The validated organic acid mixture analysis model can be used to optimize R. eutropha culture conditions, in order to meet targets for PHA production and/or polymer HV content. By modifying the growth medium made from treated industrial waste, such as palm oil mill effluent, more PHA can be produced.


Polyhydroxyalkanoate Ralstonia eutropha Organic acid Mixture model 



The authors thank Mr. John Quimby and Ms. Karen Pepper for their contributions to this manuscript. This work was supported by a grant from the government of Malaysia and the Malaysian Office of Science, Technology and Innovation (MOSTI). The result of this grant, the Malaysia-MIT Biotechnology Partnership Program (MMBPP), is a collaboration between MIT, SIRIM Berhad, Universiti Putra Malaysia, and Universiti Sains Malaysia. The authors would like to thank the members of this program for their collegial collaborations.

Supplementary material

253_2010_2699_MOESM1_ESM.doc (44 kb)
Online Resource 1 (DOC 44 kb)
253_2010_2699_MOESM2_ESM.doc (66 kb)
Online Resource 2 (DOC 66 kb)
253_2010_2699_MOESM3_ESM.doc (29 kb)
Online Resource 3 (DOC 29 kb)
253_2010_2699_MOESM4_ESM.doc (62 kb)
Online Resource 3 (DOC 62 kb)


  1. Ahmad AL, Ismail S, Bhatia S (2003) Water recycling from palm oil mill effluent (POME) using membrane technology. Desalination 157:87–95CrossRefGoogle Scholar
  2. Ahn WS, Park SJ, Lee SY (2000) Production of poly(3-hydroxybutyrate) by fed-batch culture of recombinant Escherichia coli with a highly concentrated whey solution. Appl Environ Microbiol 66:3624–3627CrossRefGoogle Scholar
  3. Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472Google Scholar
  4. Basiron Y (2007) Palm oil production through sustainable plantations. Eur J Lipid Sci Tech 109:289–295CrossRefGoogle Scholar
  5. Bhubalan K, Lee WH, Loo CY, Yamamoto T, Tsuge T, Doi Y, Sudesh K (2008) Controlled biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) from mixtures of palm kernel oil and 3HV-precursors. Polym Degrad Stabil 93:17–23CrossRefGoogle Scholar
  6. Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonas oleovorans as a source of poly(beta-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54:1977–1982Google Scholar
  7. Chakraborty P, Gibbons W, Muthukumarappan K (2009) Conversion of volatile fatty acids into polyhydroxyalkanoate by Ralstonia eutropha. J Appl Microbiol 106:1996–2005Google Scholar
  8. Chen GQ, Wu Q (2005) The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials 26:6565–6578CrossRefGoogle Scholar
  9. Choi J, Lee SY (2000) Economic considerations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacterial fermentation. Appl Microbiol Biotechnol 53:646–649CrossRefGoogle Scholar
  10. Chua ASM, Takabatake H, Satoh H, Mino T (2003) Production of polyhydroxyalkanoates (PHA) by activated sludge treating municipal wastewater: effect of pH, sludge retention time (SRT), and acetate concentration in influent. Water Res 37:3602–3611CrossRefGoogle Scholar
  11. Cornibert J, Marchessault RH (1972) Physical properties of poly-β-hydroxybutyrate. IV. Conformational analysis and crystalline structure. J Mol Biol 71:735–756CrossRefGoogle Scholar
  12. Doi Y, Tamaki A, Kunioka M, Soga K (1988) Production of copolyesters of 3-hydroxybutyrate and 3-hydroxyvalerate by Alcaligenes eutrophus from butyric and pentanoic acids. Appl Microbiol Biotechnol 28:330–334CrossRefGoogle Scholar
  13. Doi Y, Kitamura S, Abe H (1995) Microbial synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 28:4822–4828CrossRefGoogle Scholar
  14. Frazzetto G (2003) White biotechnology—the application of biotechnology to industrial production holds many promises for sustainable development, but many products still have to pass the test of economic viability. EMBO Rep 4:835–837CrossRefGoogle Scholar
  15. Füchtenbusch B, Wullbrandt D, Steinbüchel A (2000) Production of polyhydroxyalkanoic acids by Ralstonia eutropha and Pseudomonas oleovorans from an oil remaining from biotechnological rhamnose production. Appl Microbiol Biotechnol 53:167–172CrossRefGoogle Scholar
  16. Hassan MA, Shirai Y, Kusubayashi N, Karim MIA, Nakanishi K, Hashimoto K (1996) Effect of organic acid profiles during anaerobic treatment of palm oil mill effluent on the production of polyhydroxyalkanoates by Rhodobacter sphaeroides. J Ferment Bioeng 82:151–156CrossRefGoogle Scholar
  17. Hassan MA, Nawata O, Shirai Y, Rahman NAA, Yee PL, Bin Ariff A, Ismail M, Karim A (2002) A proposal for zero emission from palm oil industry incorporating the production of polyhydroxyalkanoates from palm oil mill effluent. J Chem Eng Jpn 35:9–14CrossRefGoogle Scholar
  18. Jendrossek D, Handrick R (2002) Microbial degradation of polyhydroxyalkanoates. Annu Rev Microbiol 56:403–432CrossRefGoogle Scholar
  19. Khanna S, Srivastava AK (2005) A simple structured mathematical model for biopolymer (PHB) production. Biotechnol Prog 21:830–838CrossRefGoogle Scholar
  20. Kunioka M, Doi Y (1990) Thermal degradation of microbial copolyesters: poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate). Macromolecules 23:1933–1936CrossRefGoogle Scholar
  21. Liu HY, Hall PV, Darby JL, Coats ER, Green PG, Thompson DE, Loge FJ (2008) Production of polyhydroxyalkanoate during treatment of tomato cannery wastewater. Water Environ Res 80:367–372CrossRefGoogle Scholar
  22. Loo CY, Lee WH, Tsuge T, Doi Y, Sudesh K (2005) Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from palm oil products in a Wautersia eutropha mutant. Biotechnol Lett 27:1405–1410CrossRefGoogle Scholar
  23. Madison LL, Huisman GW (1999) Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63:21–53Google Scholar
  24. Marangoni C, Furigo A, de Aragdo GMF (2002) Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Ralstonia eutropha in whey and inverted sugar with propionic acid feeding. Process Biochem 38:137–141CrossRefGoogle Scholar
  25. Mergaert J, Anderson C, Wouters A, Swings J, Kersters K (1992) Biodegradation of polyhydroxyalkanoates. FEMS Microbiol Rev 9:317–321Google Scholar
  26. Misra SK, Valappil SP, Roy I, Boccaccini AR (2006) Polyhydroxyalkanoate (PHA)/inorganic phase composites for tissue engineering applications. Biomacromolecules 7:2249–2258CrossRefGoogle Scholar
  27. Nikel PI, Pettinari MJ, Mendez BS, Galvagno MA (2005) Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strain using agroindustrial byproducts. Int Microbiol 8:243–250Google Scholar
  28. Nikel PI, de Almeida A, Melillo EC, Galvagno MA, Pettinari MJ (2006) New recombinant Escherichia coli strain tailored for the production of poly(3-hydroxybutyrate) from agroindustrial by-products. Appl Environ Microbiol 72:3949–3954CrossRefGoogle Scholar
  29. Nowruzi K, Elkamel A, Scharer JM, Cossar D, Moo-Young M (2008) Development of a minimal defined medium for recombinant human interleukin-3 production by Streptomyces lividans 66. Biotechnol Bioeng 99:214–222CrossRefGoogle Scholar
  30. Scandola M, Ceccorulli G, Pizzoli M, Gazzano M (1992) Study of the crystal phase and crystallization rate of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Macromolecules 25:1405–1410CrossRefGoogle Scholar
  31. Sim SJ, Snell KD, Hogan SA, Stubbe J, Rha C, Sinskey AJ (1997) PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo. Nat Biotechnol 15:63–67CrossRefGoogle Scholar
  32. Steinbüchel A, Füchtenbusch B (1998) Bacterial and other biological systems for polyester production. Trends Biotechnol 16:419–427CrossRefGoogle Scholar
  33. Sudesh K, Abe H, Doi Y (2000) Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 25:1503–1555CrossRefGoogle Scholar
  34. Tan IKP, Kumar KS, Theanmalar M, Gan SN, Gordon B (1997) Saponified palm kernel oil and its major free fatty acids as carbon substrates for the production of polyhydroxyalkanoates in Pseudomonas putida PGA1. Appl Microbiol Biotechnol 47:207–211CrossRefGoogle Scholar
  35. Wang J, Yu J (2001) Kinetic analysis on formation of poly(3-hydroxybutyrate) from acetic acid by Ralstonia eutropha under chemically defined conditions. J Ind Microbiol Biot 26:121–126CrossRefGoogle Scholar
  36. Wilde E (1962) Untersuchungen über wachstum und speicherstoffsynthese von hydrogenomonas. Arch Mikrobiol 43:109–137CrossRefGoogle Scholar
  37. Williams SF, Martin DP, Horowitz DM, Peoples OP (1999) PHA applications: addressing the price performance issue: I. Tissue engineering. Int J Biol Macromol 25:111–121CrossRefGoogle Scholar
  38. Wu Q, Wang Y, Chen GQ (2009a) Medical application of microbial biopolyesters polyhydroxyalkanoates. Artif Cell Blood Sub Biot 37:1–12CrossRefGoogle Scholar
  39. Wu TY, Mohammad AW, Jahim JM, Anuar N (2009b) A holistic approach to managing palm oil mill effluent (POME): Biotechnological advances in the sustainable reuse of POME. Biotechnol Adv 27:40–52CrossRefGoogle Scholar
  40. Yee PL, Hassan MA, Shirai Y, Wakisaka M, Karim MIA (2003) Continuous production of organic acids from palm oil mill effluent with sludge recycle by the freezing-thawing method. J Chem Eng Jpn 36:707–710CrossRefGoogle Scholar
  41. York GM, Lupberger J, Tian J, Lawrence AG, Stubbe J, Sinskey AJ (2003) Ralstonia eutropha H16 encodes two and possibly three intracellular Poly[D-(−)-3-hydroxybutyrate] depolymerase genes. J Bacteriol 185:3788–3794CrossRefGoogle Scholar
  42. Young AL (2003) Biotechnology for food, energy, and industrial products—new opportunities for bio-based products. Environ Sci Pollut R 10:273–276Google Scholar
  43. Zaldivar J, Nielsen J, Olsson L (2001) Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 56:17–34CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Yung-Hun Yang
    • 1
    • 2
  • Christopher J. Brigham
    • 1
  • Charles F. Budde
    • 3
  • Paolo Boccazzi
    • 1
  • Laura B. Willis
    • 1
    • 4
  • Mohd Ali Hassan
    • 5
  • Zainal Abidin Mohd Yusof
    • 6
  • ChoKyun Rha
    • 4
  • Anthony J. Sinskey
    • 1
    • 7
    • 8
    Email author
  1. 1.Department of BiologyMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Department of Microbial EngineeringKonkuk UniversitySeoulRepublic of Korea
  3. 3.Department of Chemical EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  4. 4.Biomaterials Science and Engineering LaboratoryMassachusetts Institute of TechnologyCambridgeUSA
  5. 5.Department of Biotechnology and Biomolecular ScienceUniversiti Putra MalaysiaSerdangMalaysia
  6. 6.Research & Technology DivisionSIRIM BerhadShah AlamMalaysia
  7. 7.Division of Health Sciences TechnologyMassachusetts Institute of TechnologyCambridgeUSA
  8. 8.Engineering Systems DivisionMassachusetts Institute of TechnologyCambridgeUSA

Personalised recommendations