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Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria

  • Applied Microbial and Cell Physiology
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Abstract

Ultra-high molecular weight polyhydroxyalkanoates (PHAs) with low polydispersity index (PDI = 1.3) were produced in a novel, pilot scale application of mixed cultures of nitrogen-fixing bacteria. The number average molecular weight (M n) of the poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was determined to be 2.4 × 106 and 2.5 × 106 g mol−1, respectively. Using two types of carbon sources, biomass contents of the P(3HB) and P(3HB-co-3HV) were 18% and 30% (PHA in dry biomass), respectively. The extracted polymers were analysed for their physical properties using analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC) and gel permeation chromatography (GPC). NMR confirmed the formation of homopolymer and copolymer. DSC showed a single melting endotherm peak for both polymers, with enthalpies that indicated crystallinity indices of 44% and 37% for P(3HB) and P(3HB-co-3HV), respectively. GPC showed a sharp unimodal trace for both polymers, reflecting the homogeneity of the polymer chains. The work described here emphasises the potential of mixed colony nitrogen-fixing bacteria cultures for producing biodegradable polymers which have properties that are very similar to those from their pure-culture counterparts and therefore making a more economically viable route for obtaining biopolyesters.

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References

  • Abe H, Doi Y (2001) Crystalline morphology, thermal properties, and enzymatic degradability of (R)-3-hydroxybutyric acid-based copolyesters. Riken rev 42:7–10

    CAS  Google Scholar 

  • Albuquerque MGE, Eiroa M, Torres C, Nunes BR, Reis MAM (2007) Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses. J Biotechnol 130:411–421

    Article  CAS  PubMed  Google Scholar 

  • American Public Health Association (1998) Standard methods for the examination of water and wastewater. American Public Health Association, Washington, DC

    Google Scholar 

  • Anderson AJ, Williams DR, Taidi B, Dawes EA, Ewing DF (1992) Studies on copolyester synthesis by Rhodococcus ruber and factors influencing the molecular mass of polyhydroxybutyrate accumulated by Methylobacterium extorquens and Alcaligenes eutrophus. FEMS Microbiol Rev 103:93–101

    Article  CAS  Google Scholar 

  • Baltieri RC, Mei LHI, Bartoli J (2003) Study of the influence of plasticizers on the thermal and mechanical properties of poly(3-hydroxybutyrate) compounds. Macromol Symp 197:33–44

    Article  CAS  Google Scholar 

  • Baptist JN (1962) Process for preparing poly-β-hydroxybutyric acid. Grace WR & Co, US patent 3044942

  • Barham PJ, Keller A, Otun EL, Holmes PA (1984) Crystallization and morphology of a bacterial thermoplastic: poly-3-hydroxybutyrate. J Mater Sci 19:2781–2794

    Article  CAS  Google Scholar 

  • Bengtsson S, Werker A, Welander T (2008) Production of polyhydroxyalkanoates by glycogen accumulating organisms treating a paper mill wastewater. Water Sci Technol 58:323–330

    Article  CAS  PubMed  Google Scholar 

  • Beun JJ, Paletta F, van Loosdrecht MCM, Heijnen JJ (2000) Stoichiometry and kinetics of poly-β-hydroxybutyrate metabolism in aerobic, slow growing, activated sludge cultures. Biotechnol Bioeng 67:379–389

    Article  CAS  PubMed  Google Scholar 

  • Blauhut W, Gierlinger W, Strempfl F (1993) Process for obtaining a polyhydroxyalkanoate from the cell material of a microorganism. PCD Polymere Gesellschaft mbH (Schwechat-Mannsworth, AT), US patent 5213976

  • Braunegg G, Lefebvre G, Genser KF (1998) Polyhydroxyalkanoates, biopolyesters from renewable resources: physiological and engineering aspects. J Biotechnol 65:127–161

    Article  CAS  PubMed  Google Scholar 

  • Dai Y, Yuan Z, Jack K, Keller J (2007) Production of targeted poly(3-hydroxyalkanoates) copolymers by glycogen accumulating organisms using acetate as sole carbon source. J Biotechnol 129:489–497

    Article  CAS  PubMed  Google Scholar 

  • Dai Y, Lambert L, Yuan Z, Keller J (2008) Characterisation of polyhydroxyalkanoate copolymers with controllable four-monomer composition. J Biotechnol 134:137–145

    Article  CAS  PubMed  Google Scholar 

  • Dennis D, Liebig C, Holley T, Thomas KS, Khosla A, Wilson D, Augustine B (2003) Preliminary analysis of polyhydroxyalkanoate inclusions using atomic force microscopy. FEMS Microbiol Lett 226:113–119

    Article  CAS  PubMed  Google Scholar 

  • Dennis MA, Cotter ML, Slade AH, Gapes DJ (2004) The performance of a nitrogen-fixing SBR. Water Sci Technol 50:269–278

    Article  CAS  PubMed  Google Scholar 

  • Dias JML, Lemos PC, Serafim LS, Oliveira C, Eiroa M, Albuquerque MGE, Ramos AM, Oliveira R, Reis MAM (2006) Recent advances in polyhydroxyalkanoate production by mixed aerobic cultures: from substrate to final product. Macromol Biosci 6:885–906

    Article  CAS  PubMed  Google Scholar 

  • Dionisi D, Beccari M, Di Gregorio S, Majone M, Papini MP, Vallini G (2005) Storage of biodegradable polymers by an enriched microbial community in a sequencing batch reactor operated at high organic load rate. J Chem Technol Biotechnol 80:1306–1318

    Article  CAS  Google Scholar 

  • Dionisi D, Majone M, Vallini G, Di Gregorio S, Beccari M (2006) Effect of the applied organic load rate on biodegradable polymer production by mixed microbial cultures in a sequencing batch reactor. Biotechnol Bioeng 93:76–88

    Article  CAS  PubMed  Google Scholar 

  • Dionisi D, Majone M, Vallini G, Di Gregorio S, Beccari M (2007) Effect of the length of the cycle on biodegradable polymer production and microbial community selection in a sequencing batch reactor. Biotechnol Prog 23:1064–1073

    CAS  PubMed  Google Scholar 

  • Doi Y, Steinbüchel A (2008) Biodegradable polymer (Biopol®). In: Asrar J, Grurys KJ (eds) Biopolymers. Polyester III—applications and commercial products. Wiley, Weinheim, pp 53–68

    Google Scholar 

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

    Article  CAS  Google Scholar 

  • Doi Y, Kunioka M, Nakamura Y, Soga K (1986b) Nuclear magnetic resonance studies on poly(β-hydroxybutyrate) and a copolyester of β-hydroxybutyrate and β-hydroxyvalerate from Alcaligenes eutrophus H16. Macromolecules 19:2860–2864

    Article  CAS  Google Scholar 

  • Dong L, An Y, Li G, Mo Z, Feng Z (2000) Miscibility, crystallisation kinetics, and morphology of poly(β-hydroxybutyrate) and poly(methyl methacrylate) blends. J Poly Sci Part B Poly Phys 38:1860–1867

    Article  Google Scholar 

  • El Taweel SH, Stoll B, Höhne GWH, Mansour AA, Seliger H (2004) Stress–strain behavior of blends of bacterial polyhydroxybutyrate. J Appl Poly Sci 94:2528–2537

    Article  CAS  Google Scholar 

  • Hoshino A, Tsuji M, Fukuda K, Nonagase M, Sawada H, Kimura M (2002) Changes in molecular structure of biodegradable plastics during degradation in soils estimated by FTIR and NMR. Soil Sci Plant Nutr 48:469–473

    Article  CAS  Google Scholar 

  • Jan S, Roblot C, Courtois J, Courtois B, Barbotin JN, Séguin JP (1996) 1H NMR spectroscopic determination of poly 3-hydroxybutyrate extracted from microbial biomass. Enzyme Microbiol Technol 18:195–201

    Article  CAS  Google Scholar 

  • Khanna S, Srivastava AK (2005) Recent advances in microbial polyhydroxyalkanoates. Process Biochem 40:607–619

    Article  CAS  Google Scholar 

  • Kim BS, Chang HN (1998) Production of poly(3-hydroxybutyrate) from starch by Azotobacter chroococcum. Biotechnol Lett 20:109–112

    Article  CAS  Google Scholar 

  • Kunioka M, Doi Y (1990) Thermal degradation of microbial copolyesters: poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate). Macromolecules 23:1933–1936

    Article  CAS  Google Scholar 

  • Kusaka S, Abe H, Lee SY, Doi Y (1997) Molecular mass of poly[(R)-3-hydroxybutyric acid] produced in a recombinant Escherichia coli. Appl Microbiol Biotechnol 47:140–143

    Article  CAS  PubMed  Google Scholar 

  • Kusaka S, Iwata T, Doi Y (1998) Microbial synthesis and physical properties of ultra-high-molecular-weight poly[(R)-3-hydroxybutyrate]. J Macromol Sci, Rev Macromol Chem Phys 35:319–335

    Google Scholar 

  • Kusaka S, Iwata T, Doi Y (1999) Properties and biodegradability of ultra-high-molecular-weight poly[(R)-3-hydroxybutyrate] produced by a recombinant Escherichia coli. Int J Biol Macromol 25:87–94

    Article  CAS  PubMed  Google Scholar 

  • Lawrence AG, Choi JH, Rha CK, Stubbe JA, Sinskey AJ (2005) In vitro analysis of the chain termination reaction in the synthesis of poly-(R)-β-hydroxybutyrate by the class III synthase from Allochromatium vinosum. Biomacromolecules 6:2113–2119

    Article  CAS  PubMed  Google Scholar 

  • Lee SY, Choi JI, Wong HH (1999) Recent advances in polyhydroxyalkanoate production by bacterial fermentation: mini-review. Int J Biol Macromol 25:31–36

    Article  CAS  PubMed  Google Scholar 

  • Lemos PC, Serafim LS, Reis MAM (2006) Synthesis of polyhydroxyalkanoates from different short-chain fatty acids by mixed cultures submitted to aerobic dynamic feeding. J Biotechnol 122:226–238

    Article  CAS  PubMed  Google Scholar 

  • Macrae RM, Wilkinson JF (1958) Poly-beta-hydroxybutyrate metabolism in washed suspensions of Bacillus cereus and Bacillus megaterium. J Gen Microbiol 19:210–222

    Article  CAS  PubMed  Google Scholar 

  • Ojumu TV, Yu J, Solomon BO (2004) Production of polyhydroxyalkanoates, a bacterial biodegradable polymer. Afr J Biotechnol 3:18–24

    Article  CAS  Google Scholar 

  • Poirier Y, Erard N, Petétot JMC (2001) Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid β-oxidation. Appl Environ Microbiol 67:5254–5260

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Quagliano JC, Miyazaki SS (1997) Effect of aeration and carbon/nitrogen ratio on the molecular mass of the biodegradable polymer poly-β-hydroxybutyrate obtained from Azotobacter chroococcum 6B. Appl Microbiol Biotechnol 48:662–664

    Article  CAS  Google Scholar 

  • Ramsay BA, Ramsay J, Berger E, Chavarie C, Braunegg G (1992) Separation of poly-beta-hydroxyalkanoic acid from microbial biomass. Ecole Polytech (CA), US patent 5110980

  • Randriamanhefa S, Renard E, Rin P, Langlois V (2003) Fourier transform infrared spectroscopy for screening and quantifying production of PHAs by Pseudomonas grown on sodium octanoate. Biomacromolecules 4:1092–1097

    Article  CAS  Google Scholar 

  • Reid NM, Slade AH, Stuthridge TR (2006) Process for production of biopolymers from nitrogen deficient wastewater. New Zealand Forest Research Institute Limited (NZ), US patent 6987011

  • Reis MAM, Serafim LS, Lemos PC, Ramos AM, Aguiar FR, Van Loosdrecht MCM (2003) Production of polyhydroxyalkanoates by mixed microbial cultures. Bioprocess Biosyst Eng 25:377–385

    Article  CAS  PubMed  Google Scholar 

  • Ren Q, Sierro N, Kellerhals M, Kessler B, Witholt B (2000) Properties of engineered poly-3-hydroxyalkanoates produced in recombinant Escherichia coli strains. Appl Environ Microbiol 66:1311–1320

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Riis V, Mai W (1988) Gas chromatographic determination of poy-β-polyhydroxybutyric acid in microbial biomass after hydrochloric acid propanolysis. J Chromatogr 445:285–289

    Article  CAS  Google Scholar 

  • Saad GR, Mansour AA, Hamed AH (1997) Dielectric investigation of cold crystallization of poly(3-hydroxybutyrate). Polymer 38:4091–4096

    Article  CAS  Google Scholar 

  • Salehizadeh H, Van Loosdrecht MCM (2004) Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnological importance. Biotecnol Adv 22:261–279

    Article  CAS  Google Scholar 

  • Scandola M, Pizzoli M, Ceccorulli G, Cesáro A, Paoletti S, Navarini L (1988) Viscoelastic and thermal properties of bacterial poly(d-(-)-β-hydroxybutyrate). Int J Biol Macromol 10:373–377

    Article  CAS  Google Scholar 

  • Senior PJ, Ritchie GAF, Beech GA, Awes EA (1972) The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii. Biochem J 128:1193–1201

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Senior PJ, Wright LF, Barry A (1982) Extraction process. ICI Ltd, US patent 4324907

  • Serafim LS, Lemos PC, Oliveira R, Reis MAM (2004) Optimization of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions. Biotechnol Bioeng 87:145–160

    Article  CAS  PubMed  Google Scholar 

  • Serafim LS, Lemos PC, Torres C, Reis MAM, Ramos AM (2008a) The influence of process parameters on the characteristics of polyhydroxyalkanoates produced by mixed cultures. Macrmol Biosci 8:355–366

    Article  CAS  Google Scholar 

  • Serafim LS, Lemos PC, Albuquerque MGE, Reis MAM (2008b) Strategies for PHA production by mixed cultures and renewable waste materials. Appl Microbiol Biotechnol 81:615–628

    Article  CAS  PubMed  Google Scholar 

  • Song JJ, Yoon SC, Yu SM, Lenz RW (1998) Differential scanning calorimetric study of poly(3-hydroxyoctanoate) inclusions in bacterial cells. Int J Biol Macromol 23:165–173

    Article  CAS  PubMed  Google Scholar 

  • Sprent JJ, Sprent P (1990) Nitrogen fixing organisms: pure and applied aspects. Cambridge University Press, Cambridge

    Book  Google Scholar 

  • Waser P, Rueping M, Seebach D, Duchardt E, Schwalbe H (2001) On the solution structure of PHB: preparation and NMR analysis of isotopically labeled oligo [(R)-3-hydroxybutanoic acids] (OHBs). Helv Chim Acta 84:1821–1845

    Article  CAS  Google Scholar 

  • Yoon SC, Choi MH (1999) Local sequence dependence of polyhydroxyalkanoic acid degradation in Hydrogenophaga pseudoflava. J Biol Chem 274:37800–37808

    Article  CAS  PubMed  Google Scholar 

  • Zhao K, Deng Y, Chen JC, Chen GQ (2003) Polyhydroxyalkanoate (PHA) scaffolds with good mechanical properties and biocompatibility. Biomaterials 24:1041–1045

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank Stephanie Hölldampf for extracting the polyesters from the biomass and Stefan Hill, Michael Witt and Marc Gaugler for assistance with NMR, GPC and DSC experiments, respectively.

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  1. Scion, Private Bag 3020, Rotorua, New Zealand

    Meeta Patel, Daniel J. Gapes, Roger H. Newman & Peter H. Dare

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  1. Meeta Patel
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  2. Daniel J. Gapes
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Correspondence to Daniel J. Gapes.

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Patel, M., Gapes, D.J., Newman, R.H. et al. Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria. Appl Microbiol Biotechnol 82, 545–555 (2009). https://doi.org/10.1007/s00253-008-1836-0

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