Production and characterization of poly 3-hydroxybutyrate-co-3-hydroxyvalerate in wheat starch wastewater and its potential for nanoparticle synthesis


Polyhydroxyalkanoates (PHAs) are polymers with biodegradable and biocompatible properties accumulated in a wide variety of bacterial strains. In the present study, active sludge, wheat starch wastewater (WSW), and oil wastewater were used for the isolation and screening of PHA-accumulating bacteria. WSW was then implemented as a cheap and economical culture medium for the production of PHAs by the selected isolate. The extracted PHA was characterized, and the capability of produced biopolymer for preparing nanoparticles was evaluated. Based on the results, 96 different bacterial isolates were obtained, of which the strains isolated from WSW demonstrated the highest PHA-accumulation capability. The maximum PHA content of 3.07 g/l (59.50% of dry cell weight) was obtained by strain N6 in 21 h. The selected strain was identified by molecular approaches as Bacillus cereus. Afterward, the physicochemical characterization of an accumulated biopolymer was specified as a PHBV copolymer. Finally, spherical homogenous PHBV nanoparticles with a size of 137 nm were achieved. The PHBV nanoparticles showed a suitable small size and good zeta potential for medical applications. Hence, it can be concluded that isolated wild strain (B. cereus) has the potential exploitation capability for cost-effective PHBV production using the WSW.

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Data availability

All isolated strains are preserved in Persian Type Culture Collection (PTCC) and data are available by N. Sinaei.


  1. 1.

    Luengo JM, Garcia B, Sandoval A, Naharro G, Olivera ER (2003) Bioplastics from microorganisms. Curr Opin Microbiol 6:251–260.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Akaraonye E, Keshavarz T, Roy I (2010) Production of polyhydroxyalkanoates: the future green materials of choice. J Chem Technol Biotechnol 85:732–743.

    CAS  Article  Google Scholar 

  3. 3.

    Peña C, López S, García A, Espín G, Romo-Uribe A, Segura D (2014) Biosynthesis of poly-βhydroxybutyrate (PHB) with a high molecular mass by a mutant strain of Azotobacter vinelandii (OPN). Ann Microbiol 64:39–47.

    CAS  Article  Google Scholar 

  4. 4.

    Batcha AFM, Prasad DMR, Khan MR, Abdullah H (2014) Biosynthesis of poly(3- hydroxybutyrate) (PHB) by Cupriavidus necator H16 from jatropha oil as carbon source. Bioprocess Biosyst Eng 37:943–951.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Song R, Murphy M, Li C, Ting K, Soo C, Zheng Z (2018) Current development of biodegradable polymeric materials for biomedical applications. Drug Des Dev Ther 12:3117–3145.

    CAS  Article  Google Scholar 

  6. 6.

    Mohan S, Oluwafemi OS, Kalarikkal N, Thomas S, Songca SP (2016) Biopolymers– application in nanoscience and nanotechnology. Recent Adv Biopolymers 47.

  7. 7.

    Ozdil D, Aydin HM (2014) Polymers for medical and tissue engineering applications. J Chem Technol Biotechnol 89(12):1793–1810.

    CAS  Article  Google Scholar 

  8. 8.

    Philip S, Keshavarz T, Roy I (2007) Polyhydroxyalkanoates: biodegradable polymers with a range of applications. J Chem Technol Biotechnol 82:233–247.

    CAS  Article  Google Scholar 

  9. 9.

    Kerketta A, Vasanth D (2019) Madhuca indica flower extract as cheaper carbon source for production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) using Ralstonia eutropha. Process Biochem 87:1–9.

    CAS  Article  Google Scholar 

  10. 10.

    Chen GQ, Wu Q (2005) The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials 26(33):6565–6578.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Rivera-Briso AL, Serrano-Aroca Á (2018) Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate): enhancement strategies for advanced applications. Polymers 10(7):732–759

    Article  Google Scholar 

  12. 12.

    Halami PM (2008) Production of polyhydroxyalkanoate from starch by the native isolate Bacillus cereus CFR06. World J Microbiol Biotechnol 24:805–812.

    CAS  Article  Google Scholar 

  13. 13.

    Sharma P, Bajaj B (2015) Cost-effective substrates for production of poly-β-hydroxybutyrate by a newly isolated Bacillus cereus PS-10. J Environ Biol 36:1297–1304

    CAS  PubMed  Google Scholar 

  14. 14.

    Naheed N, Jamil N (2014) Optimization of biodegradable plastic production on sugar cane molasses in Enterobacter sp. SEL2. Braz J Microbiol 45:417–426.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    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 9:2(1):34.

  16. 16.

    Sathiyanarayanan G, Kiran GS, Selvinc J, Saibaba G (2013) Optimization of polyhydroxybutyrate production by marine Bacillus megaterium MSBN04 under solid state culture. Int J Biol Macromol 60:253–261.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Haas R, Jin B, Zepf FT (2008) Production of poly(3-hydroxybutyrate) from waste potato starch. Biosci Biotechnol Biochem 72:253–256.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Dahman Y, Ugwu CU (2014) Production of green biodegradable plastics of poly(3-hydroxybutyrate) from renewable resources of agricultural residues. Bioprocess Biosyst Eng 37:1561–1568.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Novackova I, Kucera D, Porizka J, Pernicova I, Sedlacek P, Koller M, Kovalcik A, Obruca S (2019) Adaptation of Cupriavidus necator to levulinic acid for enhanced production of P(3HB-co-3HV) copolyesters. Biochem Eng J 151:107350.

    CAS  Article  Google Scholar 

  20. 20.

    Farrag Y, Montero B, Rico M, Barral L, Bouza R (2018) Preparation and characterization of nano and micro particles of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) via emulsification/solvent evaporation and nanoprecipitation techniques. J Nanopart Res 20:71.

    CAS  Article  Google Scholar 

  21. 21.

    Wei YH, Chen WC, Huang CK, Wu HS, Sun YM, Lo CW, Janarthanan OM (2011) Screening and evaluation of Polyhydroxybutyrate-producing strains from indigenous isolate Cupriavidus taiwanensis strains. Int J Mol Sci 12:252–265.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Miller GL (1959) Use of dinitrosaIicyIic acid reagent for determination of reducing sugar. Anal Chem 31:426–428.

    CAS  Article  Google Scholar 

  23. 23.

    Singh G, Kumari A, Mittal A, Yadav A, Aggarwal NK (2013) Poly β-Hydroxybutyrate production by Bacillus subtilis NG220 using sugar industry waste water. BioMed Research International Article ID 952641.

  24. 24.

    Law J, Slepecky RA (1961) Assay of poly-beta-hydroxybutyric acid. J Bacteriol 82(1):33–36.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Hahn SK, Chang YK, Kim BS, Lee KM, Chang HN (1993) The recovery of poly(3-hydroxybutyrate) by using dispersions of sodium hypochlorite solution and chloroform. Biotechnol Tech 7(3):209–212.

    CAS  Article  Google Scholar 

  26. 26.

    Shrivastava A, Mishraa SK, Shethia B, Pancha I, Jain D, Mishraa S (2010) Isolation of promising bacterial strains from soil and marine environment for polyhydroxyalkanoates (PHAs) production utilizing Jatropha biodiesel byproduct. Int J Biol Macromol 47:283–287.

    CAS  Article  Google Scholar 

  27. 27.

    Preethi R, Sasikala P, Aravind J (2012) Microbial production of polyhydroxyalkanoate (PHAs) utilizing fruit waste as a substrate. Res in Biotech 3(1):61–69

    Google Scholar 

  28. 28.

    Contreras AR, Koller M, Dias MMS, Monfort MC, Braunegg G, Calvo MSM (2013) High production of poly (3-hydroxybutyrate) from a wild Bacillus megaterium bolivian strain. J Appl Microbiol 114(5):1378–1387.

    CAS  Article  Google Scholar 

  29. 29.

    Öner M, Çöl AA, Pochat-Bohatier C, Bechelany M (2016) Effect of incorporation of boron nitride nanoparticles on the oxygen barrier and thermalproperties of poly(3-hydroxybutyrate-cohydroxyvalerate). RSC Adv 6:90973–90981.

    CAS  Article  Google Scholar 

  30. 30.

    Vilos C, Morales FA, Solar PA, Herrera NS, Gonzalez-Nilo FD, Aguayo DA, Mendoza HL, Comer J, Bravo ML, Gonzalez PA, Kato S, Cuello MA, Alonso C, Bravo EJ, Bustamante EI, Owen GI, Velasquez LA (2013) Paclitaxel-PHBV nanoparticles and their toxicity to endometrial and primary ovarian cancer cells. Biomaterials 34(16):4098–4108.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Liu H, Pancholi M, Stubbs J III, Raghavan D (2010) Influence of hydroxyvalerate composition of polyhydroxy butyrate valerate (PHBV) copolymer on bone cell viability and in vitro degradation. J Appl Polym Sci 116:3225–3231.

    CAS  Article  Google Scholar 

  32. 32.

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

    CAS  Article  Google Scholar 

  33. 33.

    Moorkoth D, Nampoothiri KM (2016) Production and characterization of poly(3-hydroxy butyrate-co-3hydroxyvalerate) (PHBV) by a novel halotolerant mangrove isolate. Bioresour Technol 201:253–260.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Mohapatra S, Mohanta PR, Sarkar B, Daware A, Kumar C, Samantaray DP (2015) Production of Polyhydroxyalkanoates (PHAs) by Bacillus strain isolated from waste water and its biochemical characterization. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 87(2):459–466.

  35. 35.

    Bhuwal AK, Singh G, Aggarwal NK, Goyal V, Yadav A (2014) Poly-β-hydroxybutyrate production and management of cardboard industry effluent by new Bacillus sp. NA10. Bioresour. Bioprocess. 1, 9.

  36. 36.

    Aslim B, Yüksekdağ ZN, Beyatli Y (2002) Determination of PHB growth quantities of certain bacillus species isolated from soil. Turk.E.J. Biotechnol. 2:24–30

  37. 37.

    Labuzek S, Radecka I (2001) Biosynthesis of PHB tercopolymer by Bacillus cereus UW85. J Appl Microbiol 90:353–357.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Yilmaz M, Soran H, Beyatli Y (2005) Determination of poly-b-hydroxybutyrate (PHB) production by some Bacillus spp. World J Microbiol Biotechnol 21:565–566.

    CAS  Article  Google Scholar 

  39. 39.

    Tufail S, Munir S, Jamil N (2017) Variation analysis of bacterial polyhydroxyalkanoates production using saturated and unsaturated hydrocarbons. Braz J Microbiol 48(4):629–636.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Liu Z, Wang Y, He N, Huang J, Zhu K, Shao W, Wang H, Yuan W, Li Q (2011) Optimization of polyhydroxybutyrate (PHB) production by excess activated sludge and microbial community analysis. J Hazard Mater 185:8–16.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Masood F, Hasan 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 20:865–871.

    CAS  Article  Google Scholar 

  42. 42.

    Rivera-Briso AL, Aachmann FL, Moreno-Manzano V, Serrano-Aroca Á (2020) Graphene oxide nanosheets versus carbon nanofibers: enhancement of physical and biological properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) films for biomedical applications. Int J Biol Macromol 143:1000–1008.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Lombardo D, Kiselev MA, Caccamo MT (2019) Smart nanoparticles for drug delivery application: development of versatile nanocarrier platforms in biotechnology and nanomedicine. J Nanomater 2019:1–26.

    CAS  Article  Google Scholar 

  44. 44.

    Decuzzi P, Ferrari M (2007) The role of specific and non-specific interactions in receptor-mediated endocytosis of nanoparticles. Biomaterials 28:2915–2922.

    CAS  Article  PubMed  Google Scholar 

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The authors are thankful to the Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia, for providing laboratory facilities for nanoparticle study. We are also grateful to Ms. Mohseni, the head of the Persian Type Culture Collection (PTCC), for identifying the isolated strains.

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Design of study was accomplished by Davood Zare, Neda Sinaei, and Mehrdad Azin. Data and draft of manuscript were collected by Neda Sinaei and finalized by Davood Zare and Mehrdad Azin. All authors read and approved the final manuscript.

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Correspondence to Davood Zare.

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Sinaei, N., Zare, D. & Azin, M. Production and characterization of poly 3-hydroxybutyrate-co-3-hydroxyvalerate in wheat starch wastewater and its potential for nanoparticle synthesis. Braz J Microbiol (2021).

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  • Polyhydroxyalkanoates
  • Poly (3-hydroxybutyrate-co-3-hydroxyvalerate)
  • Wheat starch wastewater
  • Bacillus cereus
  • Nanoparticle
  • Medical application