, Volume 19, Issue 2, pp 515–524 | Cite as

Preparation of poly(3-hydroxybutyrate-co-hydroxyvalerate) films from halophilic archaea and their potential use in drug delivery

  • Ozkan Danis
  • Ayse Ogan
  • Pınar Tatlican
  • Azade Attar
  • Emrah Cakmakci
  • Bulent Mertoglu
  • Meral Birbir
Original Paper


Halophilic archaea offer a potential source for production of polyhydroxyalkanoates (PHAs). Hence, the experiments were carried out with five extremely halophilic archaeal isolates to determine the highest PHA-producing strain. PHA production of each isolates was separately examined in cheap carbon sources such as corn starch, sucrose, whey, apple, melon and tomato wastes. Corn starch was found to be a fairly effective substrate for PHA production. Among the strains studied here, the strain with the highest capability for PHA biosynthesis was found to be 1KYS1. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that 1KYS1 closely related to species of the genus Natrinema. The closest phylogenetic similarity was with the strain of Natrinema pallidum JCM 8980 (99 %). PHA content of 1KYS1 was about 53.14 % of the cell dry weight when starch was used as a carbon source. The formation of large and uniform PHA granules was confirmed by transmission electron microscopy and the biopolymer was identified as poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV). PHBV produced by 1KYS1 was blended with low molar mass polyethylene glycol (PEG 300) to prepare biocompatible films for drug delivery. Rifampicin was used as a model drug and its release from PHBV films was investigated at pH 7.4, 37 °C. It was found that PHBV films obtained from 1KYS1 were very effective for drug delivery. In conclusion, PHBV of 1KYS1 may have a potential usage in drug delivery applications.


Halophilic archaea poly(3-hydroxybutyrate-co-hydroxyvalerate) PHBV Drug delivery Rifampicin 


  1. Aktan CK, Yapsakli K, Mertoglu B (2012) Inhibitory effects of free ammonia on Anammox bacteria. Biodegradation 23:751–762CrossRefPubMedGoogle Scholar
  2. Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472PubMedCentralPubMedGoogle Scholar
  3. Arahal DR, Dewhirst FE, Paster BJ, Volcani BE, Ventosa A (1996) Phylogenetic analyses of some extremely halophilic archae isolated from dead sea water. Determined on the basis of their 16S rRNA sequences. Appl Environ Microbiol 62:3779–3786PubMedCentralPubMedGoogle Scholar
  4. Bailey M, Birbir DG (1993) A study of the extremely halophilic microorganisms found on commercially brine-cured cattle hides. J Am Leather Chem Assoc 88:285–293Google Scholar
  5. Benola G, Ventosa A, Megias M, Berraquero F (1984) The sensitivity of Halobacteria to antibiotics. FEMS Microbiol Lett 21:341–345CrossRefGoogle Scholar
  6. Birbir M, Ogan A, Calli B, Mertoglu B (2004) Enzyme characteristics of extremely halophilic archaeal community in Tuzkoy Salt Mine, Turkey. World J Microb Biot 20:613–621CrossRefGoogle Scholar
  7. Birbir M, Calli B, Mertoglu B, Bardavid RE, Oren A, Ogmen MN, Ogan A (2007) Extremely halophilic Archaea from Tuz Lake, Turkey, and the adjacent Kaldirim and Kayacik salterns. W J Microb Biotech 23:309–316CrossRefGoogle Scholar
  8. Bloembergen S, Holden DA (1986) Studies of composition and crystallinity of bacterial poly(β-hydroxybutyrate-co-β-hydroxyvalerate). Macromolecules 19:2865–2871CrossRefGoogle Scholar
  9. Bonartsev AP, Myshkina VL, Nikolaeva DA, Furina EK, Makhina TA, Livshits VA, Boskhomdzhiev AP, Ivanov EA, Iordanskii AL, Bonartseva GA (2007) Biosynthesis, biodegradation, and application of poly(3-hydroxybutyrate) and its copolymers—natural polyesters produced by diazotrophic bacteria. In: Mendez-Villas A (ed) Communicating current research and educational topics and trends in applied microbiology, 2007th edn. Formatex, Spain, pp 295–307Google Scholar
  10. Brown AD (1963) The peripheral structures of Gram-negative bacteria. IV. The cation-sensitive dissolution of the cell membrane of the halophilic bacterium Halobacterium halobium. Biochim Biophys Acta 75:425–435CrossRefPubMedGoogle Scholar
  11. Cai Y, Lv J, Feng J (2013) Spectral characterization of four kinds of biodegradable plastics: poly (lactic acid), poly (butylenes adipate-co-terephthalate), poly (hydroxybutyrate-co-hydroxyvalerate) and poly (butylenes succinate) with FTIR and raman spectroscopy. J Polym Environ 21:108–114CrossRefGoogle Scholar
  12. Chan RTH, Marcal H, Russel RA, Holden PJ, Foster LJR (2011) Application of polyethylene glycol to promote cellular biocompatibility of polyhydroxybutyrate films. Int J Polymer Sci 2011:1–9Google Scholar
  13. Choi J, Lee SY (1999) Efficient and economical recovery of poly(3-hydroxybutyrate) from recombinant Escherichia coli by simple digestion with chemicals. Biotechnol Bioeng 62:546–553CrossRefPubMedGoogle Scholar
  14. Di Donato P, Fiorentino G, Anzelmo G, Tommonaro G, Nicolaus B, Poli A (2011) Re-use of vegetable wastes as cheap substrates for extremophile biomass production. Waste Biomass Valor 2:103–111CrossRefGoogle Scholar
  15. Dussault HP (1955) An improved technique for staining red halophilic bacteria. J Bacteriol 70:484–485PubMedCentralPubMedGoogle Scholar
  16. Gonzalez C, Gutierrez C, Ramirez C (1978) Halobacterium vallismortis sp. nov. An amylolytic and carbohydrate-metabolizing, extremely halophilic bacterium. Can J Microbiol 24:710–715CrossRefPubMedGoogle Scholar
  17. Grant WD, Kamekura M, McGenity TJ, Ventosa A (2001) Order Halobacteriales. In: Boone DR, Castenholz RW, Garrity GM (eds) Bergey’s manual of systematic bacteriology, 2nd edn. Springer, New York, pp 294–334Google Scholar
  18. Gursel I, Hasirci V (1995) Properties and drug release behavior of PHB and various PHBV copolymer microcapsules. J Microencapsul 12:185–193CrossRefPubMedGoogle Scholar
  19. Gursel I, Yagmurlu F, Korkusuz F, Hasırcı V (2002) In vitro antibiotic release from poly(3-hydroxybutyrate-co-3-hydroxyvalerate) rods. J Microencapsul 19:153–164CrossRefGoogle Scholar
  20. Han J, lu Q, Zhou L, Zhou J, Xiang H (2007) Molecular characterization of the phaECHm genes, required for biosynthesis of poly(3-hydroxybutyrate) in the extremely halophilic archaeon Haloarcula marismortui. Appl Environ Microbiol 73:6058-6065Google Scholar
  21. Han J, Hou J, Liu H, Cai S, Feng B, Zhou J, Xiang H (2010) Wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type III synthases. Appl Environ Microbiol 76:7811–7819PubMedCentralCrossRefPubMedGoogle Scholar
  22. Hasirci V (2000) Biodegradable biomedical polymers: a review of degradation of and in vivo response to polylactides and polyhydroxyalkanoates. In: Wise DL (ed) Biomaterials and bioengineering handbook Marcel Dekker Inc, New York, pp 141–155Google Scholar
  23. Hong K, Sun S, Tian W, Chen GQ, Huang W (1999) A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy. Appl Microbiol Biotechnol 51:523–526CrossRefGoogle Scholar
  24. Huang TY, Duan KJ, Huang SY, Chen CW (2006) Production of polyhydroxyalkanoates from inexpensive extruded rice bran and starch by Haloferax mediterranei. J Ind Microbiol Biotechnol 33:701–706CrossRefPubMedGoogle Scholar
  25. Kassab AC, Xu K, Denkbas EB, Dou Y, Zhao S, Piskin E (1997) Rifampicin carrying polyhydroxybutyrate as a potential chemoembolization agent. J Biomater Sci Polymer Edn 8:947–961CrossRefGoogle Scholar
  26. Koller M, Hesse P, Bona R, Kutschera C, Atlic A, Braunegg G (2007) Potential of various archae- and eubacterial strains as industrial polyhydroxyalkanoate producers from whey. Macromol Biosci 7:218–226CrossRefPubMedGoogle Scholar
  27. Koller M, Altic A, Gonzales-Garcia Y, Kutschera C, Braunegg G (2008) Polyhydroxyalkanoate (PHA) biosynthesis from whey lactose. Macromol Symp 272:87–92CrossRefGoogle Scholar
  28. Law JH, Slepecky RA (1961) Assay of poly-β-hydroxybutyric acid. J Bacteriol 82:33–36PubMedCentralPubMedGoogle Scholar
  29. Lee SY (1996) Bacterial polyhydroxyalkanoates. Biotechnol Bioeng 49:1–14CrossRefPubMedGoogle Scholar
  30. Lee SY, Yim KS, Chang HN, Chang YK (1994) Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli. J Biotechnol 32:203–211CrossRefPubMedGoogle Scholar
  31. Lillo JG, Rodriguez-Valera F (1990) Effects of culture conditions on poly(β-hydroxybutyric acid) production by Haloferax mediterranei. Appl Environ Microbiol 56:2517–2521PubMedCentralPubMedGoogle Scholar
  32. Lu Q, Han J, Zhou L, Zhou J, Xiang H (2008) Genetic and biochemical characterization of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthase in Haloferax mediterranei. J Bacteriol 190:4173–4180PubMedCentralCrossRefPubMedGoogle Scholar
  33. Luengo J, Garcia B, Sandoval A, Naharro G, Oliver E (2003) Bioplastics from microorganisms. Current Opin Biotechnol 6:51–260Google Scholar
  34. Margesin R, Schinner F (2001) Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles 5:13–83CrossRefGoogle Scholar
  35. Ogan A, Danis O, Gozuacik A, Cakmar E, Birbir M (2012) Production of cellulase by immobilized whole cells of Haloarcula. Appl Biochem Microbiol 48:440–443CrossRefGoogle Scholar
  36. Oren A (1994) Characterization of the halophilic archaeal community in saltern crystallizer ponds by means of polar lipid analysis. Int J Salt Lake Res 3:15–29CrossRefGoogle Scholar
  37. Oren A, Ventosa A, Grant WD (1997) Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47:233–238CrossRefGoogle Scholar
  38. Poli A, Di Donato P, Abbamondi GR, Nicolaus B (2011) Synthesis, production, and biotechnological applications of exopolysaccharides and polyhydroxyalkanoates by archaea. Archaea 2011:1–13CrossRefGoogle Scholar
  39. Quesada E, Ventosa A, Valera FR, Cormenzana AR (1982) Types and properties of some bacteria isolated from hypersaline soils. J Appl Bacteriol 53:155–161CrossRefGoogle Scholar
  40. Quillaguamán J, Hashim S, Bento F, Mattiasson B, Hatti-Kaul R (2005) Poly(β-hydroxybutyrate) production by a moderate halophile, Halomonas boliviensis LC1 using starch hydrolysate as substrate. J Appl Microbiol 99:151–157CrossRefPubMedGoogle Scholar
  41. Quillaguamán J, Delgado O, Mattiasson B, Hatti-Kaul R (2006) Poly(β-hydroxybutyrate) production by a moderate halophile, Halomonas boliviensis LC1. Enzyme Microb Tech 38:148–154CrossRefGoogle Scholar
  42. Quillaguamán J, Guzmán H, Van-Thuoc D, Hatti-Kau R (2010) Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects. Appl Microbiol Biotechnol 85:1687–1696CrossRefPubMedGoogle Scholar
  43. Reddy CSK, Ghai R, Kalia VC (2003) Polyhydroxyalkanoates: an overview. Bioresour Technol 87:137–149CrossRefPubMedGoogle Scholar
  44. Rodriguez-Valera F, Lillo AG (1992) Halobacteria as producers of polyhydroxyalkanoates. FEMS Microbiol Rev 103:181–186CrossRefGoogle Scholar
  45. Rodriguez-Valera F, Juez G, Kushner DJ (1983) Halobacterium mediterranei spec. nov., a new carbohydrate-utilizing extreme halophile. Syst Appl Microbiol 4:369–381CrossRefPubMedGoogle Scholar
  46. Savenkova L, Gercberga Z, Nikolaeva V, Dzene A, Bibers I, Kalnin M (2000) Mechanical properties and biodegradation characteristics of PHB-based films. Process Biochem 35:573–579CrossRefGoogle Scholar
  47. Steinbüchel A, Aerts K, Babel W, Folner C, Leibergesell M, Wieczorek R (1995) Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. Can J Microbiol 41(Suppl. 1):94–105CrossRefPubMedGoogle Scholar
  48. Tian J, He A, Lawrence AG, Liu P, Watson N, Sinskey AJ, Stubble J (2005) Analysis of transient polyhydroxybutyrate production in Wautersia eutropha H16 by quantitative Western Analysis and transmission electron microscopy. J Bacteriol 187:3825–3832PubMedCentralCrossRefPubMedGoogle Scholar
  49. Van-Thuoc D, Huu-Phong T, Thi-Binh N, Thi-Tho N, Minh-Lam D, Quillaguamán J (2012) Polyester production by halophilic and halotolerant bacterial strains obtained from mangrove soil samples located in Northern Vietnam. Microbiologyopen 1:395–406PubMedCentralCrossRefPubMedGoogle Scholar
  50. Verlinden RAJ, Hill DJ, Kenward MA, Williams CD, Radecka I (2007) Bacterial synthesis of biodegradable polyhydroxyalkanoates. J Appl Microbiol 102:1437–1449CrossRefPubMedGoogle Scholar
  51. Vreeland RH (1993) Taxonomy of Halophilic Bacteria. In: Vreeland RH, Hochstein LI (eds) The biology of halophilic bacteria. CRS Press, Baco Raton, pp 115–134Google Scholar
  52. Vroman I, Tighzert L (2009) Biodegradable polymers. Materials 2:307–344CrossRefGoogle Scholar

Copyright information

© Springer Japan 2015

Authors and Affiliations

  • Ozkan Danis
    • 1
  • Ayse Ogan
    • 1
  • Pınar Tatlican
    • 1
  • Azade Attar
    • 2
  • Emrah Cakmakci
    • 1
  • Bulent Mertoglu
    • 3
  • Meral Birbir
    • 1
  1. 1.Faculty of Arts and SciencesMamara UniversityIstanbulTurkey
  2. 2.Faculty of Chemical and Metallurgical EngineeringYıldız Technical UniversityIstanbulTurkey
  3. 3.Faculty of EngineeringMarmara UniversityIstanbulTurkey

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