Skip to main content
Log in

Fed-Batch Strategies for Production of PHA Using a Native Isolate of Halomonas venusta KT832796 Strain

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

In this study, polyhydroxyalkanoates (PHA) accumulation by Halomonas venusta KT832796, a moderate halophilic bacteria isolated from marine source was studied. Both nutritional requirements and process parameters for submerged cultivation of the organism in bioreactor have been standardized. From the shake flask studies, glucose and ammonium citrate as carbon and nitrogen source produced maximum PHA at a ratio 20 with 3.52 g/L of dry cell weight and 70.56% of PHA content. However, ammonium sulfate as the nitrogen source was found to be more suitable for fed-batch cultivation. Several feeding strategies including pH-based fed-batch and variants of pulse feeding were studied to improve the PHA levels. pH-based feeding, although improved PHA level to 26 g/L, most of the carbon flux was diverted towards biomass formation; hence, the percent PHA was only 39.15% of the dry cell weight. Maximum PHA of 33.4 g/L, which corresponded to 88.12% of the dry cell, was obtained from high concentration single pulse method. There was a net 8.65-fold increase in PHA using this feeding strategy when compared to batch studies. According to our knowledge, this is the highest amount of PHA reported for a Halomonas venusta strain.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

7References

  1. Oren, A. (2002). Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. Journal of Industrial Microbiology & Biotechnology, 28, 56–63.

    Article  CAS  Google Scholar 

  2. Ventosa, A., Nieto, J. J., & Oren, A. (1998). Biology of moderately halophilic aerobic bacteria. Microbiology and Molecular Biology Reviews, 62, 504–544.

    CAS  Google Scholar 

  3. DasSarma, S., & DasSarma, P. (2012). Halophiles. In Encyclopedia of life sciences. Chichester: John Wiley & Sons, Ltd.

    Google Scholar 

  4. Oren, A. (2010). Industrial and environmental applications of halophilic microorganisms. Environmental Technology, 31, 825–834.

    Article  CAS  Google Scholar 

  5. Gao, S., & Zhang, L. H. (2014). The synthesis of poly-β-Hydroxybutyrate by moderately Halophilic bacteria Halomonas venusta. Advanced Materials Research, 1033–1034, 306–310.

  6. García-Torreiro, M., Lu-Chau, T. A., & Lema, J. M. (2016). Effect of nitrogen and/or oxygen concentration on poly(3-hydroxybutyrate) accumulation by Halomonas boliviensis. Bioprocess and Biosystems Engineering, 39, 1365–1374.

    Article  Google Scholar 

  7. Koller, M., Atli, A., Dias, M., Reiterer, A., & Braunegg, G. (2010). Microbial PHA production from waste raw materials. In G. Q. Chen (Ed.), Plastics from bacteria: natural functions and applications, microbiology monographs (Vol. 14, pp. 85–119). Berlin: Springer- Verlag.

    Chapter  Google Scholar 

  8. Quillaguamán, J., Guzmán, H., Van-Thuoc, D., & Hatti-Kaul, R. (2010). Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects. Applied Microbiology and Biotechnology, 85(6), 1687–1696.

    Article  Google Scholar 

  9. Vijayendra, S. V. N., & Shamala, T. R. (2014). Film forming microbial biopolymers for commercial applications—a review. Critical Reviews in Biotechnology, 8551(4), 338–357.

    Article  Google Scholar 

  10. Ray, S., & Kalia, V. C. (2017). Biomedical applications of polyhydroxyalkanoates. Biomaterials from Nature for Advanced Devices and Therapies, 57(3), 261–269.

    CAS  Google Scholar 

  11. Berlanga, M., Miñana-galbis, D., Domènech, Ò., & Guerrero, R. (2012). Enhanced polyhydroxyalkanoates accumulation by Halomonas spp. in artificial biofilms of alginate beads. International Microbiology, 15, 191–199.

    CAS  Google Scholar 

  12. Quillaguam, J., Delgado, O., Mattiasson, B., & Hatti-kaul, R. (2006). Poly(β-hydroxybutyrate) production by a moderate halophile, Halomonas boliviensis LC1. Enzyme and Microbial Technology, 38, 148–154.

    Article  Google Scholar 

  13. Van-thuoc, D., Huu-phong, T., Thi-binh, N., Thi-tho, N., Minh-lam, D., & Quillaguaman, J. (2012). Polyester production by halophilic and halotolerant bacterial strains obtained from mangrove soil samples located in Northern Vietnam. Microbiology Open, 1(14), 395–406.

    Article  CAS  Google Scholar 

  14. Chen, C. W., Don, T., & Yen, H. (2006). Enzymatic extruded starch as a carbon source for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Haloferax mediterranei. Process Biochemistry, 41, 2289–2296.

    Article  CAS  Google Scholar 

  15. Tan, D., Xue, Y. S., Aibaidula, G., & Chen, G. Q. (2011). Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01. Bioresource Technology, 102(17), 8130–8136.

    Article  CAS  Google Scholar 

  16. Koller, M. (2017). Production of polyhydroxyalkanoate (PHA) biopolyesters by extremophiles. MOJ Polymer Science, 1(2), 1–19.

    Article  Google Scholar 

  17. Chen, X., Yin, J., Ye, J., Zhang, H., Che, X., Ma, Y., Li, M., Wu, L.-P., & Chen, G.-Q. (2017). Engineering Halomonas bluephagenesis TD01 for non-sterile production of poly(3-hydroxybutyrate- co -4-hydroxybutyrate). Bioresource Technology, 244, 534–541.

    Article  CAS  Google Scholar 

  18. Selvakumar, K., Srinivasan, G., Baskar, V., & Madhan, R. (2011). Production and isolation of polyhydroxyalkanoates from Haloarcula marismortui MTCC 1596 using cost effective osmotic lysis methodology. European Journal of Experimental Biology, 1(3), 180–187.

    CAS  Google Scholar 

  19. Salgaonkar, B. B., & Bragança, J. M. (2017). Utilization of sugarcane bagasse by Halogeometricum borinquense strain E3 for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Bioengineering, 4(50), 1–18.

    Google Scholar 

  20. Alsafadi, D., & Al-Mashaqbeh, O. (2017). A one-stage cultivation process for the production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) from olive mill wastewater by Haloferax mediterranei. New Biotechnology, 34, 47–53.

    Article  CAS  Google Scholar 

  21. Ienczak, J. L., De, G. M. F., Schmidell, W., & De Aragão, G. M. F. (2013). High-cell-density culture strategies for polyhydroxyalkanoate production: a review. Journal of Industrial Microbiology and Biotechnology, 40(3–4), 275–286.

    Article  CAS  Google Scholar 

  22. Sabbagh, F., & Idayu, I. (2017). Production of poly-hydroxyalkanoate as secondary metabolite with main focus on sustainable energy. Renewable and Sustainable Energy Reviews, 72, 95–104.

    Article  CAS  Google Scholar 

  23. Huschner, F., Grousseau, E., Brigham, C. J., Plassmeier, J., Popovic, M., Rha, C., & Sinskey, A. J. (2015). Development of a feeding strategy for high cell and PHA density fed-batch fermentation of Ralstonia eutropha H16 from organic acids and their salts. Process Biochemistry, 50(2), 165–172.

    Article  CAS  Google Scholar 

  24. 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 and Biosystems Engineering, 36, 1463–1474.

    Article  CAS  Google Scholar 

  25. Rodriguez-contreras, A., Koller, M., Dias, M. M. S., Calafell-Monfort, M., Braunegg, G., & Marques-Calvo, M. S. (2013). High production of poly( 3-hydroxybutyrate ) from a wild Bacillus megaterium Bolivian strain. Journal of Applied Microbiology, 114(5), 1378–1387.

    Article  CAS  Google Scholar 

  26. Ahn, W. S., Park, S. J., Lee, S. Y., Ahn, W. O. O. S. U. K., & Park, S. I. J. A. E. (2000). Production of poly( 3-Hydroxybutyrate ) by fed-batch culture of recombinant Escherichia coli with a highly concentrated whey solution. Applied and Environmental Microbiology, 66(8), 3624–3627.

    Article  CAS  Google Scholar 

  27. Garcia, A., Segura, D., Galindo, E., & Castillo, T. (2014). High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process. Biochemical Enginerring Journal, 82, 117–123.

    Article  CAS  Google Scholar 

  28. Shamala, T. R., Rohinishree, Y. S., & Vijayendra, S. V. N. (2014). Biosynthesis of multiple biopolymers by Sinorhizobium meliloti CFR 14 in high cell density cultures through fed batch fermentation. Biocatalysis and Agricultural Biotechnology, 3(4), 316–322.

    Article  Google Scholar 

  29. Kawata, Y., Ando, H., Matsushita, I., & Tsubota, J. (2014). Efficient secretion of (R)-3-hydroxybutyric acid from Halomonas sp. KM-1 by nitrate fed-batch cultivation with glucose under microaerobic conditions. Bioresource Technology, 156, 400–403.

    Article  CAS  Google Scholar 

  30. Quillaguamán, J., Doan-van, T., Guzman, H., Guzman, D., Martin, J., Everest, A., & Hatti-kaul, R. (2008). Poly(3-hydroxybutyrate) production by Halomonas boliviensis in fed-batch culture. Applied Microbiology and Biotechnology, 78, 227–232.

    Article  Google Scholar 

  31. Law, J. H., & Slepecky, R. A. (1961). Assay of poly-β-hydroxybutyric acid. Journal of Bacteriology, 82(1), 33–36.

    CAS  Google Scholar 

  32. Williamson, D. H., & Wilkinson, J. F. (1958). The isolation and estimation of the poly-β-hydroxy-butyrate inclusions of Bacillus species. Journal of General Microbiology, 19(1), 198–209.

    Article  CAS  Google Scholar 

  33. Mojaveryazdi, F. S., Muhamad, I. I., Rezania, S., & Benham, H. (2014). Importance of glucose and Pseudomonas in producing degradable plastics. Sciences & Engineering, 69(5), 7–10.

    Google Scholar 

  34. Quillaguamán, J., Muñoz, M., Mattiasson, B., & Hatti-kaul, R. (2007). Optimizing conditions for poly(β-hydroxybutyrate) production by Halomonas boliviensis LC1 in batch culture with sucrose as carbon source. Applied Microbiology and Biotechnology, 74, 981–986.

    Article  Google Scholar 

  35. Strazzullo, G., Gambacorta, A., Monica, F., Immirzi, B., Romano, I., Calandrelli, V., & Nicolaus, B. (2008). Chemical-physical characterization of polyhydroxyalkanoates recovered by means of a simplified method from cultures of Halomonas campaniensis. World Journal of Microbiology and Biotechnology, 24, 1513–1519.

    Article  CAS  Google Scholar 

  36. Van-Thuoc, D., Huu-Phong, T., Minh-Khuong, D., & Hatti-Kaul, R. (2015). Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by a moderate halophile Yangia sp. ND199 using glycerol as a carbon source. Applied Biochemistry and Biotechnology, 175(6), 3120–3132.

    Article  CAS  Google Scholar 

  37. Salgaonkar, B. B., & Braganca, J. M. (2015). Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Halogeometricum borinquense strain E3. International Journal of Biological Macromolecules, 78, 339–346.

    Article  CAS  Google Scholar 

  38. Salgaonkar, B. B., Mani, K., & Braganca, J. M. (2013). Characterization of polyhydroxyalkanoates accumulated by a moderately halophilic salt pan isolate Bacillus megaterium strain H16. Journal of Applied Microbiology, 114(5), 1347–1356.

    Article  CAS  Google Scholar 

  39. Kawata, Y., & Aiba, S. (2010). Poly(3-hydroxybutyrate) production by isolated Halomonas sp. KM-1 using waste glycerol. Bioscience, Biotechnology, and Biochemistry, 74(1), 175–177.

    Article  CAS  Google Scholar 

  40. Lee, S. Y. (1996). Bacterial polyhydroxyalkanoates. Biotechnology and Bioengineering, 49(1), 1–14.

    Article  CAS  Google Scholar 

  41. Page, W. J., & Cornish, A. (1993). Growth of Azotobacter vinelandii UWD in fish peptone medium and simplified extraction of poly-B-hydroxybutyrate. Applied and Environmental Microbiology, 59(12), 4236–4244.

    CAS  Google Scholar 

  42. Wang, F., Lee, S., & Y. (1997). Poly(3-hydroxybutyrate) production with high productivity and high polymer content by a fed-batch culture of Alcaligenes latus under nitrogen limitation. Applied and Environmental Microbiology, 63(9), 3703–3706.

    CAS  Google Scholar 

  43. Wei, Y., Chen, W., Huang, C., Wu, H., & Sun, Y. (2011). Screening and evaluation of polyhydroxybutyrate-producing strains from indigenous osolate Cupriavidus taiwanensis strains. International Journal of Molecular Sciences, 12, 252–265.

    Article  CAS  Google Scholar 

  44. Cui, Y., Shi, Y.-P., & Gong, X.-Y. (2017). Production of polyhydroxyalkanoates and extracellular polymeric substances by Haloferax mediterranei via kinetic model analysis. RSC Advances, 7, 18953–18961.

    Article  CAS  Google Scholar 

  45. Khanna, S., & Srivastava, A. K. (2005). Recent advances in microbial polyhydroxyalkanoates. Process Biochemistry, 40(2), 607–619.

    Article  CAS  Google Scholar 

  46. Rathi, D., Amir, H. G., Abed, R. M. M., Kosugi, A., Arai, T., Sulaiman, O., & Hashim, R. (2012). Polyhydroxyalkanoate biosynthesis and simplified polymer recovery by a novel moderately halophilic bacterium isolated from hypersaline microbial mats. Journal of Applied Microbiology, 114, 384–395.

    Article  Google Scholar 

  47. Kulkarni, S. O., Kanekar, P. P., Jog, J. P., Patil, P. A., Nilegaonkar, S. S., Sarnaik, S. S., & Kshirsagar, P. R. (2011). Characterisation of copolymer, poly(hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) produced by Halomonas campisalis (MCM B-1027), its biodegradability and potential application. Bioresource Technology, 102(11), 6625–6628.

    Article  Google Scholar 

  48. Simon-Colin, C., Raguénès, G., Cozien, J., & Guezennec, J. G. (2008). Halomonas profundus sp. nov., a new PHA-producing bacterium isolated from a deep-sea hydrothermal vent shrimp. Journal of Applied Microbiology, 104(5), 1425–1432.

    Article  CAS  Google Scholar 

  49. Kawata, Y., Shi, L. H., Kawasaki, K., & Shigeri, Y. (2012). Taxonomic characterization and metabolic analysis of the Halomonas sp. KM-1, a highly bioplastic poly(3-hydroxybutyrate)-producing bacterium. Journal of Bioscience and Bioengineering, 113(4), 456–460.

    Article  CAS  Google Scholar 

  50. Van-Thuoc, D., Quillaguama, J., Mamo, G., & Mattiasson, B. (2008). Utilization of agricultural residues for poly(3-hydroxybutyrate) production by Halomonas boliviensis LC1. Journal of Applied Microbiology, 104, 420–428.

    CAS  Google Scholar 

  51. Sarma, M. V. R. K., Gautam, A., Kumar, L., Saharan, K., Kapoor, A., Shrivastava, N., & Bisaria, V. S. (2013). Bioprocess strategies for mass multiplication of and metabolite synthesis by plant growth promoting pseudomonads for agronomical applications. Process Biochemistry, 48(9), 1418–1424.

    Article  CAS  Google Scholar 

  52. Berlanga, M., & Guerrero, R. (2016). Living together in biofilms: the microbial cell factory and its biotechnological implications. Microbial Cell Factories, 15, 1–11.

    Article  Google Scholar 

  53. Zhao, D., Cai, L., Wu, J., & Li, M. (2013). Improving polyhydroxyalkanoate production by knocking out the genes involved in exopolysaccharide biosynthesis in Haloferax mediterranei. Applied Microbiology and Biotechnology, 97(1), 3027–3036.

    Article  CAS  Google Scholar 

  54. Kim, B. S., Lee, S. C., Lee, S. Y., Chang, H. N., Chang, Y. K., & Woo, S. I. (1994). Production of poly(3-hydroxybutyric-co-3-hydroxyvaleric acid) by fed-batch culture of Alcaligenes eutrophus with substrate control using on-line glucose analyzer. Enzyme and Microbial Technology, 16(7), 556–561.

    Article  CAS  Google Scholar 

  55. Ryu, H. W., Hahn, S. K., Chang, Y. K., & Chang, H. N. (1997). Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation. Biotechnology and Bioengineering, 55(1), 28–32.

    Article  CAS  Google Scholar 

  56. Phong, T. H., Khuong, D. M., Hop, D. V., Thuoc, D. V., Giay, C., Cai, L., & Giay, C. (2017). Different fructose feedind strategies for poly(3-hydroxybutyrate) production by Yangia sp. ND199. Journal of Science and Technology, 55(2), 195–201.

    Google Scholar 

  57. Berlanga, M., Domènech, Ò., & Guerrero, R. (2014). Biofilm formation on polystyrene in detached vs. planktonic cells of polyhydroxyalkanoate-accumulating Halomonas venusta. International Microbiology, 17(4), 25–212.

    Google Scholar 

  58. Mozumder, M. S. I., De Wever, H., Volcke, E. I. P., & Garcia-Gonzalez, L. (2014). A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production. Process Biochemistry, 49(3), 365–373.

    Article  CAS  Google Scholar 

  59. Pham, T. H., Webb, J. S., & Rehm, B. H. A. (2004). The role of polyhydroxyalkanoate biosynthesis by Pseudomonas aeruginosa in rhamnolipid and alginate production as well as stress tolerance and biofilm formation. Microbiology, 150(10), 3405–3413.

    Article  CAS  Google Scholar 

  60. Rodríguez-Contreras, A., Koller, M., Miranda-de Sousa Dias, M., Calafell-Monfort, M., Braunegg, G., & Marqués-Calvo, M. S. (2013). High production of poly(3-hydroxybutyrate) from a wild Bacillus megaterium Bolivian strain. Journal of Applied Microbiology, 114(5), 1378–1387.

    Article  Google Scholar 

  61. García-torreiro, M., Lú-chau, T. A., Steinbüchel, A., & Juan, M. (2016). Waste to bioplastic conversion by the moderate halophilic bacterium Halomonas boliviensis. Chemical Engineering Transactions, 49, 163–168.

    Google Scholar 

  62. Suzuki, T., Yamane, T., & Shimizu, S. (1986). Kinetics and effect of nitrogen source feeding on production of poly-b-hydroxybutyric acid by fed-batch culture. Applied Microbiology and Biotechnology, 24, 366–369.

    Article  CAS  Google Scholar 

  63. Kulpreecha, S., Boonruangthavorn, A., Meksiriporn, B., & Thongchul, N. (2009). Inexpensive fed-batch cultivation for high poly(3-hydroxybutyrate ) production by a new isolate of Bacillus megaterium. Journal of Bioscience and Bioengineering, 107(3), 240–245.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to express their gratitude to the Director, CSIR-CFTRI for providing facilities to carry out the research work. Ms. Angelina would like to thank the funding agency of UGC-Maulana Azad National Fellowship for financially supporting the research work.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Sarma Mutturi or S. V. N Vijayendra.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Stanley, A., Punil Kumar, H.N., Mutturi, S. et al. Fed-Batch Strategies for Production of PHA Using a Native Isolate of Halomonas venusta KT832796 Strain. Appl Biochem Biotechnol 184, 935–952 (2018). https://doi.org/10.1007/s12010-017-2601-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-017-2601-6

Keywords

Navigation