Extraction and Characterization of Polyhydroxybutyrate (PHB) From Bacillus flexus MHO57386.1 Isolated From Marine Sponge Oceanopia arenosa (Rao, 1941)

Polyhydroxybutyrate (PHB) is the most widely studied biodegradable plastic that does not release any toxins or residues in the environment like petroleum based plastics. This work has been undertaken to screen PHB accumulating microorganisms from marine sponges and a total of sixteen isolates were collected and purified. Screening of isolated strains was done by Nile blue staining and observed under Leica LSCM to confirm the production of PHB. Yellow pigmented AB8a isolate from Oceanopia arenosa scored positive for PHB accumulation and subjected to morphological, biochemical and phylogenetic characterization. The biopolymer was extracted by dispersion of sodium hypochlorite and chloroform solution and characterized by FT-IR and 1H NMR for the confirmation as PHB. The highest PHB production (70.25%/100 ml) was achieved at pH 7.0 by applying dextrose as medium at incubation temperature 30°C and 150 rpm agitation speed. The FTIR spectrum of the PHB sample showed major peaks at 3457, 1692, 1550, 1454, 1420, 1190 and 1050 cm-1, whereas the remaining peaks are closely laid between 3450 cm-1 and 600 cm-1. 1H NMR spectrum of PHA isolated from dextrose media indicated characteristic signals of PHB. The spectrum also revealed the presence of three groups of signals characteristic of PHB by the doublet at 1.3 ppm attributed to the methyl group coupled to one proton; and the spectrum of the quadruplet at 2.57 ppm, the methylene group adjacent to an asymmetric carbon atom bearing a single proton and the multiplet at 5.28 ppm indicated signals of PHB. The PHB accumulated bacterium identified as Bacillus flexus strain based on characterization studies and 16S rRNA sequence analysis and confirmed the presence of intracellular accumulated polymer substantiated as PHB.

Keywords:

Bacillus flexus, Biopolymers, FTIR 1HNMR, Oceanopia arenosa,

PDF

___

Annandale, N. (1914). Fauna symbiotica indica. 5. Some sponges commonly associated with oysters and mussels in Madras Harbour and the Chilka Lake. Records of the Indian Museum, 10: 149-158. https://doi.org/10.5962/bhl.part.5625
Arun, A., Arthi, R., Shanmugabalaji, V. & Eyini, M. (2009). Microbial production of poly-beta-hydroxybutyrate by marine microbes isolated from various marine environments. Bioresource Technology, 100(7): 2320-2323. https://doi.org/10.1016/j.biortech.2008.08.037
Aslim, B., Yuksekdag, Z. & Beyatli, Y. (2002). Determination of PHB Growth Quantities of Certain Bacillus Species Isolated From Soil. Turkish Electronic Journal of Biotechnology, Special Issue, 24-30.
Balakrishna Pillai, A., Jaya Kumar, A., Thulasi, K. & Kumarapilla, H. (2017). Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai. Brazilian Journal of Microbiology, 48(3): 451-460. https://doi.org/10.1016/j.bjm.2017.01.005
Bauer, A. W., Kirby, W. M. M. & Turck, M. (1966). Antibiotic Susceptibility Testing by a Standardized Single Disk Method. American Journal of Clinical Pathology, 45(4_ts): 493-496. https://doi.org/10.1093/AJCP/45.4_TS.493
Baumann, P., Baumann, L., Mandel, M. & Allen, R. D. (1971). Taxonomy of marine bacteria: Beneckea nigrapulchrituda sp. n. Journal of Bacteriology, 108(3): 1380–1383. https://doi.org/10.1128/JB.108.3.1380-1383.1971
Beveridge, T. J. (2001).Use of the Gram stain in microbiology. Biotechnic & Histochemistry, 76(3): 111–118. https://doi.org/10.1080/bih.76.3.111.118
Bhuwal, A. K., Singh, G., Aggarwal, N. K., Goyal, V. & Yadav, A. (2014). Poly-β-hydroxybutyrate production and management of cardboard industry effluent by new Bacillus sp. NA10. Bioresources and Bioprocessing, 1(1): 9. https://doi.org/10.1186/s40643-014-0009-5
Bowerbank, J. S. (1864). A monograph of the British Spongidae, Vol 1. London: The Ray Society.
Castilho, L. R., Mitchell, D. A. & Freire, D. M. G. (2009). Production of polyhydroxyalkanoates (PHAs) from waste materials and by-products by submerged and solid-state fermentation. Bioresource Technology, 100(23): 5996-6009. https://doi.org/10.1016/j.biortech.2009.03.088
Chen, G. Q. (2009). A microbial polyhydroxyalkanoates (PHA) based bio and materials industry. Chemical Society Reviews, 38(8): 2434-2446. https://doi.org/10.1039/B812677C
Dendy, A. (1889). Report on a second collection of sponges from the Gulf of Manaar. Annals and Magazine of Natural History, 3(6): 73-99. https://doi.org/10.1080/00222938909460303
Dendy, A. (1905). Report on the sponges collected by Professor Herdman, at Ceylon, in 1902 (pp. 57-246). In: Herdman, W.A. (Ed.), Report to the Government of Ceylon on the Pearl Oyster Fisheries of the Gulf of Manaar. 3 (Supplement 18). (Royal Society: London).
Divyashree, M. S., Rastogi, N. K. & Shamala, T. R. (2009b). A simple kinetic model for growth and biosynthesis of polyhydroxyalkanoate in Bacillus flexus. New Biotechnology, 26(1-2): 92-98. https://doi.org/10.1016/j.nbt.2009.04.004
Divyashree, M. S., Shamala, T. R. & Rastogi, N. K. (2009a). Isolation of polyhydroxyalkanoate from hydrolyzed cells of Bacillus flexus using aqueous two-phase system containing polyethylene glycol and phosphate. Biotechnology and Bioprocess Engineering, 14: 482-489. https://doi.org/10.1007/s12257-008-0119-z
El-Sheekh, M. M., El-Abd, M. A. & El-Diwany, A. I. (2015). Poly-3 hydroxybutyrate production by Bacillus flexus ME-77 using some industrial wastes. Rendiconti Lincei, 26(2): 109-119. https://doi.org/10.1007/s12210-014-0368-z
Enkicknap, J. J., Kelly, M., Peraud, O. & Hill, R. T. (2006) Characterization of a culturable alphaproteobacterial symbiont common to many marine sponges and evidence for vertical transmission via sponge larvae. Applied and Environmental Microbiology, 72(5): 3724–3732. https://doi.org/10.1128/aem.72.5.3724-3732.2006
Flora, G., Bhatt, K. & Tuteja, U. (2010). Optimization of culture conditions for poly A-Hydroxybutyrate production from isolated Bacillus species. Journal of Cell and Tissue Research, 10(2): 2235-2242.
Gandhimathi, R., Arunkumar, M., Selvin, J., Thangavelu, T., Sivaramakrishnan, S., Kiran, G. S., Shanmughapriya, S. & Natarajaseenivasan, K. (2008). Antimicrobial potential of sponge associated marine actinomycetes. Journal of Medical Mycology, 18(1): 16-22. https://doi.org/10.1016/j.mycmed.2007.11.001
Getachew, A. & Woldesenbet, F. (2016). Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low cost agricultural waste material. BMC Research Notes, 9: 509. https://doi.org/10.1186/s13104-016-2321-y
Gopi, M., Kumaran, S., Kumar, T. T., Deivasigamani, B., Alagappan, K. & Prasad, S. G. (2012). Antibacterial potential of sponge endosymbiont marine Enterobacter sp. at Kavaratti Island, Lakshadweep archipelago. Asian Pacific Journal of Tropical Medicine, 5(2): 142-146. https://doi.org/10.1016/S1995-7645(12)60013-3
Gouda, M. K., Swellam, A. E. & Omar, S. H. (2001).Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon & nitrogen sources. Microbiological Research, 156(3): 201-207. https://doi.org/10.1078/0944-5013-00104
Grothe, E., Moo-Young, M. & Chisti, Y. (1999). Fermentation optimization for the production of poly-(B-hydroxybutyric acid) microbial thermoplastic. Enzyme and Microbial Technology, 25(1): 132–141.
Halami, P. (2007). Production of polyhydroxyalkanoate from starch by the native isolate Bacillus cereus CFR06. World Journal of Microbiology and Biotechnology, 24: 805–812. https://doi.org/10.1007/s11274-007-9543-z
Hall, T. A. (1999). BioEdit A User-Friendly Biological Sequence Alignment Editor & Analysis Program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41: 95-98.
Hooper, J. N. A. & Van Soest, R., W., M. (2002). Systema Porifera: A guide to the classification of sponges. Kluwer Academic/Plenum Publishers, New York, USA. 1-1099.
Hooper, J. N. A. (2003). Sponguide. Guide to sponge collection & identification. Queensl and Museum, Australia., 1-26. https://doi.org/10.1128/AEM.72.3.2118-2125.2006
Kalaivani, R. & Sukumaran, V. (2015). Enhancement of technique for optimized production of PHA from marine bacteria, utilizing cheaply available carbon sources at Thanjavur district, India. International Journal of Current Microbiology and Applied Sciences, 4(4): 408-417.
Kansiz, M., Jacobe, H. B. & McNaughton, D. (2000). Quantitative Determination of the Biodegradable Polymer Poly (β-hydroxybutyrate) in a Recombinant Escherichia coli Strain by Use of Mid-Infrared Spectroscopy and Multivariative Statistics. Applied and Environmental Microbiology, 66(8): 3415-3420. https://doi.org/10.1128/AEM.66.8.3415-3420.2000
Kim, T. K., Hewavitharana, A. K., Shaw P. N. & Fuerst J. A. (2006). Discovery of a new source of rifamycin antibiotic in marine sponge actinobacteria by phylogenetic prediction. Applied and Environmental Microbiology, 72(3): 2118-2125.
Kirk, R. G & Ginzburg, M. (1972). Ultrastructure of two species of halobacterium. Journal of Ultrastructure Research, 41(1-2): 80-94. https://doi.org/10.1016/S0022-5320(72)90040-8
Koller, M. (2018). Biodegradable and Biocompatible Polyhydroxy-alkanoates (PHA): Auspicious Microbial Macromolecules for Pharmaceutical and Therapeutic Applications. Molecules, 23(2): 362. https://doi.org/10.3390/molecules23020362
Koopmans, M., Rijswijk, P. V., Martens, D., Egovora- Zachernyuk, T. A. Middelburg, J. J. & Wijffels, R. H. (2011). Carbon conversion and metabolic rates in two marine sponges. Marine Biology, 158: 9-20. https://doi.org/10.1007/s00227-010-1538-x
Kreig, N. & Holt, J. (1984). Bergey’s manual of systematic Bacteriology. Williams and Wilkins, Baltimore.
Lie, J. & Zhou, J. (2002). A marine natural product database. Journal of Chemical Information and Computer Sciences, 42(3): 742-744. https://doi.org/10.1021/ci010111x
Lillie, R. D. (1977). H.J. Conn’s biological stains: A handbook on the nature and uses of the dyes employed in the biological laboratory. (9th ed.). The Williams and Wilkins Co., Baltimore.
Madison, L. & Huisman, G. (1999). Metabolic engineering of poly (3-hydroxyalkanoates): from DNA to plastic. Microbiology and Molecular Biology Reviews, 63(1): 21-53.
Montagu, G. (1814). An essay on sponges, with descriptions of all the species that have been discovered on the coast of Great Britain. Memoirs of the Wernerian Natural History Society, 2(1): 67-122.
Numata, K. & Morisaki, K. (2015). Screening of marine bacteria to synthesize polyhydroxyalkanoate from lignin: contribution of lignin derivatives to biosynthesis by Oceanimonas doudoroffii. ACS Sustainable Chemistry & Engineering, 3(4): 569-573. https://doi.org/10.1021/acssuschemeng.5b00031
Oliveira, F., Freire, D. & Castilho, L. (2004). Production of poly (3-hydroxybutyrate) by solid-state fermentation with Ralstonia eutropha. Biotechnology Letters, 26: 1851-1855. https://doi.org/10.1007/s10529-004-5315-0
Oliver, J. D. & Colwell, R. R. (1973). Extractable lipids of gram-negative marine bacteria: phospholipid composition. Journal of Bacteriology, 114(3): 897–908. https://doi.org/10.1128/JB.114.3.897-908.1973
Ostle, A. G. & Holt, J. G. (1982). Nile blue A as a fluorescent stain for poly-3-hydroxybutyrate. Applied and Environmental Microbiology, 44(1): 238-241. https://doi.org/10.1128/aem.44.1.238-241.1982
Poli, A., Di Donato, P., Abbamondi, G. R. & Nicolaus, B. (2011). Synthesis, production, and biotechnological applications of exopolysaccharides and polyhydroxyalkanoates by Archaea. Archaea, 2011: 693253. https://doi.org/10.1155/2011/693253
Poli, A., Finore, I., Romano, I., Gioiello, A., Lama, L. & Nicolaus, B. (2017). Microbial diversity in extreme marine habitats and their biomolecules. Microorganisms, 5(2): 25. https://doi.org/10.3390/microorganisms5020025
Rachanamol, R. S., Lipton, A. P., Thankamani, V., Sarika, A. R. & Selvin, J. (2014). Molecular characterization and bioactivity profile of the tropical sponge associated with bacterium Shewanella algae VCDB. Helgoland Marine Research, 68: 263-269. https://doi.org/10.1007/s10152-014-0386-3
Ramadas, N., Singh, S., Soccol, C. & Pandev, A. (2009). Polyhydroxybutyrate production using agro-industrial residue as substrate by Bacillus sphaericus NCIM 5149. Brazilian Archives of Biology and Technology, 52(1): 17-23. https://doi.org/10.1590/S1516-89132009000100003
Rao, H. S. (1941). Indian and Ceylon sponges of the Naturhistoriska Riksmuseet, Stockholm, collected by K. Fristedt. Rec. Indian Museum, 43: 417-469.
Rehm, B. H. A. (2010). Bacterial polymers: biosynthesis, modifications and applications. Nature Reviews Microbiology, 8: 578-92. https://doi.org/10.1038/nrmicro2354
Ridley, S. O. & Dendy, A. (1886). Preliminary report on the Monaxonida collected by H.M.S. Challenger. Part I. Annals & Magazine of Natural History, 18: 325-351, 470-493.
Ridley, S. O. (1884). Spongiida. In: Report on the Zoological Collections made in the Indo-Pacific Ocean during the Voyage of H.M.S. ‘Alert’, 1881-2. (British Museum (Natural History): London). 366-482, 582-630.
Rohini, D., Phadni, S. & Rawal, S. K. (2006). Synthesis & characterization of poly beta hydroxybutyrate from Bacillus thuringiensis. Indian Journal of Biotechnology, 5: 276-283.
S. E., El-Shiek, H. H., Elabd, M. A. & Shehab, A. M. (2014). Screening, optimization and extraction of polyhydroxyalkanoates from Bacillus thuringienesis. Journal of Advances in Biology & Biotechnology, 1(1): 40-54. https://doi.org/10.9734/JABB/2014/12286
Saharan, B. S., Grewal, A. & Kumar, P. (2014). Biotechnological Production of Polyhydroxyalkanoates: A review on trends and latest developments. Chinese Journal of Biology, 2014: 802984. https://doi.org/10.1155/2014/802984
Selvakumar, D. & Dhevendaran, K. (2016). Antagonistic activity of marine sponges associated Actinobacteria. Journal of Coastal Life Medicine, 4(6): 465-474. https://doi.org/10.12980/JCLM.4.2016J6-17
Selvin, J., Thangavelu, T., Kiran, G. S., Gandhimathi, R. & Priya, S. S. (2009). Culturable heterotrophic bacteria from the marine sponge Dendrilla nigra: isolation & phylogenetic diversity of action bacteria. Helgoland Marine Research, 63: 239-247. https://doi.org/10.1007/s10152-009-0153-z
Shah, K. R. (2012). FT-IR analysis of polyhydroxyalkanoates by novel Bacillus sp. AS 3-2 from soil of Kadi region, North Gujarat, India. Journal of Biochemical Technology, 3(4): 380-383.
Singh, P. & Parmar, N., (2011). Isolation and characterization of two novel polyhydroxybutyrate (PHB) - producing bacteria. African Journal of Biotechnology, 10(24): 4907-4919.
Singla, N., Bansal, N., Gupta, V. & Chander, J. (2013). Outbreak of Salmonella typhi enteric fever in sub-urban area of North India: a public health perspective. Asian Pacific Journal of Tropical Medicine, 6: 167-168. https://doi.org/10.1016/S1995-7645(13)60017-6
Sivaprakasam, S., Mahadevan, S., Sekar, S. & Rajakumar, S. (2008). Biological treatment of tannery wastewater by using salt-tolerant bacterial strains. Microbial Cell Factories, 7: 15. https://doi.org/10.1186/1475-2859-7-15
Spiekermann, P., Rehm, B. & Kalscheuer, R. (1999). A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Archives of Microbiology, 71: 73-80. https://doi.org/10.1007/s002030050681
Tamdogan, N. & Sidal, U. (2011). Investigation of Poly-β-Hydroxybutyrate (PHB) Production by Bacillus subtilis ATCC 6633 under different conditions. Kafkas Universitesi Veteriner Fakultesi Dergisi, 17(Supplement A): S173-S176. https://doi.org/10.9775/kvfd.2011.2474
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30: 2725-2729. https://doi.org/10.1093/molbev/mst197
Thomas, P. A. (1986). Demospongiae of the Gulf of Mannar and Palk Bay (pp. 205-365). In: James, P. S. B. R. (Ed.), Recent Advances in Marine Biology. New Delhi, India: Today and Tomorrow Printers and Publishers.
Valappil, S., Misra, S. & Boccaccini, A. (2007). Large-scale production & efficient recovery of PHB with desirable material properties, from the newly characterized Bacillus cereus SPV. Journal of Biotechnology, 132: 251–258. https://doi.org/10.1016/j.jbiotec.2007.03.013
Velho-Pereira, S. & Furtado, I. (2012). Antibacterial activity of halophilic bacterial bionts from marine invertebrates of Mandapam, India. Indian Journal of Pharmaceutical Sciences, 74(4): 331-338. https://doi.org/10.4103/0250-474x.107065
Wei, Y., Chen, W., Huang, C., Wu, H., Sun, Y., Lo, C. & Janarthanan, O. (2011). Screening and Evaluation of Polyhydroxybutyrate- Producing Strains from Indigenous Isolate Cupriavidus taiwanensis Strains. International Journal of Molecular Sciences, 12(1): 252-265. https://doi.org/10.3390/ijms12010252