Sains Malaysiana 48(12)(2019): 2663–2673

http://dx.doi.org/10.17576/jsm-2019-4812-07

Polyphasic Identification of Amylolytic Bacteria Producing Bioplastic Poly-β-hydroxybutyrate (PHB)

(Pengenalpastian Polifasa Bakteria Amilolisis Menghasilkan Bioplastik Poli-ß-hidroksibutirat (PHB))

NUR ARFA YANTI¹*, L. SEMBIRING², S. MARGINO³, NURHAYANI H. MUHIDDIN⁴, SITTI WIRDHANA AHMAD¹ & ARDIANSYAH¹

¹Department of Biology, Faculty of Mathematics and Natural Science, Halu Oleo University, Kendari Southeast Sulawesi, Indonesia

²Faculty of Biology, Gadjah Mada University, Yogyakarta, Indonesia

³Faculty of Agricultural, Gadjah Mada University, Yogyakarta, Indonesia

⁴Faculty of Mathematics and Natural Science, Makassar State University, Makassar, South Sulawesi, Indonesia

Received: 27 April 2019/Accepted: 25 September 2019

ABSTRACT

The final goal of this study is to make a modern systematic-based inventory of amylolytic bacterial isolates producing of bioplastic Poly-β-hydroxybutyrate (PHB) from sago starch substrate. The identity of three local bacterial isolates was examined in this study, using a polyphasic approach. A data set based on phenotypic characteristics, namely morphological, physiological, biochemical and chemical character, namely whole cells protein profiles using SDS-PAGE method, together with phylogenetic studies based on 16S rRNA sequences was used to identified by polyphasic approach. Phenotypic characteristics of 3 local bacterial isolates and 4 reference strains to members of genus Bacillus was analyzed by numerical analysis using MVSP 3,1 program to determine the value of similarity. Based on the preliminary characterization of the profile matching method showed that the three isolates of bacteria producing PHB namely PSA10, PPK5 and PPK6 are members of the genus Bacillus. The results of numerical analysis based on phenotypic characteristic and chemical character of the three bacterial isolates producing PHB with reference strains showed that the PSA10 isolate bacterial identical with Bacillus megaterium, PPK5 isolate identical with Bacillus subtilis and PPK6 isolate identical with Bacillus cereus, and these results also support by the molecular phylogenetic analysis. Therefore, the polyphasic taxonomy is an effective approach to uncover the identity of the novel bacterial isolates.

Keywords: Amylolytic bacteria; bioplastic; numeric-phenetic; PHB; polyphasic

ABSTRAK

Matlamat akhir kajian ini adalah untuk membuat inventori berasaskan sistematik moden daripada pencilan bakteria amilolisis yang menghasilkan bioplastik Poli-ß-hidroksibutirat (PHB) daripada substrat kanji sagu. Identiti tiga pencilan bakteria tempatan telah diperiksa dalam kajian ini, menggunakan pendekatan polifasa. Satu set data berdasarkan ciri-ciri fenotip, iaitu sifat morfologi, fisiologi, biokimia dan kimia, iaitu profil protein sel keseluruhan menggunakan kaedah SDS-PAGE, bersama-sama dengan kajian filogenetik berdasarkan urutan 16S rRNA digunakan untuk mengenal pasti dengan pendekatan polifasa. Ciri-ciri fenotip 3 pencilan bakteria tempatan dan 4 rujukan kepada ahli genus Bacillus dianalisis dengan analisis berangka menggunakan MVSP 3,1 program untuk menentukan nilai keserupaan. Berdasarkan pencirian permulaan kaedah pencocokan profil menunjukkan bahawa tiga pencilan bakteria yang menghasilkan PHB iaitu PSA10, PPK5 dan PPK6 adalah ahli genus Bacillus. Hasil analisis berangka berdasarkan pencirian fenotip dan sifat kimia ketiga pencilan bakteria yang menghasilkan PHB dengan strain rujukan menunjukkan bahawa PSA10 seiras dengan Bacillus megaterium, PPK5 seiras dengan Bacillus subtilis dan PPK6 seiras dengan Bacillus cereus, dan keputusan ini juga disokong oleh analisis filogenetik molekul. Oleh itu, taksonomi polifasa adalah pendekatan yang berkesan untuk mendedahkan identiti novel pencilan bakteria.

Kata kunci: Bakteria amilolisis; bioplastik; numerik-fenetik; PHB; polifasa

REFERENCES

Apun, K., Jong, B.C. & Salleh, M.D. 2000. Screening and isolation of a cellulolytic and amylolytic Bacillus from sago pith waste. J. Gen. App. Microbiol. 46: 263-267.

Aremu, M.O., Layokun, S.K. & Solomon, B.O. 2010. Production of poly(3-hydroxybutyrate) from cassava starch hydrolysate by Pseudomonas aeruginosa NCIB 950. American J. Scientific and Industrial Research 1(3): 421-426.

Berber, I. 2004. Characterization of Bacillus species by numerical analysis of their SDS-PAGE protein profiles. J. Cell Mol. Biol. 3: 33-37.

Berber U. & E. Yenidunya. 2005. Identification of alkaliphilic Bacillus species isolated from Lake Van and its surroundings by computerized analysis of extracellular protein profiles. Turk. J. Biol. 29: 181-188.

Blackwood, K.S., Turenne, C.Y., Harmsen, D. & Kabani, A.M. 2004. Reassessment of sequence-based targets for identification of Bacillus species, J. Clinical Microbiology 42(4): 1626-1630.

Borsodi, A.K., Makk, J., Rusznya´k, A., Vajna, B., Taba, G. & Ma´rialigeti, K. 2007. Phenotypic characterization and molecular taxonomic studies on Bacillus and related isolates from Phragmites australis periphyton. Aquatic Botany 86: 243-252.

Chee, J.Y., Yoga, S.S., Lau, N.S., Ling, S.C., Abed, R.M.M. & Sudesh, R. 2010. Bacterially produced polyhydroxyalkanoate (PHA): Converting renewable resources into bioplastics. In Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, edited by Méndez-Vilas, A. Spain: Formatex Research Center. pp. 1395-1404.

Daroonpunt, R., Tanaka, N., Uchino, M. & Tanasupawat, S. 2018. Characterization and screening of lipolytic bacteria from Thai fermented fish. Sains Malaysiana 47(1): 91-97.

Espino, T.M. & Tambalo, R.D. 1997. Isolation, screening and characterization of high yielding α-amylase producing bacteria. Annual Reports of IC Biotech. 20: 744-754.

Felsenstein, J. 1995. Phylip (Phylogeny Inference Package) Version 3.57c. Washington: University of Washington.

Ghazi, F., Henni, D.E., Benmechernene & Kihal, M. 2009. Phenotypic and whole cell protein analysis by SDS-PAGE for identification of dominants lactic acid bacteria isolated from Algerian raw milk. World J. of Dairy and Food Sciences 4(1): 78-87.

Halami, P.M. 2008. Production of polyhydroxyalkanoate from starch by the native isolate Bacillus cereus CFR06. World J. Microbiol. and Biotechnol. 24: 805-812.

Holt, J.G., Krieg, N.R., Sneath, P.H.A., Stanley, J.T. & Williams, S.T. 1994. Bergey's Manual of Determinative Bacteriology. 9th ed. Baltimore: Lipincot, Williams and Wilkins.

Hong, Y., Yang, H.S., Li, J., Han, S.K., Chang, H.C. & Kim, H.Y. 2014. Identification of lactic acid bacteria in salted Chinese cabbage by SDS-PAGE and PCR-DGGE, J. Sci. Food Agric. 30(2): 296-300.

Jamili, Yanti, N.A. & Susilowati, P.E. 2016a. Diversity and the role of yeast in spontaneous cocoa bean fermentation from Southeast Sulawesi, Indonesia. Biodiversitas 17(1): 90-95.

Jamili, Yanti, N.A. & Susilowati, P.E. 2016b. The indigenous lactic acid bacteria from fermented cocoa bean and its role in cocoa bean fermentation. Research Journal of Pharmaceutical, Biological and Chemical Sciences 7(5): 2268-2276.

Kovach Computing Service. 1990. Multi Variate Statistical Package, Version 3.1, Users' Manual.: http://www.kovcomp. com//.

Lenz, R.W. & Marchessault, R.H. 2005. Bacterial polyesters: Biosynthesis, biodegradable plastics and biotechnology. Biomacromolecules 6(1): 1-8.

Lekota, K.E., Hassim, A., Mafofo, J., Rees, J., Muchadeyi, C.F., Van Heerden, H. & Madoroba, E. 2016. Polyphasic characterization of Bacillus species from anthrax outbreaks in animals from South Africa and Lesotho. The Journal of Infection in Developing Countries 10(8): 814-823.

Logan, N.A., Forsyth, G., Lebbe, L., Goris, J., Heyndrickx, M., Balcaen, A., Verhelst, A., Falsen, E., Ljungh, A. Hansson, H.B. & De Vos, P. 2002. Polyphasic identification of Bacillus and Brevibacillus strains from clinical, dairy and industrial specimens and proposal of Brevibacillus invocatus sp. nov. Inter. J. Syst. Evol. Microbiol. 52: 953-966.

Mainville, I., Robert, N., Lee, B. & Farnworth, E.R. 2006. Polyphasic characterization of the lactic acid bacteria in kefir. Syst. and App. Microbiol. 29: 59-68.

Mishra, S. & Behera, N. 2008. Amylase activity of a starch degrading bacteria isolated from soil receiving kitchen wastes. African J. Biotech. 7(18): 3326-3331.

O'Donnel, A.G., Norris, J.R., Berkeley, R.C.W., Claus, D., Kaneko, T., Logan, N.A. & Nozak, R. 1980. Characterization of Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, and Bacillus amyloliquefaciens by pyrolysis gas-liquid chromatography, deoxyribonucleic acid-deoxyribonucleic acid hybridization, biochemical tests and API Systems. Inter. J. Syst. Bacteriol. 30(2): 448-459.

Ostle, A.G. & Holt, J.G. 1982. Nile blue A as a fluorescent stain for poly-β-hydroxybutyrate. Applied and Environmental Microbiology 44(1): 238-241.

Priest, F.G., Goodfellow, M. & Todd, C. 1988. A numerical classification of the genus Bacillus. J. Gen. Microbiol. 134: 1837-1882.

Randazzo, C.L., Scifo, G.O., Tomaselli, F. & Caggia, C. 2009. Polyphasic characterization of bacterial community in fresh cut salads. International Journal of Food Microbiology 128: 484-490.

Ray, S. & Kalia, V.C. 2016. Microbial cometabolism and polyhydroxyalkanoate co-polymers. Indian Journal of Microbiology 57(1): 39-47. DOI 10.1007/s12088-016- 0622-4.

Saitou, N. & Nei, M. 1987. The neighbour-joining method: A new method for reconstructing phylogenetic trees. Molecular Biol. Evol. 4: 406-426.

Santos, O.D., De Resende, M.C., De Mello, A.L., Frazzon, A.P. & D'Azevedo, P.A. 2012. The use of whole-cell protein profile analysis by SDS-PAGE as an accurate tool to identify species and subspecies of coagulase-negative staphylococci. APMIS 120(1): 39-46.

Sarkono, Moeljopawiro, S., Setiaji, B. & Sembiring, L. 2016. Analysis of whole cell protein profiles by SDSPAGE to identify indigenous celluloseproducer acetic acid bacteria. Indonesian Journal of Biotechnology 21(2): 86-92.

Scopes, R.K. 1987. Protein Purification. Principles and Practice. 2nd ed. New York: Springer Verlag.

Sembiring, L. 2009. Molecular phylogenetic classification of streptomycetes isolated from the rhizosphere of tropical legume (Paraserianthes falcataria) (L.) Nielsen. Hayati Journal of Biosciences 16(3): 100-108.

Shivakumar, S. 2012. Polyhydroxybutyrate (PHB) production using agro-industrial residue as substrate by Bacillus thuringiensis IAM 12077. Inter. J. ChemTech. Res. 4(3): 1158-1162.

Singh, M., Patel, K.S. &. Kalia, V.C. 2009. Bacillus subtilis as potential producer for polyhydroxyalkanoates. Microbial Cell Factories 8: 38-49.

Smibert, R.M. & Krieg, N.R. 1994. General characterization. In Manual of Methods for General Bacteriology, edited by Gerhardt, P., Murray, R.G.E., Wood, W.A. & Krieg, N.R. Washington D.C.: American Society for Microbiology.

Slepecky, R.A. &. Hemphill, H.E. 2006. The genus Bacillus-Non medical. Prokaryotes 4: 530-562.

Shivalkar, Y.K. & Prabha, R. 2017. Polyhydroxybutyrate as bio-degradable plastic - A review. IOSR Journal of Environmental Science, Toxicology and Food Technology 11(1): 10-12.

Song, J., Lee, S.C., Kang, J.W., Baek, H.J. & Suh, J.W. 2004. Phylogenetic analysis of Streptomyces spp. isolated from potato scab lesions in Korea on the basis of 16S rRNA gene and 16S-23S rDNA internally transcribed spacer sequences. Inter. J. Syst. Evol. Microbiol. 54: 203-209.

Vandamme, P., Pot, B., Gillis, M., De Vos, P., Kersters, K. & Swings. J. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiological Rev. 60(2): 407-438.

Vauterin, L., Swings, J. & Priest, F.G. 1993. Protein electrophoresis and classification. In Handbook of New Bacterial Systematics, edited by Goodfellow, M. & O'Donnell, A.G. London: Academic Press Ltd. pp. 252-280.

Visnuvardhan, R.S., Thirumala, M., Kishore, R.T.V. & Mahmood, S.K. 2008. Isolation of bacteria producing polyhydroxyalkanoates (PHA) from municipal sewage sludge. World J. Microbiol. Biotechnol. 10: 1-7.

Wang, W. & Sun, M. 2009. Phylogenetic relationships between Bacillus species and related genera inferred from 16S rDNA sequences. Braz. J. Microbiol. 40: 505-521.

Yanti, N.A., Sembiring, L., Margino, S. & Muhiddin, N.H. 2013. A study on production of poly-β-Hydroxybutyrate bioplastic from sago starch by indigenous amylolytic bacteria. Indonesian J. Biotech. 18(2): 144-150.

Yanti, N.A., Sembiring, L. & Margino, S. 2009. Amilolytic bacteria producing of the raw material of bioplastic (in Indonesian). Berk. Penel. Hayati. 3C: 95-99.

*Corresponding author; email: nur.yanti@uho.ac.id