Isolation of Bacillus cereus C042 capable of producing polyhydroxybutyrate by using  native rice bran waste materials from rice bran oil production as carbon source

Wimol Chobchuenchom

Authors

Wimol Chobchuenchom Faculty of Medical Technology, Rangsit University, Patumthani 12000, Thailand

Keywords:

polyhydroxyalkanoates (PHAs), biopolymer, native rice bran waste materials

Abstract

A replacement of polyhydroxyalkanoates (PHAs) for existing petroleum-based plastics is limited due to their high production cost. Thus, increased production of PHAs on cheap substrates is an important challenge to support the commercialization. Normally, the pre-treatment of cheap substrates is required which results in increased costs and time. In this study, native rice bran waste materials from rice bran oil production, using the cold press method which generates various fatty acids, were used as the main carbon source for poly(3-hydroxybutyrate) (P(3HB)) production. Fifty soil samples obtained from various locations in Thailand, including industrial sites, refuse sites, garden and community areas, were screened for PHAs-producing microorganisms. Each soil sample was diluted in distilled water and the soil suspension was poured on MSA agar supplemented with 3%(w/v) native rice bran waste materials. The microorganisms that grew on the medium were further screened for PHAs production by using Sudan Black B staining. The production of P(3HB) was confirmed by using Gas Chromatography-Mass Spectrometry (GC-MS) by comparing to standard P(3HB). The P(3HB) content was measured by using GC and percentage of P(3HB) content per cell dry weight was calculated. It was found that 55 of 336 isolates (16.4%) which grew were positive for PHAs. The isolate designated C042 showed the highest potential for PHAs production and was used for further experiments. Biopolymer was extracted from C042 dried cells by using chloroform. From GC-MS analysis, it was found that biopolymer was P(3HB) by comparing the mass spectrum to standard P(3HB). The isolate C042 was gram positive bacilli with spore forming abilities and was further identified as Bacillus cereus by using 16S RNA sequencing method. The percentage of P(3HB) production per cell dry weight of Bacillus cereus C042 was 11.6(w/w) when the cells were grown on MSA agar supplemented with 21%(w/v) native rice bran waste materials.

References

Anil Kumar, P. K., Shamala, T. R., Kshama, L., Prakash, M. H., Joshi, G. J., Chandrashekar, A., … Divyashree, M. S. (2007). Bacterial synthesis of poly(hydroxybutyrate- co-hydroxyvalerate) using carbohydrate-rich mahua (Madhuca sp.) flowers. Journal of Applied Microbiology, 103(1), 204–209. DOI: 10.1111/j.1365-2672.2006.03221.x

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), 1-11. DOI: 10.1186/s40643-014-0009-5

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. DOI: 10.1016/j.biortech.2009.03.088

Chaudhry, W. N., Jamil, N., Ali, I., Ayaz, M. H., & Hasnain, S. (2011). Screening for polyhydroxyalkanoate (PHA)-producing bacterial strains and comparison of PHA production from various inexpensive carbon sources. Annals of Microbiology, 61(3), 623–629. DOI: 10.1007/s13213-010-0181-6

Chee, J.-Y., Tan, Y., Samian, M.-R., & Sudesh, K. (2010). Isolation and characterization of a Burkholderia sp. USM (JCM15050) capable of producing polyhydroxyalkanoate (PHA) from triglycerides, fatty acids and glycerols. Journal of Polymers and the Environment, 18(4), 584–592. DOI: 10.1007/s10924-010-0204-1

Chen, G. Q., Wu, Q., Wang, Y. W., & Zheng, Z. (2005). Application of microbial polyesters-polyhydroxyalkanoates as tissue engineering materials. Key Engineering Materials, 288–289, 437–440. DOI: 10.4028/www.scientific.net/KEM.288-289.437

Choi, J., & Lee, S. Y. (1999). Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation. Applied Microbiology and Biotechnology, 51(1), 13–21. DOI: 10.1007/s002530051357

Gamal, R. F., Abdelhady, H. M., Khodair, T. A., El-Tayeb, T. S., Hassan, E. A., & Aboutaleb, K. A. (2013). Semi-scale production of PHAs from waste frying oil by Pseudomonas fluorescens S48. Brazilian Journal of Microbiology, 44(2), 539–549. DOI: 10.1590/S1517-83822013000200034

Halami, P. M. (2008). Production of polyhydroxyalkanoate from starch by the native isolate Bacillus cereus CFR06. World Journal of Microbiology and Biotechnology, 24(6), 805–812. DOI: 10.1007/s11274-007-9543-z

Huang, T.-Y., Duan, K.-J., Huang, S.-Y., & Chen, C. W. (2006). Production of polyhydroxyalkanoates from inexpensive extruded rice bran and starch by Haloferax mediterranei. Journal of Industrial Microbiology & Biotechnology, 33(8), 701–706. DOI: 10.1007/s10295-006-0098-z

Jiun-Yee, C., Yifen, T., Mohd-Razip, S., & Kumar, S. (2010). Isolation and characterization of a Burkholderia sp. USM (JCM15050) capable of producing polyhydroxyalkanoate (PHA) from triglycerides, fatty acids and glycerols. Journal of Polymers and the Environment, 18(4), 584-592. DOI: 10.1007/s10924-010-0204-1

Khanna, S., & Srivastava, A. K. (2005). Recent advances in microbial polyhydroxyalkanoates. Process Biochemistry, 40(2), 607–619. DOI: 10.1016/j.procbio.2004.01.053

Morgan-Sagastume, F., Valentino, F., Hjort, M., Cirne, D., Karabegovic, L., Gerardin, F., … Werker, A. (2014). Polyhydroxyalkanoate (PHA) production from sludge and municipal wastewater treatment. Water Science & Technology, 69(1), 177-184. DOI: 10.2166/wst.2013.643

Ngozi, O. P., & Adeniyi, O. (2012). Characterization of polyhydroxyalkanoate (PHA) produced by Bacillus species isolated from garden soil. New York Science Journal, 5(12), 159-163. DOI: 10.7537/marsnys051212.26

Queirós, D., Rossetti, S., & Serafim, L. S. (2014). PHA production by mixed cultures: a way to valorize wastes from pulp industry. Bioresource Technology, 157, 197–205. DOI: 10.1016/j.biortech.2014.01.099

Sangkharak, K., & Prasertsan, P. (2012). Screening and identification of polyhydroxyalkanoates producing bacteria and biochemical characterization of their possible application. The Journal of General and Applied Microbiology, 58(3), 173–182. DOI: 10.2323/jgam.58.173

Saranya Devi, E., Vijayendra, S. V. N., & Shamala, T. R. (2012). Exploration of rice bran, an agro-industry residue, for the production of intra- and extra-cellular polymers by Sinorhizobium meliloti MTCC 100. Biocatalysis and Agricultural Biotechnology, 1(1), 80–84. DOI: 10.1016/j.bcab.2011.08.014

Shamala, T. R., Vijayendra, S. V. N., & Joshi, G. J. (2012). Agro-industrial residues and starch for growth and co-production of polyhydroxyalkanoate copolymer and a -amylase by Bacillus sp. CFR-67. Brazilian Journal of Microbiology, 43(3), 1094–1102. DOI: 10.1590/S1517-83822012000300036

Singh, M., Patel, S. K., & Kalia, V. C. (2009). Bacillus subtilis as potential producer for polyhydroxyalkanoates. Microbial Cell Factories, 8(1), 38-48. DOI: 10.1186/1475-2859-8-38

Valappil, S. P., Peiris, D., Langley, G. J., Herniman, J. M., Boccaccini, A. R., Bucke, C., & Roy, I. (2007). Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon sources by a newly characterized Bacillus spp. Journal of Biotechnology, 127(3), 475–487. DOI: 10.1016/j.jbiotec.2006.07.015

Valappil, S. P., Rai, R., Bucke, C., & Roy, I. (2008). Polyhydroxyalkanoate biosynthesis in Bacillus cereus SPV under varied limiting conditions and an insight into the biosynthetic genes involved. Journal of Applied Microbiology, 104(6), 1624–1635. DOI: 10.1111/j.1365-2672.2007.03678.x

Wiggam, M. I., O’Kane, M. J., Harper, R., Atkinson, A. B., Hadden, D. R., Trimble, E. R., & Bell, P. M. (1997). Treatment of diabetic ketoacidosis using normalization of blood 3-hydroxybutyrate concentration as the endpoint of emergency management. A randomized controlled study. Diabetes Care, 20(9), 1347–1352.