Production of biopolyester poly(3-hydroxybutyrate) by Bacillus cereus RCL 02, a leaf endophyte of Ricinus communis L.

  • Rituparna Das Microbiology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700 019, West Bengal, India
  • Arundhati Pal Post Graduate Department of Botany, Serampore College, 9, William Carey Road, Serampore, Hooghly 712 201, West Bengal, India
  • Amal K. Paul Microbiology Laboratory, Department of Botany, University of Calcutta,35, Ballygunge Circular Road, Kolkata 700 019, West Bengal, India


Endophytic bacteria colonizing the internal tissues of plants have attracted the attention of scientific communities in recent years for production of biodegradable polyesters like polyhydroxyalkanotaes (PHAs). A newly characterized bacterium, Bacillus cereus RCL 02 (GenBank accession no. KX458035), isolated from surface sterilized leaves of Ricinus communis L. has been explored for the production of poly(3-hydroxybutyrate) [P(3HB)], the most common PHA. As revealed by scanning electron microscopy, P(3HB) accumulating cells developed swellings or blebs and released the native granules as a function of autolysis. During growth in glucose containing mineral salts medium under batch fermentation, the isolate produced P(3HB) accounting 68% of its cell dry weight (CDW). Glucose and yeast extract when used in the ratio of 5:1, significantly influenced intracellular biopolyester accumulation (72.2%, CDW and 2.54 g/L). A further increase of polymer production (81%, CDW and 3.17 g/L) was accomplished in presence of 1.5 mM manganese as exogenous metal stress. Moreover, supplementation of the growth medium with non-conventional carbon sources especially refined sugarcane molasses further enhanced the production of both biomass (9.44 g/L) as well as polyester (83.6%, CDW and 7.89 g/L). These finding emphasises exploration of endophytic bacteria of oleaginous plants in general and R. communis L. in particular as potential but hitherto an under exploited bioresource for commercial production of biodegradable polyesters.


Steinbuchel A. Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example. Macromolecular Bioscience. 2001; 1(1): 1-24.

Castro-Sowinski S, Burdman S, Matan O, Okon Y. Natural functions of bacterial polyhydroxyalkanoates, in: Chen GQ (Eds.), Plastics from bacteria. Springer, Berlin, Heidelberg. 2010; pp 39-61.

Strobel G, Daisy B, Castillo U, Harper J. Natural products from endophytic microorganisms. Journal of Natural Products. 2004; 67(2): 257-68.

Catalan AI, Ferreira F, Gill PR, Batista S. Production of polyhydroxyalkanoates by Herbaspirillum seropedicae grown with different sole carbon sources and on lactose when engineered to express the lacZ lacY genes. Enzyme and Microbial Technology. 2007; 40(5): 1352-7.

Kamnev AA, Antonyuk LP, Tugarova AV, Tarantilis PA, Polissiou MG, Gardiner PHE. Fourier transform infrared spectroscopic characterization of heavy metal-induced metabolic changes in the plant-associated soil bacterium Azospirillum brasilense Sp7. Journal of Molecular Structure. 2002; 610(1-3): 127-31.

Das R, Pal A, Mandal S, Paul AK. Screening and production of biodegradable polyester poly(3-hydroxybutyrate) by bacteria endophytic to Brassica nigra L. British Biotechnology Journal. 2015; 7(3): 134-46.

Das R, Dey A, Pal A, Paul AK. Influence of growth conditions on production of poly(3-hydroxybutyrate) by Bacillus cereus HAL 03 endophytic to Helianthus annuus L. Journal of Applied Biology and Biotechnology. 2016; 4(4): 75-84.

Rajkumar M, Freitas H. Influence of metal resistant plant growth-promoting bacteria on the growth of Ricinus communis in soil contaminated with heavy metal. Chemosphere. 2008; 71(5): 834-42.

Singh B, Thakur A, Kaur S, Chadha BS, Kaur A. Acetylcholinesterase inhibitory potential and insecticidal activity of an endophytic Alternaria sp. from Ricinus communis. Applied Biochemistry and Biotechnology. 2012; 168(5): 991-1002.

Ryu HW, Hahn SK, Chang YK, Chang HN. Production of poly (3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phosphate limitation. Biotechnology and Bioengineering. 1997; 55(1): 28-32.

Wang F, Lee SY. 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. 1997; 63(9): 3703-6.

Fidler S, Dennis D. Polyhydroxyalkanoate production in recombinant Escherichia coli. FEMS Microbiology Letters. 1992; 103(2-4): 231-5.

Macrae RM, Wilkinson JF. Poly-β-hydroxybutyrate metabolism in washed suspension of Bacillus cereus and Bacillus megaterium. Journal of General Microbiology. 1958; 19(1): 210-22.

Chen GQ. Occurrence of poly-D(-)-3-hydroxyalkanoates in the genus Bacillus. FEMS Microbiology Letters. 1991; 84(2): 173-6.

Wu Q, Huang H, Hu G, Chen J, Ho KP, Chen GQ. Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media. Antonie van Leeuwenhoek. 2001; 80(2): 111-8.

Valappil SP, Misra SK, Boccaccini AR, Keshavarz T, Bucke C, Roy I. Large-scale production and efficient recovery of PHB with desirable material properties, from the newly characterized Bacillus cereus SPV. Journal of Biotechnology. 2007; 132(3): 251-8.

Fukui T, Doi Y. Effcient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain. Applied Microbiology and Biotechnology. 1998; 49(3): 333-6.

Lee WH, Loo CY, Nomura CT, Sudesh K. Biosynthesis of polyhydroxyalkanoate copolymers from mixtures of plant oils and 3-hydroxyvalerate precursors. Bioresource Technology. 2008; 99(15): 6844-51.

Gouda MK, Swellam AE, Omar SH. Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources. Microbiological Research. 2001; 156(3): 201-7.

Ramadas NV, Singh SK, Soccol CR, Pandey A. Polyhydroxybutyrate production using agro-industrial residue as substrate by Bacillus sphaericus NCIM 5149. Brazilian Archives of Biology and Technology. 2009; 52(1): 17-23.

Mothes G, Schnorpfeil C, Ackermann JU. Production of PHB from crude glycerol. Engineering in Life Sciences. 2007; 7(5): 475-9.

Getachew A, Woldesenbet F. Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low cost agricultural waste material. BMC Research Notes. 2016; 9(1): 509.

Ramsay BA, Lomaliza K, Chavarie C, Dube B, Bataille P, Ramsay JA. Production of poly-(β-hydroxybutyric-co-β-valeric) acids. Applied and Environmental Microbiology. 1990; 56(7): 2093-8.

Law JH, Slepeckey RA. Assay of Polyhydroxybutyric acid. Journal of Bacteriology. 1961; 82(1): 32-6.

Benoit TG, Wilson GR, Baugh CL. Fermentation during growth and sporulation of Bacillus thuringiensis HD-1. Letters in Applied Microbiology. 1990; 10(1): 15-18.

Kominek LA, Halvorson HO. Metabolism of poly-β-hydroxybutyrate and acetoin in Bacillus cereus. Journal of Bacteriology. 1965; 90(5): 1251-9.

Page WJ. Production of poly-β-hydroxybutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources. Applied Microbiology and Biotechnology. 1992; 38(1): 117.

Narayanan A, Ramana KV. Polyhydroxybutyrate production in Bacillus mycoides DFC1 using response surface optimization for physico-chemical process parameters. 3 Biotech. 2012; 2(4): 287-96.

Gomma EZ. Production of polyhydroxyalkanoates (PHAs) by Bacillus subtilis and Escherichia coli grown on cane molasses fortified with ethanol. Brazilian Archives of Biology and Technology. 2014; 57(1): 145-54.

Biswas A, Paul AK. Synthesis and accumulation of polyhydroxyalkanoic acids by Halomonas maura HMA 102 isolated from thalassohaline environment. International Journal of Applied Biotechnology and Biochemistry. 2012; 2(2): 137-51.

Kamnev AA, Tugarova AV, Antonyuk LP. Endophytic and epiphytic strains of Azospirillum brasilense respond differently to heavy metal stress. Microbiology. 2007; 76(6): 809-11.

Pal A, Paul AK. Accumulation of polyhydroxyalkanoates by rhizobacteria underneath nickel-hyperaccumulators from serpentine ecosystem. Journal of Polymers and the Environment. 2012; 20(1): 10-16.

Beaulieu M, Beaulieu Y, Melinard J, Pandian S, Goulet J. Influence of ammonium salts and cane molasses on growth of Alcaligenes eutrophus and production of polyhydroxybutyrate. Applied and Environmental Microbiology. 1995; 61(1): 165-9.