Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon sources by a newly characterized Bacillus spp.
Introduction
Polyhydroxyalkanoates are polyesters of 3-, 4-, 5- and 6-hydroxyalkanoic acids, produced by a variety of bacterial species under nutrient-limiting conditions with excess carbon (Anderson and Dawes, 1990). These water-insoluble storage polymers are biodegradable, exhibit thermoplastic properties and can be produced from renewable carbon sources. The composition of the polymer synthesised is governed by two main factors, i.e. the bacterial strain being used and the carbon source utilized to grow the bacteria.
The material properties and hence the application of the PHAs vary depending on the monomer composition. Polyhydroxybutyrate, P(3HB), is the most well known and well characterized PHA. However, industrial applications of P(3HB) have been hampered owing to its low thermal stability and excessive brittleness upon storage (Lee, 1996). The copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), P(3HB-co-3HV), is more flexible and tougher than P(3HB). It can be used to make various products, including films, coated paper and board, compost bags, disposable food service ware and moulded products such as bottles and razors and in biomedical applications (Lee, 1996). Recently, the homopolymer of 4-hydroxybutyrate, P(4HB), has been found to be useful in the biomedical applications (Martin and Williams, 2003). P(4HB) was used for tissue engineered heart valve scaffold and viable ovine blood vessels (Chen and Wu, 2005). Also, a high molecular weight copolymer of 3HB and 4HB [P(3HB-co-4HB)] containing 0–100 mol% 4HB can be produced by Comamonas acidovorans with a controlled degradation rate (Saito and Doi, 1994), making them ideal candidates for biomedical applications such as tissue engineering (Martin and Williams, 2003). Another copolymer containing 3HB and 3-hydroxyhexanoate (3HHx), P(3HB-co-3HHx), has been produced by recombinant E. coli containing the PHA biosynthetic genes from Ralstonia eutropha (Dennis et al., 1998). P(3HB-co-3HHx) containing 10–17 mol% of 3HHx fractions possess much higher stretching ability (850%) than P(3HB-co-3HV) containing 20 mol% of 3HV (Doi et al., 1995).
P(3HB) was first identified and isolated from the genus Bacillus (Lemoigne, 1926). In 1958 Macrae and Wilkinson, 1958, reported that P(3HB) served as an intracellular carbon and energy reserve in Bacillus cereus and Bacillus megaterium. Slepecky and Law, 1961, further determined the role of the polymer in sporulation in B. megaterium. Species of the genus Bacillus produce a range of different PHAs. B. cereus produces 3HB-rich polymers containing 2–4-mol% of 3HHx units and a terpolymer containing 3HHx and 3-hydroxyoctanoate (3HO) units (Caballero et al., 1995). Also, B. cereus UW85 when fed with ɛ-caprolactone, produces a tercopolymer with 3HB, 3HV and 3HHx (Labuzek and Radecka, 2001). Bacillus spp. INT005 produces P(3HB), P(3HB-co-3HV), P(3HB-co-3HHx) and P(3HB-co-6HHx-co-3HHx) from different carbon sources (Tajima et al., 2003). This indicates that the genus Bacillus has the potential for the production of novel, yet to be discovered, PHAs and a range of mole compositions of already known copolymers of PHAs.
This paper reports the identification and characterization of a newly acquired PHA producing Bacillus spp. using microbial and molecular techniques. PHA production has been attempted using a novel medium and conditions which prevent degradation of the PHA produced have been explored. Further, the ability of this new strain of Bacillus spp. to utilize a variety of different carbon sources and produce a range of different PHAs has been studied. Finally, all the polymers produced have been characterized.
Section snippets
Chemicals
All chemicals were obtained from Sigma–Aldrich Company Ltd. (Dorset, England) except yeast extract which is from DIFCO (BD UK Ltd., Oxford, UK). GC and GC–MS analyses were performed using analytical grade reagents. FT-IR grade KBr was used for the FT-IR analysis.
Bacterial strain
The Bacillus spp. was obtained from the University of Westminster, London, UK, culture collection. Stock cultures were grown at 30 °C in nutrient broth [containing (g L−1): ‘Lab-Lemco’ powder 1.0; yeast extract, 2.0; peptone, 5.0; sodium
Species identification of the PHA-producing Bacillus spp.
Various strains of Bacillus obtained from the University of Westminster culture collection were initially screened for the production of PHA using Nile Blue A analysis as described previously (Law and Slepecky, 1961). Of the Bacillus spp. investigated the one used for this work exhibited the strongest orange fluorescence compared to others and was selected for further studies. Genomic DNA isolated from the Bacillus spp. was used for amplification of the 16S rRNA gene by PCR at NCIMB, Aberdeen,
Discussion
A PHA producing Bacillus spp. was identified to be a B. cereus using molecular and microbial tests and was named B. cereus SPV. B. cereus SPV was able to use a large variety of carbon sources such as sugars and fatty acids for PHA production, a great advantage for the commercial production of the polymer. The Kannan and Rehacek medium used in this work has for the first time been successfully used for PHA production by Bacillus. Also, the study of the time dependence of P(3HB) production by B.
Acknowledgements
We thank Dr. Helen Hails, University College London, UK for her discussion on the mechanism of o-methylation during the methanolysis reaction of 4HB, prior to the GC–MS analysis. We thank Dr. Colin Bedford, University of Westminster, UK for his help in carrying out the methanolysis reaction. This work was supported by the EPSRC, UK grant no. EP/C515617/1. S.P. Valappil was also financially supported by a scholarship from the University of Westminster, London, UK.
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