Short CommunicationProduction of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) under photoautotrophy and heterotrophy by non-heterocystous N2-fixing cyanobacterium
Introduction
The microbial bioplastic poly-3-hydroxybutyrate (PHB) has tensile strength and thermal properties comparable to the petroleum-based plastic polypropylene; however, PHB has a much lower elongation of less than 6% length/length (Lee, 1996, Verlinden et al., 2007).
Superior levels of elongation and elasticity compared to those of PHB can be obtained from the co-polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV hereafter), which comprises 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV) monomers (Balaji et al., 2013, Verlinden et al., 2007). PHBV also melts at a lower temperature and exhibits less crystallinity relative to PHB (Lee, 1996, Verlinden et al., 2007). A wide variety of mole proportion (mol%) between HB and HV in PHBV resulted in diverse material properties suitable for various applications (Chanprateep, 2010, Lee, 1996).
PHBV has been commercially produced using heterotrophic bacteria that consume costly composite organic substrates (Chanprateep, 2010). Thus, a new PHBV producer that does not require organic substrates such as photosynthetic microbes is desirable. Still, PHBV production by phototrophic microbes has yet to be demonstrated.
Under photoautotrophy, several species of cyanobacteria produced PHB (Drosg et al., 2015, Kaewbai-Ngam et al., 2016, Koller and Maršálek, 2015). Increased PHB accumulation in cyanobacteria has been observed in cells deprived of combined inorganic nitrogen (–N) or phosphorus (–P) (Drosg et al., 2015, Kaewbai-Ngam et al., 2016, Koller and Maršálek, 2015). Previously, the photoautotrophic cyanobacterium Oscillatoria limosa was found to produce poly-3-hydroxyvalerate (PHV) (Stal et al., 1990). Nevertheless, production of the co-polymer PHBV by photoautotrophic cyanobacteria has yet to be described.
In this study, whether or not there is a cyanobacterial species capable of producing PHBV under photoautotrophy was investigated. The 137 strains of cyanobacteria were screened for bioplastic production under photoautotrophy (Kaewbai-Ngam et al., 2016). The selected Oscillatoria okeni TISTR 8549 (hereafter O. okeni) was found to produce PHBV. Here, PHBV production by O. okeni was monitored. PHBV was analyzed for chemical identity and material properties.
Section snippets
Strain and culture conditions
The axenic Oscillatoria okeni TISTR 8549 was obtained from Thailand Institute of Scientific and Technological Research. The strain was isolated using ampicillin treatment (Sena et al., 2011). Approximately 5% (v/v) of a 12-d old culture was inoculated into BG-11 medium with the omission of citrate and supplemented with 20 mM HEPES-NaOH (pH 7.5) as described (Monshupanee et al., 2016). The composition of the BG11 medium is given in Supplementary information Table S1. For photoautotrophy, cells
O. okeni required nitrate for rapid growth under photoautotrophy
O. okeni was cultured under photoautotrophy. Under the N2-fixing condition (cultured in BG11 medium without nitrate), low cell growth rate was obtained at 11 mg/L/d (d4-d32, Fig. 1A). An 8-fold higher cell growth rate (88 mg/L/d, d4-d12, Fig. 1A) was obtained under non-N2-fixing condition (cultured in BG11 medium with nitrate). Thus, nitrate is required for rapid growth under photoautotrophy. The culture time taken to achieve a maximal biomass production (1136 mg/L) was 20 d in a BG11 medium with
Conclusions
This work reported PHBV synthesis under photoautotrophy using carbon dioxide (CO2) as the sole carbon source and under heterotrophy using acetate (C2H3O2) as the organic substrate. This PHBV production occurred under nitrogen deprivation. Identification of the PHBV synthetic pathway is essential for metabolic engineering to enhance PHBV yield (such as that demonstrated by Osanai et al., 2013). Optimization of organic substrates in heterotrophic culture (such as that reported by Bhati and
Acknowledgements
This work was supported by Thailand Research Fund (TRF) (RSA5980016) and Ratchadaphiseksomphot Fund of Chulalongkorn University (CU) (NRU-59-046-AM). The authors thank 90th Anniversary Fund of CU (GCUGR1125572120M), CU research assistant fund, TRF institution fund (IRG5780008), TISTR and Miss Onuma Phoraksa.
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