Characterization of newly isolated thermotolerant bacterium Cupriavidus sp. CB15 from composting and its ability to produce polyhydroxyalkanoate from glycerol

Abstract

Background: This study aimed to isolate a novel thermotolerant bacterium that is capable of synthesizing polyhydroxyalkanoate from glycerol under high temperature conditions.

Results: A newly thermotolerant polyhydroxyalkanoate (PHA) producing bacterium, Cupriavidus sp. strain CB15, was isolated from corncob compost. The potential ability to synthesize PHA was confirmed by detection of PHA synthase (phaC) gene in the genome. This strain could produce poly(3-hydroxybutyrate) [P(3HB)] with 0.95 g/L (PHA content 75.3 wt% of dry cell weight 1.24 g/L) using glycerol as a carbon source. The concentration of PHA was enhanced and optimized based on one-factor-at-a-time (OFAT) experiments and response surface methodology (RSM). The optimum conditions for growth and PHA biosynthesis were 10 g/L glycerol, 0.78 g/L NH₄Cl, shaking speed at 175 rpm, temperature at 45 °C, and cultivation time at 72 h. Under the optimized conditions, PHA production was enhanced to 2.09 g/L (PHA content of 74.4 wt% and dry cell weight of 2.81 g/L), which is 2.12-fold compared with non-optimized conditions. Nuclear magnetic resonance (NMR) analysis confirmed that the extracted PHA was a homopolyester of 3-hydyoxybutyrate.

Conclusion: Cupriavidus sp. strain CB15 exhibited potential for cost-effective production of PHA from glycerol.

Keywords: Cupriavidus sp.; Glycerol; Polyhydroxyalkanoate; Thermotolerant bacterium.

Fig. 1

PHA production by isolate strains. Each isolate was cultivated in a mineral salt medium containing 1% (w/v) glycerol at 45 °C for 72 h

Fig. 2

Morphology of Cupriavidus sp. CB15. A Morphology of the CB15 on Nutrient agar, B Nile Red staining under UV light, C Gram staining of the CB15 at 100× magnification, and D Morphology of the CB15 on MacConkey agar

Fig. 3

A Phylogenetic tree of the CB15 based on partial 16S rDNA sequences. B PCR amplification of phaC gene using G-D and G2-R primers. Genomic DNA of Cupriavidus necator H16 was used as a positive control. The tree was constructed by the neighbor-joining method in the bootstrap test (1000 replicates). Bar = 20% sequence divergence. Pseudomonas putida strain KT2440 was taken as outgroup for tree formation

Fig. 4

Optimization of PHA production by Cupriavidus sp. CB15 under submerged fermentation. CB15 was cultured in mineral salt medium containing 1% (w/v) glycerol at 45 °C for 72 h with shaking at 200 rpm. A Inoculum age, B Glycerol concentration, C Nitrogen sources, D NH₄Cl concentration, E pH, and F Shaking speed. Dry cell weight (filled with dark bar), PHA production (filled with gray bar), and PHA content (wt%) (dark triangle up) were examined. Data shown are the means and standard deviations of triplicate experiments

Fig. 5

3D surface and contour plots showing the interaction effect of A, D NH₄Cl concentration and temperature; B, E NH₄Cl concentration and shaking speed; C, F Temperature and shaking speed on DCW by Cupriavidus sp. CB15

Fig. 6

3D surface and contour plots showing the interaction effect of A, D NH₄Cl concentration and temperature; B, E NH₄Cl concentration and shaking speed; C, F Temperature and shaking speed on PHA production by Cupriavidus sp. CB15

Fig. 7

Effect of cultivation time on cell growth and PHA production. Cupriavidus sp. CB15 was cultivated in a 100 mL Erlenmeyer flask under optimal conditions. Dry cell weight (green triangle up), PHA concentration (blue square), PHA content (wt%) (red circle), residual cell weight (g/L) (pink star), glycerol concentration (g/L) (black diamond), and ammonium nitrogen concentration (mg/L) (purple triangle down) were examined. Data shown are the means and standard deviations of triplicate experiments

Fig. 8

500 MHz ¹H NMR spectrum of A P(3HB) produced by Cupriavidus sp. CB15 and B P(3HB) standard (Sigma-Aldrich, USA)