Strains, growth media, and culture condition
A soil sample from Changbai Mountain (altitude 2183 m, N 42°08′19.7″, E 128°10′8.3″), Jilin Province, China, was suspended in sterile H2O, vortexed thoroughly, and cultured in an enriched medium containing 5 g/L yeast extract, 10 g/L tryptone, and 2.5 g/L NaCl at 20°C for 2 days. A 50-μL aliquot was spread on PHB detection agar (20 g/L glucose, 2 g/L (NH4)2SO4, 13.3 g/L KH2PO4, 1.2 g/L MgSO4
.7H2O, 1.7 g/L citric acid, 1.7 g/L trace elements solution, 15 g/L agar, and 0.5 μg/mL Nile blue A (Spiekermann et al., 1999). After overnight incubation at 20°C, plates were observed under ultraviolet (UV) light. Fluorescent colonies were purified by streaking on the same agar plates, and the isolates were further characterized by staining with Sudan Black B (Lee and Choi, 2004).
Cell morphology was examined using transmission electron microscopy as follows: a 2-mL aliquot of cells in late exponential phase (optical density OD600 55–80) was fixed with 4% glutaraldehyde and 1% osmium tetroxide and dehydrated by sequential treatment with 30%, 50%, 70%, 80%, 90%, and 100% acetone. The dehydrated cells were immersed in epoxy resin and then transferred into sample boats. The resin was polymerized for 24 h at 30, 45, and 60°C. Ultra-thin sections were prepared using an ultramicrotome, stained sequentially with uranyl acetate and lead citrate for 22 min and 5 min, respectively, and observed using an H-7650 transmission electron microscope (Hitachi, Tokyo, Japan).
Biolog Gram-negative (GN) assay
Gram staining was performed as described by Magee et al. (1975). A pure culture was grown on a BUG agar plate (Biolog Catalog #70101). Biolog™ GN 96-well microtiter plates contain 95 different carbon sources, a negative control, and a tetrazolium dye (Biolog Inc., Hayward, CA, USA). Cells were swabbed from the surface of the plate and suspended to a specific density in GN Inoculating Fluid (Biolog Catalog #72101). The well of the Biolog GN2 MicroPlate (Biolog Catalog #1011) was inoculated individually with 150 μL of diluted suspensions and then incubated at 30°C for 24 h (Garland and Mills, 1991). The MicroPlate was inspected either visually or analyzed using the Biolog MicroStation™ and the results compared with the GN III Database according to the manufacturer’s instructions (Biolog Catalog #22730D).
Polymerase chain reaction (PCR) amplification of the 16S rRNA gene
The 16S rRNA gene was amplified using PCR with the universal bacterial primers 27F (5′-agagttgatcctggctcag-3′) and 1492R (5′-ggytaccttgttacgactt-3′) (Lane, 1991), and the product was sequenced by Takara Corp. (Dalian, China). The sequence was compared with 16S rRNA gene sequences in the GenBank database (http://www.ncbi.nlm.nih.gov) using the BLAST algorithm (Altschul et al., 1990). A neighbor-joining tree was constructed using the MEGA 4.0 program (Kumar et al., 2004).
Cells were harvested and treated with 10% sodium dodecyl sulfate at 100°C for 15 min. After centrifugation at 10,000 rpm for 20 min, the pellets were washed twice with H2O, dried at 40°C, and extracted with chloroform for 1 h at 60°C. Insoluble material was removed by filtration, and the soluble PHB was separated from the chloroform phase by evaporation, washed twice with methanol, filtered, dried at 60–70°C, and weighed using an electronic balance (Sartorius, BSA224S).
Nuclear magnetic resonance (NMR) spectroscopy
The NMR spectra of 1-mL samples were recorded using a BRUAK AV-400 spectrometer with a 5-mm 1H-probe, and deuterated chloroform (CDCl3) was used as a solvent at a final concentration of 10 g/L. The 1H-NMR spectrum for PHB was recorded at 400 MHz. A PHB standard was purchased from Sigma-Aldrich (St. Louis, MO, USA, #MSDS 363502).
Effect of culture conditions on cell growth
The effects on cell growth of carbon and nitrogen sources, C/N ratio, temperature, initial pH, and NaCl concentration were investigated using single-factor tests (Tang and Luo, 2008). Specifically, yeast extract, glucose, sucrose, fructose, glycerol, and ethanol were used as carbon source. Nitrogen sources included tryptone, peptone, beef extract, monosodium glutamate, urea, and NH4Cl. All media contained 1.0 g/L KH2PO4, 2.5 g/L Na2HPO4, pH 7.0, and cells were cultured at 20°C, 200 rpm. To test the effect of the C/N ratio, sucrose and monosodium glutamate served as the sole carbon and nitrogen sources and were added at the C/N ratios as follows: 1, 5, 10, 15, 20, 25, and 30. Other conditions were the same as those described above. The optimum C/N ratio was used to test the conditions described next. NaCl concentrations were varied from 0–50 g/L (5-g/L intervals), and at initial pH values from 5.0 to 9.0 (0.5 pH unit intervals). Cultures were incubated from 5°C to 40°C (5°C intervals). After 3 days, cells were collected by centrifugation, and the pellets were dried at 70°C to a constant weight.
PHB production by aerated cultures
Cells was cultured at 20°C in medium containing 1.0 g/L KH2PO4, 2.5 g/L Na2HPO4, with optimal 1% (w/v) sucrose as the carbon source and monosodium glutamate as the nitrogen source. The pH was adjusted to 7.0 with 0.5 M NaOH. A 10-mL inoculum from a CBS-1 starter culture (OD600 0.5) was added to a 150-mL minimal medium. The 250-mL flasks were incubated at 20°C for at least 3 days and shaken at 200 rpm. Samples were collected at 8-h intervals from 0–72 h, centrifuged, and dried at 70°C to a constant weight.
All results are shown as the average and standard deviation of at least three independent experiments. Student’s t-test, analysis of variance (ANOVA), and Duncan’s multiple range test were used to determine the statistical significance of differences in the nitrite-to-nitrogen conversion rate. Statistical significance was defined as P < 0.05. All analyses were performed using SPSS for Windows version 11.0.