Article Figures & Data
Figures
- Fig. 1.
(a) Growth and increase in optical density during second-step cultivation of H. pseudoflava on γ-butyrolactone (2 ml/liter) as a sole carbon source in a two-step procedure. Zero time was the beginning of the second-step cultivation. (b) Time course of 4HB monomer incorporation during the second-step cultivation of H. pseudoflava on γ-butyrolactone (2 ml/liter) as a sole carbon source in a two-step procedure. Zero time was the beginning of the second-step cultivation. A660, absorbance at 660 nm.
- Fig. 2.
Dyad sequence distribution in the C13 NMR carbonyl absorption region of the 3HB-4HB (5 mol% 3HB–95 mol% 4HB) polymer synthesized from γ-butyrolactone during two-step cultivation of H. pseudoflava. The 3HB unit was designated 3, and the 4HB unit was designated 4.
- Fig. 3.
125-MHz 13C NMR spectra of P(4HB) homopolymer (A) and P(3HB) homopolymer (B).
- Fig. 4.
Stimulation of intracellular P(3HB) degradation inH. pseudoflava by ammonium sulfate (1.0 g/liter). The cells initially contained 49% (wt/wt) P(3HB) homopolymer.
- Fig. 5.
Putative metabolic pathway for PHA synthesis from γ-butyrolactone in H. pseudoflava. The intracellular degradability of P(3HB-co-4HB) copolymers depends on the 3HB/4HB ratio of the copolyesters. TCA, tricarboxylic acid.
Tables
- Table 1.
Growth of and polyester accumulation by H. pseudoflava grown on γ-butyrolactone (0.8 ml/liter; 10.4 mM) as the sole carbon source by using the one-step cultivation processa
Culture time (h) Dry cell wt (g/liter) Polyester content (%, wt/wt) Polyester composition (mol%)b Amt of remaining nitrogen (g/liter)c Amt of remaining γ-butyrolactone (ml/liter) C/N ratio 3HB 4HB 120d 0.69 1.3 37 63 0.18 (1.4)e 0.41 (5.3)e 3.8f 144 0.69 6.1 45 55 0.16 (1.2) 0.35 (4.6) 3.8 168 0.66 8.0 44 56 0.15 (1.1) 0.34 (4.4) 4.0 192 0.64 7.8 45 55 0.16 (1.2) 0.33 (4.3) 3.6 ↵a Two additional sets of batch experiments produced similar results.
↵b Calculated from gas chromatographic data.
↵c The initial concentration of ammonium sulfate was 0.66 g/liter (5.0 mM).
↵d The culture time includes the 96-h induction period.
↵e The values in parentheses are molar concentrations.
↵f The C/N ratio was defined as the ratio of the molar concentration of γ-butyrolactone to the molar concentration of ammonium sulfate remaining in the medium.
- Table 2.
Effect of the residual P(3HB) formed during first-step cultivation of H. pseudoflava in Luria-Bertani medium for 22 h on the composition of polyesters that accumulated during second-step cultivation in which the strain was grown on γ-butyrolactone (2.2 g/liter) as the sole carbon source for 48 ha
First-step cultivation Second-step cultivation with γ-butyrolactone Addition to medium Dry cell wt (g/liter) P(3HB) content (%, wt/wt) Concn of residual P(3HB) (g/liter)b Dry cell wt (g/liter) Polyester content (%, wt/wt) Polyester composition (mol%)b Concn of residual 3HB in the polymer (g/liter) % γ-Butyrolactone 3HB 4HB Used for 4HBc Unused Noned 1.61 ± 0.02 3.23 ± 0.12 0.052 ± 0.003 2.40 ± 0.02 30.4 ± 0.1 11 ± 1 89 ± 1 0.083 ± 0.004 29 ± 2 13 ± 1 NaCl (170 mM)e 1.87 ± 0.02 11.1 ± 0.20 0.206 ± 0.003 2.25 ± 0.04 33.3 ± 0.8 9 ± 1 91 ± 1 0.067 ± 0.007 31 ± 2 11 ± 1 KCl (170 mM)e 1.73 ± 0.02 3.51 ± 0.08 0.061 ± 0.001 2.20 ± 0.08 30.6 ± 0.6 5 ± 1 95 ± 1 0.045 ± 0.006 29 ± 2 11 ± 1 ↵a Cells grown in Luria-Bertani medium were transferred to PHA synthesis mineral medium containing γ-butyrolactone (2.2 g/liter) and 0.6 g of ammonium sulfate per liter for polyester accumulation.
↵b Calculated from gas chromatographic data.
↵c Percent conversion of γ-butyrolactone (2.2 g/liter) to 4HB in the polymer.
↵d All values are averages for three different experiments.
↵e All values are averages for six different experiments.
- Table 3.
P(4HB) homopolymer synthesis during three-step cultivation of H. pseudoflavaa
Culture step Dry cell wt (g/liter) Polyester content (%, wt/wt) Polyester composition (mol%)b 3HB 4HB First 1.94 ± 0.08 10.1 ± 0.6 100 Second 1.68 ± 0.07 NDc Third 2.42 ± 0.06 19.6 ± 0.5 100 ↵a The second-step carbon-free medium contained 0.3 g of ammonium sulfate per liter. The P(3HB)-free cells recovered were transferred to γ-butyrolactone-containing PHA synthesis mineral medium (the third culture). Duplicate experiments were carried out.
↵b Calculated from gas chromatographic data.
↵c ND, not detected by gas chromatographic analysis.
- Table 4.
Activities of 3HBA and 4HBA dehydrogenases in H. pseudoflava and R. eutropha H16 grown on γ-butyrolactone under various culture conditions
Organism Culture medium Culture time (h) Total protein concn (mg/ml) Sp Act (U/mg) of: 3HBA dehydrogenase 4HBA dehydrogenase H. pseudoflava Luria-Bertani medium 22 6.83 0.254 NDc Medium containing γ-butyrolactone (second step)a 48 3.21 2.75 0.064 Medium containing 0.8 ml of γ-butyrolactone per liter (one step)b 96 1.06 ND 0.089 108 2.31 0.031 0.170 120 2.85 0.095 0.492 132 3.13 0.183 0.531 144 3.61 0.338 0.557 Medium containing 0.8 g of 4HBA per liter (one step)b 96 1.54 0.075 0.030 108 2.01 0.188 0.092 120 2.47 0.231 0.162 132 2.93 0.243 0.285 144 3.01 0.277 0.369 R. eutropha H16 Luria-Bertani medium 22 3.81 1.69 ND Medium containing γ-butyrolactone (second step)a 48 4.33 3.15 0.071
