Poly(3-Hydroxybutyrate) Biosynthesis from [U-13C6]D-Glucose by Ralstonia eutropha NCIMB 11599 and Recombinant Escherichia coli

Abstract

The use of stable isotope-labeled polymers in in situ biodegradation tests provides detailed information on the degradation process. As isotope-labeled raw chemicals are generally expensive, it is desirable to prepare polymer samples with high production yields and high isotope-labeling ratios. The biodegradable plastic poly[(R)-3-hydroxybutyrate)] (P(3HB)) is produced by microorganisms. In this study, to produce carbon 13 (13C)-labeled P(3HB) from [U-13C6]D-glucose (13C-glucose), the culture conditions needed for high production yields and high 13C-labeling ratios were investigated using Ralstonia eutropha NCIMB 11599 and recombinant Escherichia coli JM109. We found that over 10 g/L of P(3HB) could be obtained when these microorganisms were cultured in Luria-Bertani (LB3) medium containing 3 g/L NaCl and 40 g/L 13C-glucose, while 1.4–4.7 g/L of P(3HB) was obtained when a mineral salt (MS) medium containing 20 g/L 13C-glucose was used. The 13C-labeling ratio of P(3HB) was determined by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry (GC-MS), and both analytical methods yielded nearly identical results. High 13C-labeling ratios (97.6 atom% by GC-MS) were observed in the MS medium, whereas low 13C-labeling ratios (88.8–94.4 atom% by GC-MS) were observed in the LB3 medium. Isotope effects were observed for the P(3HB) content in cells cultured in the LB3 medium and the polydispersity of P(3HB).