Biosynthesis and Characterization of Polyhydroxyalkanoates with Controlled Composition and Microstructure.

Volatile fatty acids (VFA) C2:0 to C6:0 were used as the sole carbon source for poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) production with controllable composition and microstructure in Haloferax mediterranei. Feeding carbon-even VFA gave >90 mol % poly(3-hydroxybutyrate) (3HB) PHBV, while carbon-odd VFA generated >87 mol % poly(3-hydroxyvalerate) (3HV) PHBV. Bespoke random, block, and blend copolymers with 0-100 mol % 3HV were synthesized using C4:0/C5:0 mixtures. The copolymer 3HV fraction is proportional to the %C5:0 in the feed mixture, allowing control over copolymer composition. Microstructure depends on the substrate addition order: cofeeding generated random copolymers, while sequential feeding created block and blend copolymers. On average, the PHBV had an ultrahigh molecular weight of 3 × 106 g/mol. 3HV rich copolymers showed lower melting temperatures, enhanced elasticity, and ductility. H. mediterranei is ideal for large-scale production of PHBV due to its inherent bioprocessing advantages, while control over the composition and microstructure of PHBV will facilitate the production of biopolymers capable of meeting industrial criteria for specific applications.

Production of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with varied composition using different nitrogen sources with Haloferax mediterranei.

The extreme haloarchaea Haloferax mediterranei accumulates poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) without the need for specific precursors. In this study, growth kinetics and PHBV synthesis were characterised under nitrogen-excess and nitrogen-limiting conditions in ammonium and, for the first time, nitrate. With excess nitrogen, ammonium and nitrate cultures generated 10.7 g/L biomass containing 4.6 wt% PHBV and 5.6 g/L biomass with 9.3 wt% PHBV, respectively. Copolymer composition varied with the nitrogen source used: PHBV from ammonium cultures had 16.9 mol% 3-hydroxyvalerate (HV), while PHBV from nitrate cultures contained 12.5 mol% HV. Nitrogen limitation was achieved with carbon-to-nitrogen (C/N) molar ratios of 25 or higher. Nitrogen limitation reduced biomass generation and polymer concentration, but polymer accumulation increased to 6.6 and 9.4% for ammonium and nitrate, respectively, with C/N 42. PHBV composition was also affected and cultures with lower C/N ratios produced richer HV polymers. Copolymer formation was not a uniform process: HV was only detected after a minimum accumulation of 0.45 g/L PHB and lasted for a maximum of 48 h. The understanding of copolymer synthesis and the influence of culture conditions such as the nitrogen source will help in designing novel strategies for the production of PHBV with more regular structure and material properties.