Abstract

The mechanical toughness of poly(3-(R)-hydroxybutyrate) (PHB) homopolymer is known to be enhanced by the incorporation of 3-(R)-hydroxyvalerate (PHV) monomers along the polymer chain, especially when the PHV monomers are segregated within block domains, i.e. PHB-b-(PHV-co-PHB). In this paper we demonstrate the synthesis of both random and block-copolymers of PHB and PHV using CO2 and valeric acid as monomer precursors. We perform this useful conversion via the mixotrophic capabilities of Ralstonia eutropha, which permit the simultaneous operation of catabolic pathways used to degrade heterotrophic substrates along with anabolic cycles for CO2 fixation using hydrogen gas. Key to our analysis is the collection of real-time, mass-spectroscopy data from the biosynthesis off-gas, thereby allowing us to calculate the relative uptake rates of CO2, H2, O2, and valerate. With this data, we are able to understand the dynamic interplay between catabolic and anabolic cycles and how polymer synthesis responds to the availability of different substrates. Under heterotrophic conditions (i.e. without hydrogen co-feed), the catabolism of valerate results in the production of monomers for both PHB and PHV; however, under mixotrophic conditions (i.e. with hydrogen co-feed) an excess of reducing equivalents shifts monomer fluxes towards increasing PHV fractions and even the production of PHV homopolymer. Owing to the fine resolution of the off-gas measurements, we are able to make predictions for not only the size but also the composition of PHV-co-PHB domains formed during polymer synthesis. This has provided us with new insight about how mechanical toughness arises in PHB-b-(PHV-co-PHB) block copolymers. While PHB homopolymer is highly crystalline, PHV-co-PHB random copolymers are more amorphous, with a maximum in their amorphous fraction occurring at around 50 mol% PHV. As a result of the decrease in crystallinity, the modulus of PHV-co-PHB is reduced, but its elasticity is enhanced. By covalently linking a segment of PHB, which is strong but brittle, with a separate domain of PHV-co-PHB, which is soft and elastomeric, materials can be produced that are both strong and tough. Correspondingly, our results suggest that the mechanical toughness and resistance to embrittlement of PHB-b-(PHV-co-PHB) block-copolymers is greatest when the composition of the PHV-co-PHB domains approach 50 mol% PHV. However, our experiments show that achieving this target composition, while also trying to control the length of PHV-co-PHB segments can be challenging. Close inspection of the data indicates that the uptake rate of valeric acid becomes concentration limited when the doses of valeric acid become too small. Thus, mass spectroscopy analysis of biosynthesis off-gas is a valuable tool for controlling polymerization in vivo, in order to tailor the molecular structure and hence material properties of PHA biopolymers.