Elucidating the Role of CO2 in Type II Methanotrophic Growth & Polyhydroxyalkanoate (PHA) Synthesis

While methanotrophic bacteria play a key role in global methane cycles, recent studies indicate that Type II methanotrophs specifically utilize CO₂, which has further implications for greenhouse gas mitigation Type II methanotrophs assimilate carbon into biomass and biopolymer (polyhydroxybutyrate or PHB) through the serine pathway which has a high demand of CO₂ due to the presence of carboxylation reactions. Previous studies had found that CO₂ supplementation no effects on growth but the effect of CO₂ on PHB accumulation has not been studied. This study aims to understand the stoichiometric and kinetic effects of CO₂ levels on growth and PHB synthesis, the pathways of CO₂ assimilation during growth and PHB synthesis, and how to manipulate the biochemical pathways to maximize PHB production efficiency and rate. To explore these questions, we carried out a series of batch incubations to evaluate stoichiometric changes at different ratios of CH₄:CO₂. We also tracked carbon in the PHB polymer using 13-C labelling studies. We observed that increasing the supplementary CO₂ concentration caused a decrease in the net CO₂ produced without yielding a significant change neither in the growth rates nor the amount of PHB accumulated. However, NMR results revealed that the supplementary CO₂ was incorporated in the C1 and C3 positions of the PHB polymer. In the presence of added valerate (5mM), polyhydroxybutyrate-co-hydroxyvalerate (PHBV) was accumulated. The PHBV content increased with increasing CO₂ concentrations peaking at a CH₄:CO₂ ratio of 2:1. As the initial CO₂ levels increased from 0 to 30%, the HV fraction of PHBV increased from 7% to 13%. However, the PHBV content at an initial CH₄:CO₂ ratio of 1:1 was low, reflecting the energy requirement of synthesizing this polymer. Additional studies will explore the trade-off between the polymer accumulated and the energy required.