Selection and optimization strategies for production of polyhydroxybutyrate (PHB) in methanotrophic bacteria [electronic resource]
Keywords: Civil and Environmental Engineering.

Disposal of petrochemical plastics is of major environmental concern worldwide, with plastics continuing to accumulate in landfills and oceans. Polyhydroxybutyrate (PHB) is a biologically produced and biodegradable polymer that can functionally replace many recalcitrant, petroleum-based polymers. Production cost of PHB currently prevents wider adoption, with much of this cost attributable to use of agricultural sugars as a production substrate. Methane gas is attractive as an alternative substrate because it is inexpensive, widely available, and naturally converted to PHB by methanotrophic bacteria. Production of PHB by methanotrophic bacteria is well documented, but continued gains in production efficiency must be realized to allow implementation on an industrial scale. In this dissertation, a combination of high throughput screening and application of selective pressure was used to optimize production of PHB by methanotrophic bacteria. Culturing and isolation of methanotrophs is labor-intensive due to slow growth rates and the requirement of gas-phase substrate delivery. Measurement of PHB content is similarly labor-intensive, with culture and analysis time combining to limit experimental throughput. To address this issue, a novel microplate-based system was designed, enabling high-throughput culture and integrated analysis of PHB content. This system was used to optimize growth medium for PHB production in the Type II methanotroph Methylocystis parvus OBBP. Modification of copper and calcium concentrations in the growth medium lead to a five-fold increase in final PHB content in this organism, validating the efficacy of the high-throughput system and underscoring the importance of media composition for optimal PHB production. As compared to aseptic production in pure culture systems, use of diverse mixed culture for PHB production reduces sterilization costs and allows selection for improved fitness over time. In methanotrophs, PHB production has been identified only in Type II strains, a subset of total diversity. Selection for Type II methanotrophs and exclusion of other types is therefore critical to the development of effective mixed culture PHB production. Combinations of growth on ammonium, nitrate, and urea nitrogen sources were therefore investigated as potential selectors for PHB-producing methanotrophs. In enrichment cultures inoculated with activated sludge, PHB production was observed only in replicates enriched with ammonium and PHB was detected only at low concentrations. These low concentrations are attributed to competitive inhibition of methane monooxygenase by ammonium, and to accumulation of toxic hydroxylamine produced by ammonium co-oxidation. Alternating between selective growth with ammonium and rapid growth with nitrate mitigated these negative effects, allowing rapid growth and PHB production. Hydroxylamine tolerance was also tested in pure cultures as a potential selector for Type II methanotrophs, with tolerance found to vary strain-by-strain. Added reducing equivalents increased hydroxylamine tolerance in two of four strains tested, indicating that in some strains hydroxylamine reduction plays an important role in hydroxylamine detoxification. Stored PHB serves as a source of reducing equivalents in methanotrophs, and may therefore increase fitness of methanotrophs exposed to cometabolically produced hydroxylamine. Combining selection for Type II methanotrophs with selection for increasing PHB production over time would increase yields of PHB without requiring metabolic engineering. Methane limitation during PHB consumption is known to select for increasing PHB over time, with stored PHB used to supply necessary reducing equivalents in the absence of sufficient methane. In the final study, two cycling reactors were operated with alternating application of ammonium and nitrate nitrogen sources for selection of Type II methanotrophs. Methane was limited during PHB consumption in one reactor and supplied consistently in a second, control reactor. PHB production remained stable in both reactors for 35 days, and no trend towards increasing PHB production was observed during this period. PHB produced by both reactors was of high molecular weight, and molecular weight remained consistent over time despite a shift in community composition. A period of instability was observed after 35 days, during which time biomass and PHB concentrations declined for six consecutive reactor cycles. This instability is attributed to long-term exposure to products of ammonium oxidation, and may be mitigated by reducing the duration and intensity of ammonium exposure. Full exploration of the long-term effects of methane limitation on PHB production is contingent upon operational stability. Despite this instability, cycling of nitrogen sources as described is promising due to the observed robust selection, rapid growth, short cycle times, and consistent production high molecular weight PHB.