From waste to plastic [electronic resource] : selection of methanotrophic bacteria for production of poly-3-hydroxybutyrate through applied environmental stresses

Poly-3-hydroxybutyrate (PHB) is a biologically produced, biodegradable polyester that can be used to replace conventional, petrochemical-based plastics. The relatively high production cost of PHB has thus far prevented it from becoming more widely available. The production cost can be reduced by using less expensive feedstocks and non-sterile production conditions. Methane is an attractive substrate for PHB production because it is inexpensive and its use generates positive environmental externalities. This dissertation studies how natural selection can be used to obtain high PHB production in methanotrophic bacteria through the application of environmental stresses. The first study ascertains that PHB production is only found in Type II, serine pathway methanotrophs through a screen of Type I and II methanotrophs for PHB production and the presence of the phaC gene. Environmental conditions that select for Type II, serine pathway methanotrophs from a diverse inoculum of activated sludge were then evaluated. Providing nitrogen gas as the sole nitrogen source and using a dilute mineral salts media, both in the absence of copper, were found to enrich predominantly Type II methanotrophs capable of PHB production. Lower pH levels (pH = 4-5) were also found to preferentially enrich Type II methanotrophs that produced PHB. Environmental conditions can thus be used to enrich Type II methanotrophs capable of high levels of PHB production. The second study focuses on understanding the role of PHB in the Type II methanotroph Methylocystis parvus OBBP in order to understand what conditions favor PHB accumulation and utilization. Previous work has demonstrated that PHB may function as a growth substrate during periods of carbon absence. In this study, M. parvus OBBP did not replicate using stored PHB in the absence of an exogenous carbon source, even when all other nutrients were provided in sufficiency. When methane and nitrogen were present, PHB and methane were simultaneously consumed. Cells with PHB had significantly higher specific growth rates compared to cells without PHB. Addition of formate (a source of reducing power) to PHB-rich cells delayed PHB consumption but addition of glyoxylate (a source of C-2 units) did not. It is hypothesized that PHB contributes to the reducing power available for methane oxidation and carbon and nitrogen assimilation, thus facilitating survival in environments with intermittent availability of nutrients. The third study applies this knowledge of PHB metabolism to inform the design of three cyclically stressed bioreactors. Three reactors were operated as sequencing batch reactors on 24-hour cycles and were subject to cyclic nitrogen limitation. In addition to nitrogen limitation, two reactors were subject to periodic methane or oxygen limitation while nitrogen was present. Reactors were all inoculated with the same defined, Type II methanotrophic community and operated for eleven days. PHB content and the ultimate capacity of samples to produce PHB in offline incubations were monitored. The PHB content of the nitrogen-limited reactor decreased over operation, whereas PHB production increased in the nitrogen and methane-limited reactor. Communities in all three reactors were highly enriched for Methylocystis parvus OBBP, but there were differences in the minority representatives in each reactor. PHB apparently provided cells with a stronger competitive advantage under cyclic nitrogen and carbon limitation than under nitrogen limitation alone or nitrogen and oxygen limitation. The findings in this work suggest that it will be possible to maintain a culture of methanotrophic bacteria capable of producing high levels of PHB under non-sterile conditions using a combination of environmental pressures and cyclically applied stresses.

Advisor: Criddle Craig primary advisor. Thesis advisor
Francis Christopher advisor. Thesis advisor
McCarty Perry L. advisor. Thesis advisor