Synthesis and degradation of polyhydroxybutyrate (PHB) under different nutrient combinations in the alphaproteobacterial methanotroph, Methylocystis sp. Rockwell
Creators: Sharma, Hem Kanta
Publisher: name: University of Alberta Library
Year: 2023

<p>Methane emissions and plastic pollution are two distinct environmental issues that arise from human activities. The ongoing increases in methane emissions and its atmospheric concentration pose a major environmental threat as methane is a potent greenhouse gas, with a warming effect 28 times greater than carbon dioxide over a 100-year period. On the other hand, plastic pollution is a worrisome environmental concern because plastic waste can remain in the environment for hundreds of years and harm wildlife, ecosystems, and human health. Although methane emissions and plastic pollution are separate problems, addressing both issues requires a comprehensive strategy to minimize their harmful effects. One of the promising solutions to these problems involves the application of methane-oxidizing bacteria called methanotrophs. These microorganisms use methane as their energy and carbon source for cellular functions and can be used in biotechnology, bioremediation, and bioconversion. In addition to methane, methanotrophic bacteria can also utilize and convert methanol, a common industrial waste by-product, into high-value products such as biofuels and bioplastics. However, despite years of research in the methanotroph field, the paucity of knowledge on species-specific nutritional requirements hinders the scaling up of industrial methanotroph technologies. Critical questions concerning optimal nutrient (carbon, nitrogen and oxygen) combinations for faster growth and product yield remain unexplored for industrializing specific methanotrophic strains. <br>
To address this, the present work investigated the impact of nutrient combinations, culture properties and processing strategies for the bioconversion of methane and methanol into poly-3-hydroxybutyrate (PHB), a biodegradable thermoplastic polyester, by the alphaproteobacterial methanotroph Methylocystis sp. Rockwell.</p>

<p>Firstly, combinations of methane (as carbon source) and ammonium mineral salts (AMS) or nitrate mineral salts (NMS) media (as sources of nitrogen) were investigated to find nutrient combinations favoring the optimal, simultaneous, production of biomass and PHB by Methylocystis sp. Rockwell using Response Surface Methodology (RSM). While it is generally recognized that high N:C ratios favor biomass production and low ratios are necessary for PHB production, a multi-objective approach was used to determine the optimal N:C ratio of 0.016 to yield the maximum combined biomass and PHB. <br>
Secondly, the impact of the metabolic state of the inocula (well-fed vs starved) on growth and PHB management with different levels and combinations of nutrients (methane, ammonium, and oxygen) was investigated. These experiments showed that well-fed inocula can grow even under nitrogen limitation and accumulate PHB in subsequent culture. Both biomass and PHB production were higher in an oxygen rich environment. However, recurrent nitrogen limitation when using starved inocula limited subsequence growth and PHB production. These findings offer valuable insights into inoculum preparation, media formulation and process design to improve biomass and PHB production in an industrial setting. <br>
Thirdly, bioreactor experiments were conducted to demonstrate the potential of Methylocystis sp. Rockwell to consume methane and methanol to produce PHB and determine effective processing strategies for fed-batch cultivation. This study found that a methanol concentration of 22.5 mM exceeded the toxicity threshold, and confirmed that lower nitrogen concentrations were favorable for higher PHB accumulation. Adapted pulse-feeding of methanol and nitrogen in a fed-batch bioreactor led to a maximum PHB concentration of 654.56 ± 47.04 mg/L with a PHB dry cell weight content of 66.21 ± 6.64 % and a PHB productivity rate of 45.05 ± 5.77 mg/L/d after two weeks of operation. This work expanded the scope of methanol-based bioprocesses for optimizing biomass and PHB production in Methylocystis sp. Rockwell.</p>