Synthesis of medium chain-length polyhydroxyalkanoates (mcl-PHAs) is an oxygen-requiring process. Maintaining an adequate supply of dissolved oxygen (DO) is a significant engineering challenge, and can be costly in large-scale bioreactors. The objective of this work was to understand the effect(s) that low-DO environments have on mcl-PHA synthesis in Pseudomonas putida LS46. This work focuses on two main aspects:​ 1) investigating fundamental physiology in microaerophilic environments;​ and 2) understanding how the findings could be applied to process design for bioreactor operations. In the first aspect, low-DO environments were studied in lab-scale bioreactors, mimicking conditions that might be encountered in large-scale vessels. At low DO, carbon flux to mcl-PHA exhibited considerable dependence on the carbon substrate and hence the biochemical pathway used for mcl-PHA biosynthesis. It was found that significant mcl-PHA was synthesized from β-oxidation intermediates (an oxidative process), but not when precursor monomers were obtained via a de novo fatty acid biosynthetic pathway (a reductive process). In the latter case, most of the carbon was respired as CO2. For substrates that use both pathways to some degree, low DO increased the content of monomers incorporated from β-oxidation intermediates. In the second aspect of this work, an O2-limited high-cell density fed-batch strategy was developed and implemented for improved productivity, and the effects of culture rheology on process performance were assessed. Finally, a lab-scale semi-continuous sequencing-batch system was pursued to investigate long-term performance in a non-sterile environment. It was found that fatty acid carbon substrates were highly selective for mcl-PHA producing organisms, such that the productivity of this system was comparable to a similar process in a sterile environment.

Collectively, this work represents the most comprehensive study available on the effects of O2-limitation in mcl-PHA synthesis. The findings improve fundamental understanding of how P. putida LS46 metabolism responds to its environment and also have implications for process design, including:​ 1) under what conditions low-DO environments can benefit mcl-PHA synthesis;​ 2) possibilities for co-feeding substrates to improve residual growth or PHA yield;​ 3) how DO might alter monomer compositions;​ and 4) process configurations that can improve productivity under microaerophilic conditions.