Biosynthesis of medium chain length polyhydroxyalkanoates (mcl-PHAs) requires oxygen, but little is known of how mcl-PHA production is impacted in low dissolved oxygen (DO) environments, particularly when synthesized from monomers obtained via a de novo pathway. Growth and mcl-PHA synthesis characteristics under microaerophilic bioreactor conditions were observed in Pseudomonas putida LS46 when grown on carbon substrates requiring a de novo pathway, to varying extent, for mcl-PHA synthesis. Negligible PHA accumulated from glucose, acetic acid, or valeric acid under oxygen limitation (DO <= 0.15 mg L-1), while final mcl-PHA content reached 19.5% of cell dry mass (CDM) from hexanoic acid. The C6 content of the polymer obtained under low DO was 10% higher than in a DO excess (NH4-limited) control experiment, with less of the C10 monomers indicative of de novo synthesis. In all cases, the carbon-to-PHA yield was poor (< 0.1 C-mol C-mol(-1)). We conclude that, as opposed to utilizing beta-oxidation intermediates, de novo biosynthesis of mcl-PHA using P. putida LS46 is not favorable in microaerophilic environments. In addition to scale-up implications, this work also suggests that lowering the bioreactor oxygen transfer rate can decrease the contribution of monomers incorporated using de novo synthesis for carbon feedstocks with mixed composition.