| Summary: | The consumption of methane and the production of biodegradable polymers using alphaproteobacterial methanotrophs offers a promising strategy to mitigate greenhouse gas emissions and reduce non-biodegradable plastic pollution. This study identified an ideal amount of added methane and N:C ratio in 100 mL batch cultures of the alphaproteobacterial methanotroph <i>Methylocystis</i> sp. Rockwell growing in 1-L sealed bottles using Response Surface Methodology (RSM) to achieve both high biomass and high polyhydroxybutyrate (PHB) production. RSM analysis showed achievement of optimal biomass at 474.7 ± 10.1 mg/L in nitrate mineral salts (NMS) medium and 480.0 ± 65.5 mg/L biomass in ammonium mineral salts (AMS) medium with 8 mmol of methane and an N:C ratio of 0.022. However, optimal PHB concentration was achieved with 6 mmol methane at N:C ratios of 0.012 in NMS medium (149.7 ± 16.1 mg/L) and 0.022 in AMS medium (200.3 ± 5.1 mg/L). A multi-objective RSM analysis projected maxima in PHB production and %PHB cell content (based on dry weight) when using 4.88 mmol methane and N:C ratio of 0.016 in NMS cultures, and 6.28 mmol methane and the 0.016 N:C ratio in AMS cultures. Cultures grown under these projected conditions produced 173.7 mg PHB/L with 46.8% PHB cell content in NMS and 196.9 mg/L with 53.1% PHB cell content in AMS. Taken together, these analyses predicted the optimal conditions for growth and PHB production in batch cultures of <i>Methylocystis</i> sp. Rockwell and confirmed a preference for ammonium as the N-source for PHB production. This information is valuable for media formulation in industrial scale-up of <i>Methylocystis</i> sp. Rockwell in PHB production.
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