Methanotrophic bacteria are promising means of producing value-added products as they have the ability to transform methane under atmospheric temperature and pressure. Efforts to develop methanotrophs to produce value-added products are hoped to incentivize a decrease in methane flaring operations. The data collected during this study was used to inform a larger effort for design of a bio-laminar plate reactor system (BLP) that could be used in conjunction with encapsulated whole-cell technology to convert methane into a marketable multicarbon product.
Methylomicrobium buryatense 5GB1 with an engineered plasmid PA5 was studied for methane bioconversion to soluble multicarbon products. This study focused on previously identified compounds including crotonate, succinate, and acetate, and their expression in different environments. Experiments were performed in batch to study culture environments used to induce secondary metabolites, including substrate limitation, oxygen limitation and salinity flux. Methane and oxygen consumption was monitored along with soluble metabolites made by M. buryatense 5GB1 in batch reactors. Soluble products were analyzed by high performance liquid chromatogram (HPLC). Under experimental conditions studied, carbon conversion efficiency (CCE) reached a maximum of 5.2% for crotonate production during a salinity cycle with active encapsulated cells. Succinate CCE was measured at a maximum of 2.64 ± 0.34% for batch reactors receiving multiple methane injections with decreasing oxygen concentration. Acetate maximum CCE was 17.4 % for batch reactors with exchanged fresh media.
Bacterial milking was performed by exchange of liquid media with agarose-encapsulated M. buryatense 5GB1PA5 culture. Hemispheric agarose beads were suspended in media with 8% NaCl and nanopure water in 24 hour cycles. Extracellular crotonate in salinity cycled bioreactors was observed to reach a maximum of 10.9 mg/L and a CCE above 4 % in the first three cycles. Media control bioreactors undergoing media exchanges without salinity change produced maximum crotonate concentration of 0.032 mg/L and 0.02 % CCE during the first three exchanges. This observation provides evidence that salinity exchange is likely to have the capacity to increase production of microbial products.
This study found that the methods tested to increase soluble metabolite production by M. buryatense 5GB1PA5 are unlikely to meet market needs without further refinement. High variance among triplicates in tests led to limited confidence in production reliability. Benchmark CCE for crotonate, succinate and acetate were identified for an economically feasible bioreactor plant based on the in-house techno-economic cost analysis and found to be 2.6, 6.0 and over 100 % respectively. M. buryatense 5GB1PA5 produced carbon conversion below identified benchmarks in most environments tested, and all averaged CCE values fell below this target. Future efforts for methanotrophic bioconversion should be revisited only after better metabolic control is achieved. While this study found that market application is still out of reach, it provides insight for reaction intensification technology using osmotic fluctuation.