Enhanced biological fixation of methane for microbial lipid production by recombinant Methylomicrobium buryatense

Due to the success of shale gas development in the US, the production cost of natural gas has been reduced significantly, which in turn has made methane (CH₄), the major component of natural gas, a potential alternative substrate for bioconversion processes compared with other high-price raw material sources or edible feedstocks. Therefore, exploring effective ways to use CH₄ for the production of biofuels is attractive. Biological fixation of CH₄ by methanotrophic bacteria capable of using CH₄ as their sole carbon and energy source has obtained great attention for biofuel production from this resource.

In this study, a fast-growing and lipid-rich methanotroph, Methylomicrobium buryatense 5GB1 and its glycogen-knock-out mutant (AP18) were investigated for the production of lipids derived from intracellular membranes, which are key precursors for the production of green diesel. The effects of culture conditions on cell growth and lipid production were investigated in high cell density cultivation with continuous feeding of CH₄ and O₂. The highest dry cell weight observed was 21.4 g/L and the maximum lipid productivity observed was 45.4 mg/L/h obtained in batch cultures, which corresponds to a 2-fold enhancement in cell density and 3-fold improvement in lipid production, compared with previous reported data from cultures of 5GB1. A 90% enhancement of lipid content was achieved by limiting the biosynthesis of glycogen in strain AP18. Increased CH₄/O₂ uptake and CO₂ evaluation rates were observed in AP18 cultures suggesting that more carbon substrate and energy are needed for AP18 growth while producing lipids. The lipid produced by M. buryatense was estimated to have a cetane number of 75, which is 50% higher than biofuel standards requested by US and EU.

Cell growth and lipid production were significantly influenced by culture conditions for both 5GB1 and AP18. Enhanced lipid production in terms of titer, productivity, and content was achieved under high cell density culture conditions by blocking glycogen accumulation as a carbon sink in the strain AP18. Differences observed in CH₄/O₂ gas uptake and CO₂ evolution rates as well as cell growth and glycogen accumulation between 5GB1 and AP18 suggest changes in the metabolic network between these strains. This bioconversion process provides a promising opportunity to transform CH₄ into biofuel molecules and encourages further investigation to elucidate the remarkable CH₄ biofixation mechanism used by these bacteria.