Batch Experiments Demonstrating a Two-Stage Bacterial Process Coupling Methanotrophic and Heterotrophic Bacteria for...

Methane (CH₄) is a sustainable carbon feedstock for value-added chemical production in aerobic CH₄-oxidizing bacteria (methanotrophs). Under substrate-limited (e.g., oxygen and nitrogen) conditions, CH₄ oxidation results in the production of various short-chain organic acids and platform chemicals. These CH₄-derived products could be broadened by utilizing them as feedstocks for heterotrophic bacteria. As a proof of concept, a two-stage system for CH₄ abatement and 1-alkene production was developed in this study. Type I and Type II methanotrophs, Methylobacter tundripaludum SV96 and Methylocystis rosea SV97, respectively, were investigated in batch tests under different CH₄ and air supplementation schemes. CH₄ oxidation under either microaerobic or aerobic conditions induced the production of formate, acetate, succinate, and malate in M. tundripaludum SV96, accounting for 4.8-7.0% of consumed carbon from CH₄ (C-CH₄), while M. rosea SV97 produced the same compounds except for malate, and with lower efficiency than M. tundripaludum SV96, accounting for 0.7-1.8% of consumed C-CH₄. For the first time, this study demonstrated the use of organic acid-rich spent media of methanotrophs cultivating engineered Acinetobacter baylyi ADP1 'tesA-undA cells for 1-alkene production. The highest yield of 1-undecene was obtained from the spent medium of M. tundripaludum SV96 at 68.9 ± 11.6 μmol mol C_substrate ^-1. However, further large-scale studies on fermenters and their optimization are required to increase the production yields of organic acids in methanotrophs.