Batch Experiments Demonstrating a Two-Stage Bacterial Process Coupling Methanotrophic and Heterotrophic Bacteria for 1-Alkene Production From Methane

Abstract

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 Csubstrate–1. However, further large-scale studies on fermenters and their optimization are required to increase the production yields of organic acids in methanotrophs.

Characterization and genome analysis of a psychrophilic methanotroph representing a ubiquitous Methylobacter spp. cluster in boreal lake ecosystems

“…S3L5C may provide an alternative source of CH 4 -derived metabolites for biotechnological applications, especially in cold systems. For example, organic acid-rich spent media can be used to cultivate recombinant heterotrophs to generate value-added compounds [ 16 , 66 , 67 ]. However, further studies on the effect of alternative electron acceptors (e.g., nitrogen oxides) on metabolism and organic acid production of Methylobacter sp.…”

“…To determine the organic acid composition, the cultures centrifuged (15 min at 2700 × g ) and filtered through a 0.2 µm membrane (Chromafil ® Xtra PET 20/25, Macherey-Nagel, Germany) were analyzed using a Shimadzu high-performance liquid chromatography equipped with Rezex RHM-Monosaccharide H + column (Phenomenex, USA) as described in Okonkwo et al [ 38 ]. Gas composition in the headspace (i.e., CH 4 , CO 2 , and O 2 ) was measured using a Shimadzu gas chromatograph GC-2014 equipped with a thermal conductivity detector and a Carboxen-1000 60/80 column (Agilent Technologies, USA) as described in Khanongnuch et al [ 16 ]. To obtain further insight into the distribution of carbon in the cell biomass, the total carbohydrate content was evaluated using the phenol-sulfuric acid method [ 39 ].…”

“…Furthermore, MOB interacting with a high heterotroph richness in a coculture system could also enhance CH 4 oxidation as the cross-feeding mechanism might help to reduce the toxic compounds’ inhibitory effect on MOB [ 14 ]. Studies on implementing the produced extracellular by-products as biofuel precursors or as industrial platform chemicals also imply the biotechnological importance of MOB [ 16 – 18 ].…”

Pseudomonas fluorescens MFE01 uses 1-undecene as aerial communication molecule

“…The UndA and UndB enzymes that catalyze 1-undecene synthesis by oxidative decarboxylation of lauric acid were previously studied by Rui et al (2014 , 2015) . Currently, they are mainly studied in synthetic biology field for their high value in biofuel production, allowing valorization of multiple pollutants and wastes via various metabolic engineering strategies ( Herman and Zhang, 2016 ; Chatterjee et al, 2018 ; Wang et al, 2018 ; Luo et al, 2019 ; Liu and Li, 2020 ; Salmela et al, 2020 ; Khanongnuch et al, 2022 ). Bioinformatical predictions designated UndB as a membrane bound desaturase ( Rui et al, 2015 ).…”

Anode-assisted electro-fermentation with Bacillus subtilis under oxygen-limited conditions

“…The gas samples in the headspace (CO 2 , O 2 and N 2 ) were measured by gas chromatography (GC-2014, Shimadzu, Japan) equipped with a thermal conductivity detector (TCD) using the same column and methods as described in Ref. [ 30 ].…”