To explore the differences in biological gas production in the waterlogged zone of a coal seam fire-affected area, in this study the in-situ gas production experiment was conducted with the mine water from aquitard layers in coal seam fire zones in Xinjiang. The results showed that the biogas production first increased and then decreased with the increase in distance, and the highest gas production reached 216.55 mL. The changes in key metabolic pathways during the anaerobic fermentation of coal were analyzed, which showed that as the distance from the aquitard layer in the coal seam fire zone increased, the methanogenesis pathways gradually shifted from acetic acid decarboxylation and carbon dioxide reduction to acetic acid decarboxylation and methylamine methanogenesis. The significant variability in the in-situ mine water reservoir conditions contributed to the differences. In addition, the reservoir pressure and temperature increased as the distance from the fire zone became longer, and the salinity of the farthest mine water in the reverse fault was the highest due to the lack of groundwater supply. Pearson correlation analysis revealed significant correlations of microbial communities with key functional genes and the types and concentrations of ions. The ions significantly influencing microbial enzymatic metabolic activities included Al³⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Mg²⁺, PO₄^3-, and Mo⁶⁺. The differences in metabolic pathways were attributed to the integrated effects of a co-occurring environment with multiple ions. The gas production simulation experiments and metagenomic analyses provide data support for the practical application of in-situ biogas experiments, laying a foundation for engineering applications.
Biofuels ; Coal ; Methane/biosynthesis* ; Fires ; Groundwater ; Coal Mining ; Fermentation ; China ; Anaerobiosis

Methanotrophs use methane as the sole carbon and energy source, which cause slow growth, low cell density and hinder its industrial applications. One promising solution is to heterologously express methane monooxygenase (MMO) in other host cells that can be easily cultivated at high cell density. We systematically exploited the possibility of functional expression of pMMO by choosing different promoters and different host cells. The results showed that the recombinants could oxidize methane to methanol. In particular, ethanol could also be detected in the oxidized products, but the enzyme activity was instable, implying that some changes of pMMO expressed in the host cells might have occurred. In addition, SDS-PAGE analysis showed that many recombinants could express the subunits of pMMO, but the enzyme activity could not be detected. In conclusion, correct fold of pMMO in the host cells is important for its functional expression.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; Genetic Vectors ; genetics ; Methane ; metabolism ; Methanococcaceae ; enzymology ; Methanol ; metabolism ; Oxygenases ; biosynthesis ; genetics ; Promoter Regions, Genetic ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics