Anaerobic digestion, whose final products are methane and carbon dioxide, ensures energy flow and circulation of matter in ecosystems. This naturally occurring process is used for the production of renewable energy from biomass. Lactate, a common product of acidic fermentation, is a key intermediate in anaerobic digestion of biomass in the environment and biogas plants. Effective utilization of lactate has been observed in many experimental approaches used to study anaerobic digestion. Interestingly, anaerobic lactate oxidation and lactate oxidizers as a physiological group in methane-yielding microbial communities have not received enough attention in the context of the acetogenic step of anaerobic digestion. This study focuses on metabolic transformation of lactate during the acetogenic and methanogenic steps of anaerobic digestion in methane-yielding bioreactors. Methane-yielding microbial communities instead of pure cultures of acetate producers were used to process artificial lactate-rich media to methane and carbon dioxide in up-flow anaerobic sludge blanket reactors. The media imitated the mixture of acidic products found in anaerobic environments/digesters where lactate fermentation dominates in acidogenesis. Effective utilization of lactate and biogas production was observed. 16S rRNA profiling was used to examine the selected methane-yielding communities. Among Archaea present in the bioreactors, the order Methanosarcinales predominated. The acetoclastic pathway of methane formation was further confirmed by analysis of the stable carbon isotope composition of methane and carbon dioxide. The domain Bacteria was represented by Bacteroidetes, Firmicutes, Proteobacteria, Synergistetes, Actinobacteria, Spirochaetes, Tenericutes, Caldithrix, Verrucomicrobia, Thermotogae, Chloroflexi, Nitrospirae, and Cyanobacteria. Available genome sequences of species and/or genera identified in the microbial communities were searched for genes encoding the lactate-oxidizing metabolic machinery homologous to those of Acetobacterium woodii and Desulfovibrio vulgaris. Furthermore, genes for enzymes of the reductive acetyl-CoA pathway were present in the microbial communities. The results indicate that lactate is oxidized mainly to acetate during the acetogenic step of AD and this comprises the acetotrophic pathway of methanogenesis. The genes for lactate utilization under anaerobic conditions are widespread in the domain Bacteria. Lactate oxidation to the substrates for methanogens is the most energetically attractive process in comparison to butyrate, propionate, or ethanol oxidation.

Carbon-bearing compounds (glucose, sodium acetate, methanol, yeast extract, and nutrient broth) were added in different proportions to cultures to stimulate methanogenesis in a lignite incubation experiment. Their addition significantly influenced the isotopic composition of methane generated during the fermentation of lignite. Glucose was degraded mainly in the first 2 weeks of incubation, when the atmospheric air was present in the headspace and used for biomass growth. Sodium acetate, methanol, and, presumably, lignite were decomposed in the next phase, in which anaerobic conditions occurred. The simultaneous decomposition of sodium acetate and methanol (as single substrates or as a mixture) with lignite resulted in the formation of methane with δ¹³C(CH₄) values typical for methyl-type fermentation. The identification of decomposed compounds in the mixture of sodium acetate and methanol was accomplished via isotopic analysis of carbon and hydrogen in the methane. The δ²H(CH₄) values in the case of methanol biodegradation were characterized by a negative trend over time, in contrast to a positive trend observed when sodium acetate decomposed. This observation may help to identify a very good tracer for the determination of methane precursors during methyl-type fermentation.

The common products of acidogenesis, the key stage in the process of anaerobic digestion, are lactate, butyrate, propionate, and acetate. They were decomposed in the Up-flow Anaerobic Sludge Blanket bioreactors working in continuous-flow open systems. A comprehensive analysis of variations in both isotopic ratios and concentrations of organic acids in the effluents was conducted to enhance comprehension of methanogenic processes. The analysis of carbon isotope fractionation in the CO₂-CH₄ system, as evidenced by the α¹³C_CO2-CH4 factor, has indicated that acetate decarboxylation has occurred. Furthermore, a decline in CO₂ levels was observed, accompanied by the predominance of butyrate and propionate, despite the presence of acetic acid in the effluents from all the bioreactors. Butyric acid demonstrated the greatest resistance to decomposition, resulting in ¹³C-enrichment of DIC. Lactic acid was utilised almost entirely. The observations presented above were subsequently validated through statistical analysis. A comparative analysis of the δ¹³C(CH₄) and δ¹³C(CO₂) values of our study with those of other natural substrates (detritic lignite, xylite, maize silage, and cattle manure) was undertaken, and it was found that isotope fractionation differs significantly in closed (potential thermodynamic processes) and open systems (expected Rayleigh processes). In the context of open systems, the isotope fractionation factor α¹³C_CO2-CH4 during methaneogenesis has been observed to attain values that are consistent with those observed in CH₄ oxidation. The study revealed that the presence of acetate in the substrate (i.e., the M4 bioreactor) led to the generation of CO₂ with a higher proportion of light carbon isotopes. This, in turn, resulted in a shift in the isotope fractionation factor (i.e., α¹³C_CO2-CH4) to values below 1.03. Our results suggest that methanogenic pathway signatures in open, continuous-flow systems may only be partially apparent. This is because substrate depletion drives Rayleigh-type isotope enrichment, while the dominance of a single substrate and its constant inflow stabilise pathway expression and shift control towards substrate dynamics rather than intrinsic microbial changes. Our finding suggests that isotope-based diagnostics could enhance process control in biogas plants by identifying substrate-driven limitations and facilitating more efficient and stable CH₄ production.