Evaluation of acidogenesis products’ effect on biogas production performed with metagenomics and isotopic approaches

Background: During the acetogenic step of anaerobic digestion, the products of acidogenesis are oxidized to substrates for methanogenesis: hydrogen, carbon dioxide and acetate. Acetogenesis and methanogenesis are highly interconnected processes due to the syntrophic associations between acetogenic bacteria and hydrogenotrophic methanogens, allowing the whole process to become thermodynamically favorable. The aim of this study is to determine the influence of the dominant acidic products on the metabolic pathways of methane formation and to find a core microbiome and substrate-specific species in a mixed biogas-producing system. Results: Four methane-producing microbial communities were fed with artificial media having one dominant component, respectively, lactate, butyrate, propionate and acetate, for 896 days in 3.5-L Up-flow Anaerobic Sludge Blanket (UASB) bioreactors. All the microbial communities showed moderately different methane production and utilization of the substrates. Analyses of stable carbon isotope composition of the fermentation gas and the substrates showed differences in average values of δ13C(CH4) and δ13C(CO2) revealing that acetate and lactate strongly favored the acetotrophic pathway, while butyrate and propionate favored the hydrogenotrophic pathway of methane formation. Genome-centric metagenomic analysis recovered 234 Metagenome Assembled Genomes (MAGs), including 31 archaeal and 203 bacterial species, mostly unknown and uncultivable. MAGs accounted for 54%–67% of the entire microbial community (depending on the bioreactor) and evidenced that the microbiome is extremely complex in terms of the number of species. The core microbiome was composed of Methanothrix soehngenii (the most abundant), Methanoculleus sp., unknown Bacteroidales and Spirochaetaceae. Relative abundance analysis of all the samples revealed microbes having substrate preferences. Substrate-specific species were mostly unknown and not predominant in the microbial communities. Conclusions: In this experimental system, the dominant fermentation products subjected to methanogenesis moderately modified the final effect of bioreactor performance. At the molecular level, a different contribution of acetotrophic and hydrogenotrophic pathways for methane production, a very high level of new species recovered, and a moderate variability in microbial composition depending on substrate availability were evidenced. Propionate was not a factor ceasing methane production. All these findings are relevant because lactate, acetate, propionate and butyrate are the universal products of acidogenesis, regardless of feedstock

Ageing of organic matter in incubated freshwater sediments; in fer ences from C and H isotope ratios of methane

The freshwater sediments were incubated under anaerobic conditions for 570 and 879 days to in vestigate the potential variations in methanogenic pathways due to increasing sediment age and recalcitrance of organic matter. The methanogenic pathways did not shift from acetate fermentation toward CO2 reduction, as indicated by the observed variations of the isotopic composition of methane in natural conditions. It appeared, however, that the observed decrease of methane concentration (from 86 to 39%) and continuous in crease in d13C(CH4) (from –69.7 to –59.0‰) and dD(CH4) values (from –381 to –320‰) resulted mainly from exhaustion of at least one methanogenic substratein the incubated sediments. To better understand processes controlling the variations of delta exp.13C(CH4) and deltaD(CH4) values relative to ageing of organic matter, the method of principal component analysis (PCA) was used. This method offers good comparison of the relation ships between variables when a larger number of parameters control a given process in the same time period. In this study, the PCA indicated three distinctive factors that controlled decomposition of organic matter during the incubation. Factor 1 explained 33% of observed variations among the variables and had positive (0.93–0.92) loadings for electric conductivity and DIC concentration and negative loading for delta exp.13C(CH4) val ues (–0.72). Factor 2 accounted for 28% and had high posi ive loading for deltaD(CH4) value (0.86) and high negative loading for methane concentration (–0.81). Factor 3 accounted for 19% and exhibited high positive loadings for temperature (0.90) and delta exp.13C(DIC) value (0.69). Factors 1 and 2 were directly linked to the methanogenesis and indicated that bigger accumulation of bio-products in sediments is likely important for variations of delta exp.3C and deltaD of methane. This study shows that method of principal component analysis might be a useful tool while studying biogeochemical carboncy cleduring early digenesis of freshwater sediments