Metatranscriptomic Analysis Reveals the Coexpression of Hydrogen-Producing and Homoacetogenesis Genes in Dark Fermentative Reactors Operated at High Substrate Loads

Microbial communities in dark fermentationcontinuoussystems areaffected by substrate type, concentration, and product accumulation(e.g., H-2 and CO2). Metatranscriptomics andquantitative PCR (qPCR) were used to assess how high organic loadingrates (OLR) from 60 to 160 g total carbohydrates (TC)/L-d modify themicrobial community diversity and expression of key dark fermentativegenes. Overall, the microbial communities were composed of H-2-producing bacteria (Clostridium butyricum), homoacetogens (Clostridium luticellarii), and lactic acid bacteria (Enteroccocus gallinarum and Leuconostoc mesenteroides). Quantificationthrough qPCR showed that the abundance of genes encoding the formyltetrahydrofolatesynthetase (fthfs, homoacetogens) and hydrogenase(hydA, H-2-producing bacteria) was stronglyassociated with the OLR and H-2 production performance.Similarly, increasing the OLR influenced the abundance of the genetranscripts responsible for H-2 production and homoacetogenesis.To evaluate the effect of decreasing the H-2 partial pressure,silicone oil was added to the reactor at an OLR of 138 and 160 g TC/L-d,increasing the production of H-2, the copies of genes codifyingfor hydA and fthfs, and the genestranscripts related to H-2 production and homoacetogenesis.Moreover, the metatranscriptomic analysis also showed that lactate-typefermentation and dark fermentation simultaneously occurred withoutcompromising the reactor performance for H-2 production. Minimal research exists on metagenomicsand metatranscriptomicsin dark fermentation. This study is a breakthrough into dark fermentationand its metabolic pathways, which reveals the association of H-2-producing bacteria, H-2-consuming bacteria, andlactic acid bacteria.