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

Microbial communities in dark fermentation continuous systems are affected by substrate type, concentration, and product accumulation (e.g., H 2 and CO 2 ). Metatranscriptomics and quantitative PCR (qPCR) were used to assess how high organic loading rates (OLR) from 60 to 160 g total carbohydrates (TC)/L-d modify the microbial community diversity and expression of key dark fermentative genes. 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 ). Quantification through qPCR showed that the abundance of genes encoding the formyltetrahydrofolate synthetase ( fthfs , homoacetogens) and hydrogenase ( hydA , H 2 -producing bacteria) was strongly associated with the OLR and H 2 production performance. Similarly, increasing the OLR influenced the abundance of the gene transcripts 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 codifying for hydA and fthfs , and the genes transcripts related to H 2 production and homoacetogenesis. Moreover, the metatranscriptomic analysis also showed that lactate-type fermentation and dark fermentation simultaneously occurred without compromising the reactor performance for H 2 production.