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., H₂ and CO₂). 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 H₂-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, H₂-producing bacteria) was strongly associated with the OLR and H₂ production performance. Similarly, increasing the OLR influenced the abundance of the gene transcripts responsible for H₂ production and homoacetogenesis. To evaluate the effect of decreasing the H₂ partial pressure, silicone oil was added to the reactor at an OLR of 138 and 160 g TC/L-d, increasing the production of H₂, the copies of genes codifying for hydA and fthfs, and the genes transcripts related to H₂ production and homoacetogenesis. Moreover, the metatranscriptomic analysis also showed that lactate-type fermentation and dark fermentation simultaneously occurred without compromising the reactor performance for H₂ production.