Microbial ecology of a lactate-driven dark fermentation process producing hydrogen under carbohydrate-limiting conditions

Despite the high prevalence of lactic acid bacteria in dark fermentation (DF) processes, their ecological role is not yet completely elucidated, preventing their systematic use as “helpers” for hydrogen production. The aim of this study was to investigate the microbial community structure of a lactate-driven DF process that successfully produced hydrogen under carbohydrate-limiting conditions using tequila vinasse as a substrate. Microbial responses to stepwise decreases in hydraulic retention time (HRT) from 24 to 4 h were assessed by using Illumina MiSeq sequencing. HRTs above 12 h and below 6 h led to a lower hydrogen production rate (HPR; 0.2–3.3 L/L-d) and process stability (HPR variations within 25–65%), which were associated with the presence of Acetobacter lovaniensis, Clostridium luticellari, Blautia coccoides, and the high abundance of propionate and lactate. Interestingly, transient conditions from unsteady-to-steady state occurred at an HRT of 12 h, where species richness and evenness decreased remarkably. Accordingly, HRTs between 12 and 6 h resulted in higher HPRs of up to 11.7 ± 0.7 L/L-d with HPR variations of less than 10%, which closely matched with the dominance of Clostridium sp., and butyrate and acetate as the main aqueous products. Overall, the results indicate that the successfulness of exploiting the ‘unwanted’ LAB proliferation through lactate-driven DF processes requires the enrichment of lactate-consuming and hydrogen-producing bacteria, which entails the selection of proper biocatalysts and operating conditions/strategies such as the operation of DF reactors under carbohydrate-limiting conditions and low HRTs.