New insights into microbial interactions and putative competitive mechanisms during the hydrogen production from tequila vinasses

Abstract

This study aimed to characterize the prokaryotic community and putative microbial interactions involved in hydrogen (H₂) production during the dark fermentation (DF) process, applying principal components analysis (PCA) to correlate changes in operational, physicochemical, and biological variables. For this purpose, a continuous stirred-tank reactor-type digester fed with tequila vinasses was operated at 24, 18, and 12 h of hydraulic retention times (HRTs) to apply organic loading rates of 20, 36, and 54 g-COD L^-1 d^-1, corresponding to stages I, II, and III, respectively. Results indicated high population dynamics for Archaea during the DF process toward a decrease in total sequences from 6299 to 99. Concerning the Bacteria community, lactic acid bacteria (LAB) were dominant reaching a relative abundance of 57.67%, while dominant H₂-producing bacteria (HPB) decreased from 25.76% to 21.06% during stage III. Putative competitive exclusion mechanisms such as competition for substrates, bacteriocins production, and micronutrient depletion carried out by Archaea and non-H₂-producing bacteria (non-HPB), especially LAB, could negatively impact the dominance of HPB such as Ethanoligenens harbinense and Clostridium tyrobutyricum. As a consequence, low maximal volumetric H₂ production rate (672 mL-H₂ L^-1 d^-1) and yield (3.88 mol-H₂ assimilated sugars^-1) were obtained. The global scenario obtained by PCA correlations suggested that C. tyrobutyricum positively impacted H₂ molar yield through butyrate fermentation using the butyryl-CoA:acetate CoA transferase pathway, while the most abundant HPB E. harbinense decreased its relative abundance at the shortest HRT toward the dominance of non-HPB. This study provides new insights into the microbial interactions and helps to better understand the DF performance for H₂ production using tequila vinasses as substrate. KEY POINTS: • E. harbinense and C. tyrobutyricum were responsible for H₂ production. • Clostridiales used acetate and butyrate fermentations for H₂ production. • LAB won the competition for sugars against Clostridiales during DF. • Putative bacteriocins production and micronutrients depletion could favor LAB.

Keywords: Competitive exclusion; Dark fermentation; H2 production; Microbial interactions; Principal component analysis; Tequila vinasses.