Effect of Nitrogen and Iron Supplementation on the Process Performance and Microbial Community Structure of a Hydrogen-Producing Reactor Continuously Fed with Tequila Vinasse

Abstract

Dark fermentation is gaining importance as a bioprocess able to valorize different organic wastes/wastewaters through the co-production of organic acids and biogenic hydrogen (bioH 2 ). In this study, the process performance and microbial community structure of a mesophilic lab-scale fermenter inoculated with an enriched hydrogenogenic consortium and fed with undiluted tequila vinasse (TV) were evaluated for 28 days equivalent to 112 hydraulic retention times (HRT). The influence of external nutrients supply on process performance was carefully evaluated. The fermenter was operated at 6 h HRT and fed with i) TV without additional nutrients, ii) TV with additional nitrogen (0.3 g L -1 ), and iii) TV supplemented with nitrogen (0.3 g L -1 ) and iron (0.01 g L -1 ) chemical sources. Despite the inherent presence of macro- and micronutrients in TV, the use of raw TV with no additional nutrients resulted in a very low hydrogen production rate of 24 NmL-H 2 L -1 h -1 . Contrarily, a nitrogen dosing of 1.2 g-NH 4 Cl L -1 led to a sudden increase in the bioH 2 production, reaching productivity and yield values of 283 NmL-H 2 L -1 h -1 and 82 NmL-H 2 g - 1 -volatile solids added, respectively. The further addition of FeSO 4 ·7H 2 O (0.05 g L -1 ) did not improve TV-to-bioH 2 conversion. Bridging hydrogen production and organic acids with molecular analysis, 16S rRNA next-generation sequencing, indicated that maintaining a suitable C/N ratio in the fermenter could ensure balance and syntrophy between lactic acid bacteria (e.g., Sporolactobacillus , Lactobacillus ) and bioH 2 producers ( Clostridium ). Further biochemical methane potential tests with the fermentation broth demonstrated that biohythane process (bioH 2 + CH 4 ) resulted in a 7.4% higher bioenergy recovery than that derived from the single-stage anaerobic digestion.

Keywords: Biohythane, Biogas, Dark fermentation, Hydrogen-producing bacteria, hydrogen production, Nutrient supply