Co-culturing Bacillus subtilis and wastewater microbial community in a bio-electrochemical system enhances denitrification and butyrate formation

Bio-augmentation could be a promising strategy to improve processes for treatment and resource recovery from wastewater. In this study, the Gram-positive bacterium Bacillus subtilis was co-cultured with the microbial communities present in wastewater samples with high concentrations of nitrate or ammonium. Glucose supplementation (1%) was used to boost biomass growth in all wastewater samples. In anaerobic conditions, the indigenous microbial community bio-augmented with B. subtilis was able to rapidly remove nitrate from wastewater. In these conditions, B. subtilis overexpressed nitrogen assimilatory and respiratory genes including nasD, nasE, narG, narH, and narI, which arguably accounted for the observed boost in denitrification. Next, we attempted to use the ammonium- and nitrate-enriched wastewater samples bio-augmented with B. subtilis in the cathodic compartment of bioelectrochemical systems (BES) operated in anaerobic condition. B. subtilis only had low relative abundance in the microbial community, but bio-augmentation promoted the growth of Clostridium butyricum and C. beijerinckii, which became the dominant species. Both bio-augmentation with B. subtilis and electrical current from the cathode in the BES promoted butyrate production during fermentation of glucose. A concentration of 3.4 g/L butyrate was reached with a combination of cathodic current and bio-augmentation in ammonium-enriched wastewater. With nitrate-enriched wastewater, the BES effectively removed nitrate reaching 3.2 mg/L after 48 h. In addition, 3.9 g/L butyrate was produced. We propose that bio-augmentation of wastewater with B. subtilis in combination with bioelectrochemical processes could both boost denitrification in nitrate-containing wastewater and enable commercial production of butyrate from carbohydrate- containing wastewater, e.g. dairy industry discharges. These results suggest that B. subtilis bio-augmentation in our BES promotes simultaneous wastewater treatment and butyrate production.