Lactate as co-substrate to biostimulate sulfidogenic bioreactors using brewery spent grain as slow-release electron donor for acid mine drainage treatment

Bioremediation using sulfate-reducing bacteria (SRB) for treating acid mine drainage (AMD) offers benefits such as low energy consumption, minimal sludge generation, and metal recovery. However, AMD is carbon-deficient and necessitates external electron donors, thereby increasing costs. Lignocellulosic materials function as cost-effective slow-release electron donors. This study investigated lactate as a co-substrate to stimulate SRB activity in sulfate-reducing reactors supplied with brewery spent grain (BSG). Two down-flow structured bed reactors (R BSG and R co-sub ) were operated, with lactate supplementation (300 mg L −1 as COD) exclusively in R co-sub . After stable sulfide production was established, copper and zinc were sequentially introduced at 10 mg L −1 and then 50 mg L −1 of each metal to evaluate metal toxicity resistance. In the initial phase, R co-sub exhibited superior sulfide production (150.0 mg L −1 ) and a more favorable redox potential (−250 mV) compared to R BSG (90.0 mg L −1 and -100 mV). The removal of cysteine increased influent redox potential, which in turn lowered sulfide production. However, R co-sub maintained superior sulfide production throughout operation, even under metal exposure. Prolonged metal exposure eventually inhibited both reactors at concentrations of 50 mg L −1 . Batch tests with varied BSG masses indicated lower BSG loads improved sulfide production, linked to more favorable COD/SO₄ 2− ratios (1.0 and 0.3). 16S rRNA sequencing revealed SRB enrichment in both reactors compared to the inoculum, with R BSG showing higher relative abundance, although fermentative bacteria dominated. This study concludes BSG effectively serves as a long-term slow-release electron donor for sulfate reduction, and a readily degradable co-substrate significantly enhances bioreactor start-up. • Lactate enhanced sulfate-reducing bacteria during reactor start-up phase. • Brewery spent grain can be used as long-term slow-release electron donor for biological sulfate reduction. • Residual sugars in the brewery spent grain accelerated fermentative microbial growth. • The reactor supplemented with lactate had a higher sulfide production during all operation. • Microbial community analysis revealed a larger population of sulfate reducers in the absence of co-substrate.