Effects of multiple electron acceptors on microbial interactions in a hydrogen-based biofilm

To investigate interactions among multiple electron acceptors in a H ₂-fed biofilm, we operated a membrane biofilm reactor with H ₂-delivery capacity sufficient to reduce all acceptors. ClO ₄^- and O₂ were input electron acceptors in all stages at surface loadings of 0.08 ± 0.006 g/m²-d (1.0 ± 0.7 e^- meq/m²-d) for ClO₄^- and 0.51 g/m²-d (76 e^- meq/m²-d) for O ₂. SO₄^2- was added in Stage 2 at 3.77 ± 0.39 g/m²-d (331 ± 34 e^- meq/m²-d), and NO₃^- was further added in Stage 3 at 0.72 ± 0.03 g N/m²-d (312 ± 13 e^- meq/m²-d). At steady state for each stage, ClO₄^-, O₂, and NO ₃^- (when present in the influent) were completely reduced; measured SO₄^2- reduction decreased from 78 ± 4% in Stage 2 to 59 ± 4% in Stage 3, when NO₃^- was present. While perchlorate-reducing bacteria (PRB), assayed by qPCR targeting the pcrA gene, remained stable throughout, sulfate-reducing bacteria (SRB), assayed by the dsrA gene, increased almost 3 orders of magnitude when significant SO₄^2- reduction occurred in stage 2. The abundance of denitrifying bacteria (DB), assayed by the nirK and nirS genes, increased in Stage 3, while SRB remained at high numbers, but did not increase. Based on pyrosequencing analyses, β-Proteobacteria dominated in Stage 1, but ε-Proteobacteria became more important in Stages 2 and 3, when the input of multiple electron acceptors favored genera with broader electron-accepting capabilities. Sulfuricurvum (a sulfur oxidizer and NO ₃^- reducer) and Desulfovibrio (a SO₄ ^2- reducer) become dominant in Stage 3, suggesting redox cycling of sulfur in the biofilm.