Sulfate dosing strategies regulate anaerobic propionate degradation through different electron transfer pathways for enhanced methane production

In anaerobic systems, sulfate-reducing bacteria (SRB) and methanogens determine carbon conversion and energy recovery. However, how sulfate dosing strategies influence microbial interactions and electron transfer pathways remains unclear. In this study, three anaerobic sequencing batch reactors (ASBRs) fed with a defined propionate medium were operated for 110 days under different sulfate addition strategies (no addition, continuous addition, and intermittent addition with 800 mg/L sulfate). Reactor performance, microbial community structure, and functional pathways were investigated using batch activity tests, 16S rRNA gene sequencing, and PICRUSt2-based functional prediction. Compared with continuous sulfate addition, intermittent sulfate addition resulted in a 3.12-fold higher methane production during sulfate-feeding periods and a 2.36-fold higher methane production during sulfate-free periods. The microbial community retained both sulfate reduction and propionate degradation potential, as evidenced by measurable sulfate reduction and sustained propionate degradation under sulfate-limited conditions. Hydrogenotrophic methanogens ( Methanobacterium ) were enriched under sulfate-free and intermittent-dosing conditions, while continuous sulfate addition suppressed their activity. Continuous sulfate addition was associated with a higher abundance of genes related to extracellular electron transfer, suggesting the potential involvement of direct interspecies electron transfer (DIET)-associated interactions. In contrast, intermittent sulfate exposure reduced hydrogen competition and supported both hydrogen-mediated syntrophy and potential DIET-associated interactions. Overall, this study demonstrates that sulfate dosing strategies strongly influence microbial interactions and electron transfer pathways. Intermittent sulfate addition promoted greater microbial functional resilience than continuous sulfate addition, resulting in superior methane production and propionate degradation.