Stable isotope fingerprinting suggests progressive acetoclastic dominance and potential coal‑mediated DIET in algal hydrolysate‑lignite anaerobic co‑digestion.

Enhancing biogenic coalbed methane (CBM) production through algae-coal co-digestion has attracted increasing interest. However, the potential role of coal particles in mediating direct interspecies electron transfer (DIET) and the dominant methanogenic pathways in such systems remain unresolved. Laboratory-simulated coalbed bioreactors were established to investigate these questions, with stable isotope fingerprinting employed as an independent analytical tool to resolve methanogenic pathways beyond what community composition data alone can reveal. The co-digestion group with pre-activated inoculum (Group O) achieved the highest CH4 yield (1400.68 μmol/g COD) and effectively shortened the lag phase. Pre-activation selectively enriched the electroactive bacterium Clostridium alongside the methanogenic archaea Methanobacterium and Methanosarcina. Critically, the electroactive properties of Clostridium, acting in concert with coal particles, may have synergically contributed to DIET. Group O had the highest coenzyme F420 activity, which indirectly supports this result. The stable coexistence of Methanosarcina and Methanobacterium, uniquely established by inoculum pre-activation, reflected functional complementarity, with the former sustaining CH4 flux via acetoclastic methanogenesis and the latter maintaining redox balance through H2 consumption. Analysis of the apparent fractionation factor (αc) from stable isotope data suggested that the acetoclastic pathway dominated in methanogenesis in the co-digestion systems and that its contribution progressively strengthened during anaerobic digestion, supported by acetate consumption and enrichment of key acetoclastic genes (ackA, pta, cdh). These findings provide a mechanistic framework for in situ CBM bioaugmentation, in which co-digestion of algae with pre-activated inocula enriched in electroactive bacteria can simultaneously stimulate DIET potential and sustain acetoclastic methanogenic activity in coal seam environments.