Direct interspecies electron transfer-based methanogenic aggregate: A survival strategy to overcome defensive attack from type VI secretion system during syntrophic cooperation.

Physically tight structure of methanogenic aggregates formed by syntrophic microbes that exchange electrons via interspecies hydrogen/formate transfer (IHT/IFT) can activate defensive attack from type VI secretion system (T6SS), which has been recognized as the primary cause for poor stability. Direct interspecies electron transfer (DIET) may alleviate the technical bottleneck of proximity-triggered defensive attack from T6SS, since syntrophic microbes function long-distance electron transfer via electrically conductive pili (e-pili) or its displayed c-type cytochromes. Here, three up-flow anaerobic sludge blanket reactors, respectively with ethanol, propionate, and butyrate as a sole substrate, were used to culture DIET- and IHT/IFT-based aggregates. DIET-based aggregates were generally larger and exhibited a looser, porous structure compared to IHT/IFT-based aggregates. However, rheological behavior showed that they possessed higher rigidity and toughness, attributed to the structural support of the conductive pili network. 3D reconstruction and imaging of a single DIET-based aggregate by nano-industrial computed tomography showed that syntrophic microbes did not display a pronounced localized aggregation pattern. Conductivity-temperature/pH response showed that the DIET-based aggregates exhibited a metallic-like conductance similar to that found in e-pili. Meanwhile, the surface-enhanced Raman spectra showed that the intensities of characteristic peaks associated with c-type cytochromes in DIET-based aggregates were higher than those in IHT/IFT-based aggregates. Analysis of metagenomic and metaproteomic data showed that in DIET-based aggregates expression of key proteins of T6SS was suppressed. These results demonstrated that in DIET-based aggregates syntrophic microbes did not aggregate to form a physically tight structure, eluding defensive attack from T6SS and strengthening their stabilities.