Membrane protection by glycerol sustains energy metabolism and electron transfer for anaerobic digestion under cold shock.

Temperature fluctuations often expose microbial aggregates to cold shock, increasing the risk of destabilization in anaerobic digestion (AD) systems. The cell membrane is closely associated with key metabolic activities and is highly susceptible to low-temperature stress. This study suggested that cold shock-induced membrane damage was an important contributor to impaired energy metabolism and electron transfer in AD, and provided evidence that glycerol can mitigate such damage by protecting cell membranes. Specifically, cold shock reduced membrane fluidity and integrity, suppressing ATP synthesis and membrane-associated electron transfer, resulting in electron flow toward reductive byproducts such as lactate and ethanol. In contrast, glycerol enhanced cytoprotection by stabilizing membrane structure during cold shock, increasing membrane fluidity and cell viability by 18.0% and 25.4%, respectively. This effect maintained membrane-associated ATP synthesis and promoted direct interspecies electron transfer (DIET) between electroactive bacteria (Brooklawnia) and methanogens (Methanothrix, Methanobacterium). It provided additional energy and electron for acetate activation and carbon dioxide reduction, respectively, with CH4 production increasing by 9.7%. These findings suggest that glycerol-mediated membrane protection can contribute to maintaining metabolic stability and methane production in anaerobic digestion under cold-shock conditions.