Understanding the microbial processes on carbon brushes that accelerate methanogenesis of long-chain fatty acids in anaerobic digestion.

Lipids offer high energy recovery potential during anaerobic digestion (AD), but their hydrolysis generates long-chain fatty acids (LCFAs), which are difficult to biodegrade. The introduction of microbial electrolysis cells has been widely recognized as a promising strategy to enhance AD. However, it is still under debate whether the electrical circuit needs to be connected, as certain electrodes with large specific surface areas have been reported to enhance direct interspecies electron transfer (DIET) without requiring an external power supply. Here we confirmed that the carbon brush anode pre-acclimated with electroactive bacteria (EAB) was able to accelerate LCFA methanation. Although the applied potential achieved a rapid methane production, the coupling of homoacetogenesis and electrogenesis consumed part of the bioelectrohydrogen, reducing the maximum methane production rate by 5-13 %. In the AD system with only carbon brushes added, the dominant methanogens shifted from Methanosarcina in solution to Methanothrix on brushes. Pre-enriching EAB further established a composite mechanism, with DIET driven by Syntrophomonas, Geobacter and Methanothrix as the primary pathway, and interspecies hydrogen transfer mediated by Methanospirillum as a complementary process, collectively optimizing LCFA methanation. Genetic regulation underlying microbial tolerance to high LCFA concentrations was then elucidated, underscoring the critical role of combining immobilized electrodes and pre-acclimated EAB in adapting to LCFA stress and improving lipid-rich wastewater treatment.