Riboflavin-Functionalized Conductive Material Enhances a Pilot-Scaled Anaerobic Digester Fed with Cattle Manure Wastewater: Synergies on Methanogenesis and Methanosarcina barkeri Enrichment

Anaerobic digestion (AD) technology is universally acknowledged as the most economically viable and efficient approach for energy recovery from livestock manure. To validate the efficacy of riboflavin-functionalized carbon-based conductive materials (CCM-RF) in enhancing methane production at pilot scale, three pilot-scale upflow anaerobic sludge blanket (UASB) reactors were constructed and separately supplemented with carbon cloth (CC), granular activated carbon (GAC), and a combination of CC and GAC. During reactor initialization, riboflavin and a concentrated inoculum of Methanosarcina barkeri (M. barkeri) were introduced to investigate the mechanistic role of CCM-RF in promoting direct interspecies electron transfer (DIET) and optimizing treatment efficiency during anaerobic digestion of cattle manure wastewater. The results showed that all reactors improved AD performance and maintained stable operation at the OLR of 15.66 ± 1.95 kg COD/(m3·d), with a maximum OLR of 20 kg COD/(m3·d) and the HRT as short as 5 days. Among the configurations, the CC reactor outperformed the others, achieving a methane volumetric yield of 6.42 m3/(m3·d), which represents an eight-fold increase compared to conventional AD systems. Microbial community analysis revealed that, although M. barkeri was initially inoculated in large quantities, Methanothrix—a methanogen with DIET capability—eventually became the dominant species. The enrichment of Methanothrix and the simultaneous enhancement in sludge conductivity collectively verified the mechanistic role of CCM-RF in promoting CO2-reductive methanogenesis through strengthened DIET pathways. Notably, M. barkeri showed progressive proliferation under conditions of high organic loading rates (OLR) and short hydraulic retention time (HRT). This phenomenon provides a critical theoretical basis for the development of future strategies aimed at the targeted enrichment of Methanosarcina-dominant microbial consortia.