Multiple syntrophic interactions drive biohythane production from waste sludge in microbial electrolysis cells

Background: Biohythane is a new and high-value transportation fuel present as a mixture of biomethane and biohydrogen. It has been produced from different organic matters using anaerobic digestion. Bioenergy can be recovered from waste activated sludge through methane production during anaerobic digestion, but energy yield is often insufficient to sludge disposal. Microbial electrolysis cell (MEC) is also a promising approach for bioenergy recovery and waste sludge disposal as higher energy efficiency and biogas production. The systematic understanding of microbial interactions and biohythane production in MEC is still limited. Here, we report biohythane production from waste sludge in biocathode microbial electrolysis cells and reveal syntrophic interactions in microbial communities based on high-throughput sequencing and quantitative PCR targeting 16S rRNA gene. Results: The alkali-pretreated sludge fed MECs (AS-MEC) showed the highest biohythane production rate of 0.148 L.L-1-reactor.day(-1), which is 40 and 80 % higher than raw sludge fed MECs (RS-MEC) and anaerobic digestion (open circuit MEC, RS-OCMEC). Current density, metabolite profiles, and hydrogen-methane ratio results all confirm that alkali-pretreatment and microbial electrolysis greatly enhanced sludge hydrolysis and biohythane production. Illumina Miseq sequencing of 16S rRNA gene amplicons indicates that anode biofilm was dominated by exoelectrogenic Geobacter, fermentative bacteria and hydrogen-producing bacteria in the AS-MEC. The cathode biofilm was dominated by fermentative Clostridium. The dominant archaeal populations on the cathodes of AS-MEC and RS-MEC were affiliated with hydrogenotrophic Methanobacterium (98 %, relative abundance) and Methanocorpusculum (77 %), respectively. Multiple pathways of gas production were observed in the same MEC reactor, including fermentative and electrolytic H-2 production, as well as hydrogenotrophic methanogenesis and electromethanogenesis. Real-time quantitative PCR analyses showed that higher amount of methanogens were enriched in AS-MEC than that in RS-MEC and RS-OCMEC, suggesting that alkali-pretreated sludge and MEC facilitated hydrogenotrophic methanogen enrichment. Conclusion: This study proves for the first time that biohythane could be produced directly in biocathode MECs using waste sludge. MEC and alkali-pretreatment accelerated enrichment of hydrogenotrophic methanogen and hydrolysis of waste sludge. The results indicate syntrophic interactions among fermentative bacteria, exoelectrogenic bacteria and methanogenic archaea in MECs are critical for highly efficient conversion of complex organics into biohythane, demonstrating that MECs can be more competitive than conventional anaerobic digestion for biohythane production using carbohydrate-deficient substrates. Biohythane production from