Microbial effects of part-stream low-frequency ultrasonic pretreatment on sludge anaerobic digestion as revealed by high-throughput sequencing-based metagenomics and metatranscriptomics

Yu Xia, Chao Yang, Tong Zhang

Part-stream low-frequency ultrasound (LFUS) was one of the common practices for sludge disintegration in full-scale anaerobic digestion (AD) facilities. However, the effectiveness of part-stream LFUS treatment and its effect on AD microbiome have not been fully elucidated. Here we testified the effectiveness of part-stream LFUS pretreatment by treating only a fraction of feed sludge (23% and 33% total solid of the feed sludge) with 20 Hz LFUS for 70 s. State-of-the-art metagenomic and metatranscriptomic analysis was used to investigate the microbial process underpinning the enhanced AD performance by part-stream LFUS pretreatment. By pretreating 33% total solid of the feed sludge, methane yield was increased by 36.5%, while the volatile solid reduction ratio remained unchanged. RNA-seq of the microbiome at stable stage showed that the continuous dosage of easy-degradable LFUS-pretreated feed sludge had gradually altered the microbial community by selectingBacteroidaleshydrolyzer with greater metabolic capability to hydrolyze cellulosic biomass without substrate attachment. Meanwhile,Thermotogaleswith excellent cell mobility for nutrient capturing was highly active within the community. Foremost proportion of the methanogenesis was contributed by the dominantMethanomicrobialesvia carbon dioxide reduction. More interestingly, a perceivable proportion of the reverse electron flow of the community was input fromMethanoculleusspecies other than syntrophic acetate-oxidizing bacteria. In addition, metagenomic binning retrieved several interesting novel metagenomic-assembled genomes (MAGs): MAG-bin6 ofAlistipes shahiishowed exceptional transcriptional activities towards protein degradation and MAG-bin11 of CandidatusCloacimoneteswith active cellulolytic GH74 gene detected. In summary, despite the unchanged sludge digestibility, the applied part-stream LFUS pretreatment strategy was robust in adjusting the microbial pathways towards more effective substrate conversion enabled by free-living hydrolyser and beta-oxidation-capable methanogens.