Genome-centric metagenomic insights into the impact of alkaline/acid and thermal sludge pre-treatment on digestion sludge microbiome

Pre-treatment of waste activated sludge (WAS) is an effective way to destabilize sludge floc structure and release organic matter for improving sludge digestion efficiency. Nonetheless, impacts of WAS pre-treatment on digestion sludge microbiomes, as well as mechanistic insights into how sludge pre-treatment improve digestion performance, remain elusive. In this study, genome-centric metagenomic approach was employed to investigate the digestion sludge microbiome in four sludge digesters with different feeding sludge: APAD, WAS pre-treated with 0.25 mol/L alkaline/acid; HS-APAD, WAS pre-treated with 0.8 mol/L alkaline/acid; Thermal-AD, thermal pre-treated WAS; Control-AD, fresh WAS. We retrieved 254 metagenomic-assembled genomes (MAGs) to identify the key functional populations involved in methanogenic digestion process. These MAGs span 28 phyla with 69 of them as yet-to-be-cultivated lineages, and 30 novel lineages were characterized with metabolic potential associated with hydrolysis and fermentation. Interestingly, functional populations involving carbohydrate digestion were enriched in APAD and HS-APAD, while lineages related to protein and lipid fermentation were enriched in Thermal-AD, corroborating different substrates released from alkaline/acid and thermal pre-treatments. Of the major functional populations (i.e., fermenters, syntrophic acetogens and methanogens), significant correlations between genome sizes and abundance of the fermenters were observed, particularly in the APAD and HS-APAD with improved digestion performance. IMPORTANCE Wastewater treatment generates large amounts of waste activated sludge (WAS), which mainly consist of recalcitrant microbial cells and particulate organic matter. Though WAS pre-treatment is an effective way to release sludge organic matter for subsequent digestion, information on detailed impacts of the sludge pre-treatment on digestion sludge microbiome remain scarce. Our study provides unprecedented genome-centric metagenomic insights into how WAS pre-treatments change the digestion sludge microbiomes, as well as their metabolic networks. Moreover, digestion sludge microbiome could be a unique source to explore microbial dark matter. These results may shed light on future optimization of methanogenic sludge digestion and resource recovery.