Waste activated sludge (WAS) from wastewater treatment plants contains high levels of organic matter in forms of cells, extracellular polymeric substances (EPS) and macromolecules generated from cell lysis, as well as pathogens and other biohazards [1, 2]. Anaerobic digestion as a sustainable sludge treatment technology can convert these organic substances into biogas via a multiple-step process consisting of hydrolysis, fermentation, acetogenesis and methanogenesis . The organic matter (e.g., microbial cells and EPS), together with metals and other ions, in WAS form stable and complicated sludge flocs. These sludge flocs are recalcitrant to anaerobic digestion and consequently require sludge pre-treatment to destabilize their structure and release organic matter for improving digestion efficiency . Many pre-treatment techniques (e.g., thermal, ultrasonic, microwave and alkaline pre-treatments) have been developed to effectively release sludge organic matter [5, 6, 7]. For example, thermal hydrolysis (> 100 °C), ultrasonication, microwave and alkaline pre-treatments could enhance sludge fermentation by 4–20%, 1–18%, 1–15% and 3–35%, respectively, compared to their non-pretreated controls . In our previous studies, a new alkaline/acid pre-treatment and anaerobic digestion (APAD) process was developed, in which organic carbon removal achieved 52.8 ± 1.7% . By contrast, the organic carbon removals were significantly lower in other digesters under the same operational conditions but with different influent sludge, i.e., 42.4 ± 1.6% in Thermal-AD with thermal sludge pre-treatment and 30.9 ± 2.2% in Control-AD with fresh WAS . Further increasing alkaline/acid concentrations from 0.25 mol/L (APAD) to 0.8 mol/L (HS-APAD) did not show notable changes in digestion performance and community taxonomic composition in the sludge digesters . Consequently, dissolved organic compounds (DOC) derived from sludge pre-treatments, rather than salinity, could be a predominant selective pressure driving the performance and microbiome changes in both APAD and HS-APAD, compared to the Control-AD . Nonetheless, the detailed mechanistic insights into the digestion improvement and impact of WAS pre-treatment on digestion sludge microbiome remain unknown.
The conversion of WAS organic matter into biogas highly relies on the complex and tightly coupled synergistic interactions of microbial communities in digestion sludge . Previous studies based on 16S rRNA gene amplicon sequencing showed that relative abundance of bacteria and archaea in digestion sludge microbiomes generally accounted for > 95% and < 5%, respectively [10, 11], being further confirmed by recent metagenomics analyses [9, 12]. In view of the bacterial community, Bacteroidetes, Proteobacteria, Spirochaetes and Firmicutes were generally the dominant phyla in sludge digesters, and most of them were fermentative bacteria with high compositional and functional redundancy [9, 13–16]. By contrast, the slow-growing acetogenic syntrophs and methanogenic archaea in digestion sludge were limited to several lineages [9, 11]. Accumulating experimental evidences suggested that a variety of factors including feeding substrates and operational parameters (e.g., pH, temperature and ammonia) might change community composition and function of the digestion sludge microbiome [11, 12, 16, 17]. The information generated from the 16S rRNA gene-based analyses was largely limited to community composition and succession . Metagenome sequencing could theoretically obtain all microbial genome information within a sample, which might provide direct access to the metabolic potential and networks in the highly complex digestion sludge microbiome. Early metagenomic studies mainly relied on gene-centric analyses, which were biased towards existing databases [9, 19, 20]. Current advances in both high-throughput sequencing technologies and population genome binning algorithms allowed the development of genome-centric approaches for the assessment of complex microbiomes [12, 18, 21]. For example, the genome-centric metagenomics analysis was employed to recover 101 population genomes and revealed their metabolic potential and interactions in a cellulose-degrading digester . Very recently, a collection of 1,635 metagenome-assembled genomes were recovered from publicly available datasets derived from different methanogenic digesters, showing high species diversity related to methane generation . Also, metagenomics changed the pace of virus discovery by enabling the accurate identification of viral genome sequences without requiring isolation of viruses [22, 23]. In contrast to increasing metagenomic data on anaerobic digestion, no information was available on impacts of WAS pre-treatment on digestion sludge microbiome. It would be rational to assume that the genome-centric and strain-resolved metagenomic approaches could provide a systematic understanding of digestion sludge microbiomes, particularly the yet-to-be elucidated impact of WAS pre-treatment on the digestion sludge microbiome.
In this study, we employed metagenomic approach to explore prokaryotic and DNA viral community composition and function of digestion sludge microbiomes in four sludge digesters (i.e., APAD, HS-APAD, Thermal-AD and Control-AD). Co-assembly of the four metagenomes followed by genomic binning resulted in the recovery of 254 population genomes that constituted the majority of the digestion sludge microbiome. Their metabolic potential and networks were further reconstructed to reveal impacts of WAS pre-treatment on the digestion sludge microbiome. The results provided the first genome-centric insight into how WAS pre-treatment change community composition and function, as well as metabolic networks of key players and their genomic traits in the digestion sludge microbiome.