Metagenomics and metatranscriptomics suggest pathways of 3-chloroaniline degradation in wastewater reactors

Biological wastewater treatment systems are often affected by shifts in influent quality, including the input of toxic chemicals. Yet the mechanisms underlying the adaptation of activated sludge process performance are rarely studied in a controlled and replicated experimental setting, particularly when challenged with a sustained toxin input. Three replicate bench-scale bioreactors were subjected to a chemical disturbance in the form of 3-chloroaniline (3-CA) over 132 days, after an acclimation period of 58 days, while three control reactors received no 3-CA input. Ammonia oxidation was initially affected by 3-CA. Within three weeks of the experiment, microbial communities in all three treatment reactors adapted to biologically degrade 3-CA resulting in partial ammonia oxidation recovery. Combining process and microbial community data from amplicon sequencing with potential functions gleaned from assembled metagenomics and metatranscriptomics data, two putative degradation pathways for 3-CA were identified. The first pathway, determined from metagenomics data, involves a benzoate dioxygenase and subsequent meta-cleavage of the aromatic ring. The second, determined from intensive short-term sampling for gene expression data in tandem with 3-CA degradation, involves a phenol monooxygenase followed by ortho-cleavage of the aromatic ring. The relative abundances of amplicon sequence variants associated with the genera Gemmatimonas, OLB8, and Taibaiella correlated significantly with 3-CA degradation. Metagenome-assembled genome data also showed the genus OLB8 to be differentially enriched in treatment reactors, making it a strong candidate as 3-CA degrader. Using replicated reactors, this study has demonstrated the impact of a sustained stress on the activated sludge process. The unique and novel features of this study include the identification of putative pathways and potential degraders of 3-CA using long-term and short-term sampling in tandem with multiple methods in a controlled and replicated experiment.Ministry of Education (MOE)National Research Foundation (NRF)This work was funded by the Singapore Ministry of Education and the National Research Foundation Singapore under an RCE grant awarded to the Singapore Centre for Environmental Life Sciences Engineering (SCELSE)

A genome-centric metagenomics approach to explain microbial community structure in anaerobic digesters

A functioning anaerobic digestion (AD) microbiome is integral for sludge management to be successful. Comprehensive ecological insights, and a full accounting of important microbial species, can help to improve and validate operational strategies. We analysed a time-series of metagenome samples obtained from full-scale anaerobic digesters and performed genome-resolved analysis to gain insight into the microbial community structure and potential functions of the AD microbiome. Ninety samples from three full-scale digesters were collected over a period of nine months and their nucleic acids extracted and subjected to shotgun sequencing (Illumina HiSeq2500; average 70M PE reads/sample). The raw reads were quality controlled, trimmed, assembled, binned, and dereplicated to obtain metagenome-assembled genomes (MAGs). Genome quality was assessed using the MIMAG criteria. The taxonomic assignment of the recovered MAGs was conducted using GTDB-Tk, and gene-level functional annotations were obtained using the KEGG, and CAZy databases. From ninety metagenome assemblies, 14,236 MAGs were recovered, of which 37%, 16%, and 1% satisfied medium-quality (Completeness > 50% and contamination 90% and contamination 90% and contamination <5% and presence of rRNA and tRNA genes), respectively. Taxonomical classification of the MAGs with at least medium quality (n =7666) revealed that 12.9%, 37.4%, and 77.1% of them belong to a novel family, genus, and species, respectively. A co-occurrence network analysis of the community structures in three replicate digesters revealed a highly interconnected network of microorganisms, suggesting the presence of a backbone in a functional AD microbial community. Functional analysis of the recovered MAGs showed the presence of three methanogenesis pathway modules, namely, methanogenesis via CO2, acetate, and trimethylamine. In addition, a specialization was observed in the hydrolytic bacterial community using CAZy annotation. In conclusion, we have obtained a catalogue of 166 MIMAG high-quality MAGs from a time-series metagenome survey of three full-scale anaerobic digesters situated in a tropical wastewater treatment plant, leading to novel ecological insights into the AD microbial community.Ministry of Education (MOE)National Research Foundation (NRF)Submitted/Accepted versionThis research was supported by the Singapore National Research Foundation (NRF) and the Ministry of Education under the Research Centre of Excellence Programme. Soheil A. Neshat acknowledges receipt of a Singapore International Graduate Award (SINGA)