Exploring the microbial dark matter in wastewater treatment plants

Abstract

This thesis aims to explore and reveal new insights into the microbial dark matter (MDM) conditions in wastewater treatment plants (WWTPs) with the methods of big data mining, bioinformatic analysis, cultivation, and sequencing technology. Considering the composition of microbial community in WWTPs, two kinds of MDM (prokaryotes and phages) was studied in this research.

Firstly, a global meta-analysis of MDM in WWTPs was conducted by linking sequenced 16S rRNA genes with 317,542 prokaryotic genomes. Results showed that the median proportions of the genome-sequenced cells and taxa (100% identity and 100% coverage in 16S rRNA gene region) in WWTPs reached 56.3% and 34.5% for activated sludge, 48.6% and 28.5% for aerobic biofilm, and 48.3% and 28.5% for anaerobic digestion sludge, respectively. The global-scale “wanted list” for activated sludge was defined to point out targets for future research, which contained four phyla that had few representatives and 71 operational taxonomic units (OTUs) with the majority of them did not have any genome or isolate yet. Then, a 13-month community survey of 6 full-scale WWTPs in Hong Kong was conducted to expand the understanding of prokaryotes in different WWTPs by exploring their corresponding diversity and compositions, assembly mechanisms, and co-occurrence patterns. Although 6 WWTPs all located in Hong Kong with limited geographical variations, their community diversity and structure were still very different. However, there were still universal relationships between different prokaryotes that widely existed in WWTPs, such as the OTUs belonging to Nitrospira and Rhodocyclaceae. To get isolates of MDM for further research, a workflow for the isolation of new species was established and applied to activated sludge samples. This method successfully enriched important groups such as bacteria belonging to phyla of Patescibacteria and Verrucomicrobia and isolated three new species from phyla of Bacteroidetes, Proteobacteria, Actinobacteria, respectively. Besides, two (with or without unique molecular identifier) rapid and accurate sequencing methods for the full-length 16S rRNA gene were established with Nanopore sequencing platform to provide rapid identification of prokaryotic communities and powerful guidance for enrichment. Together, these two methods could provide different accuracy rate (Q10 (90%), Q20 (99%), and Q30 (99.9%), respectively) for various research aims for both mock and real samples.

Except for prokaryotes, phages are important groups for MDM. Four phage concentration methods (flocculation, ultrafiltration module, tangential flow filtration, and ultracentrifugation) for activated sludge samples were investigated, compared, and summarized. Results showed that different pretreatment methods could get different phage catalogs and only a small fraction (~1%) of phages could be annotated.

(411 words)