Network analysis of the microorganism in 25 Danish wastewater treatment plants over 7 years using high-throughput amplicon sequencing

Wastewater treatment is the world’s largest biotechnological processes and a perfect model system for microbial ecology as the habitat is well defined and replicated all over the world. Extensive investigations on Danish wastewater treatment plants using fluorescent in situ hybridization have identified 38 probe-defined core genera, which are shared among all investigated Danish plants. A large body of knowledge exists on many of the core genera, however few attempts have been made to integrate the knowledge on a system-level understanding of the process. In this work we aimed to integrate the existing knowledge on core genera with high-throughput amplicon sequencing, plant design and process data in order to identify interactions and community shaping factors.
We investigated 25 Danish full-scale wastewater treatment plants with nutrient removal over a period of 7 years with two to four samples a year, totaling over 400 samples. All samples were subjected to 16S rDNA amplicon sequencing using V13 primers on the Illumina MiSeq platform (2x300bp) to a depth of at least 20.000 quality filtered reads per sample. The OTUs were assigned taxonomy based on a manually curated version of the greengenes database, which integrates the current knowledge of the core species in wastewater treatment plants (www.midasfieldguide.org). For each sample the wastewater treatment plants provided operational data, and selected samples were subjected to extensive physio-chemical analysis and shear tests in order to link sludge and floc properties to the microbial communities. All data was subjected to extensive network analysis and multivariate statistics through R.
The 16S amplicon results confirmed the findings of relatively few core groups of organism shared by all the wastewater treatment plants, in the majority of cases consisting of few OTUs. Interestingly, the 25 plants were largely stable over the seven years and clustered into a few large groups, which could be related to plant design. Network analysis identified larger guilds of co-existing species with relatively little competition. Using the sludge and floc properties in combination with the 16S data we were able to assign basic floc properties to hitherto completely undescribed species.
This work presents a significant effort to define the healthy state of the core microbiome in Danish wastewater treatment plants and will be a useful reference for future studies of this exceptional model ecosystem.