Microbial core communities in activated sludge plants are strongly affected by immigration and geography.

Background: The microbiota in wastewater treatment plants (WWTPs) and incoming wastewater is critical for the treatment process, the preservation of natural ecosystems and human health, and for the recovery of resources and achievement of sustainability goals. Both core species and conditionally rare and abundant taxa (CRAT) are considered process-critical but little is known about identity as well as true functional and ecological importance. Here, we present a comprehensive investigation of the microbiota of 84 municipal activated sludge (AS) plants with nutrient removal treating ~ 70% of all wastewater within a confined geographical area, Denmark (43,000 km²). With the use of an ecosystem-specific database (MiDAS 5.2), species-level classification allowed us to investigate the core and CRAT species, whether they were active, and important factors determining their presence.

Results: We established a comprehensive catalog of species with names or placeholder names showing each plant contained approx. 2,500 different species. Core and CRAT represented in total 258 species, constituting around 50% of all reads in every plant. However, not all core and CRAT could be regarded as process-critical as growth rate calculations revealed that 43% did not grow in the AS plants and were present only because of continuous immigration from the influent. Analyses of regional microbiota differences and distance decay patterns revealed a stronger effect for species than genera, demonstrating that geography had a clear effect on the AS microbiota, even across a limited geographical area such as Denmark (43,000 km²).

Conclusions: The study is the first comprehensive investigation of WWTPs in a confined geographical area providing new insights in our understanding of activated sludge microbiology by introducing a concept of combining immigration and growth calculation with identifying core and CRAT to reveal the true ecosystem-critical organisms. Additionally, the clear biogeographical pattern on this scale highlights the need for more region-level studies to find regional process-critical taxa (core and CRAT), especially at species and amplicon sequence variant (ASV) level.