Characterizing the growing microorganisms at species level in 46 anaerobic digesters at Danish wastewater treatment plants: A six-year survey on microbial community structure and key drivers.

Anaerobic digestion (AD) is a key technology at many wastewater treatment plants (WWTPs) for converting primary and surplus activated sludge to methane-rich biogas. However, the limited number of surveys and the lack of comprehensive datasets have hindered a deeper understanding of the characteristics and associations between key variables and the microbial community composition. Here, we present a six-year survey of 46 anaerobic digesters, located at 22 WWTPs in Denmark, which is the first and largest known study of the microbial ecology of AD at WWTPs at a regional scale. For three types of AD (mesophilic, mesophilic with thermal hydrolysis pretreatment, and thermophilic), we present the typical value range of 12 key parameters including operational variables and performance parameters. High-resolution bacterial and archaeal community analyses were carried out at species level using amplicon sequencing of >1,000 samples and the new ecosystem-specific MiDAS 3 reference database. We detected 42 phyla, 1,600 genera, and 3,584 species in the bacterial community, where 70% of the genera and 93% of the species represented environmental taxa that were only classified based on MiDAS 3 de novo placeholder taxonomy. More than 40% of the bacterial species were found not to grow in the mesophilic and thermophilic digesters and were only present due to immigration with the feed sludge. Ammonium concentration was the main driver shaping the bacterial community while temperature and pH were main drivers for the archaea in the three types of ADs. Sub-setting for the growing microbes improved significantly the correlation analyses and revealed the main drivers for the presence of specific species. Within mesophilic digesters, feed sludge composition and other key parameters (organic loading rate, biogas yield, and ammonium concentration) correlated with specific growing species. This survey provides a comprehensive insight into community structure at species level, providing a foundation for future studies of the ecological significance/characteristics and function of the many novel or poorly described taxa.