Activated sludge prokaryote and eukaryote characterization in a pulp mill facility using amplicon sequencing

The pulp and paper industry, a major global sector, supports economies and jobs while contributing to various products. While providing valuable products, and despite Best Available Techniques (BAT) being used, managing wastewater effectively remains a key area for developing technologies and alternatives for environmental protection. Activated sludge (AS) systems are commonly used for effluent treatment, where microorganisms composition influences reactor efficiency. Current monitoring strategies show limited correlation with chemical parameters. Metabarcoding, a DNA sequencing approach, offers a promising alternative to profile microbial communities comprehensively. Utilizing MinION and Illumina sequencing, this study reveals the prokaryotic and eukaryotic microbial communities in AS from an extended aeration-activated sludge reactor from a pulp mill for the first time. Eukaryotic composition evaluated by 18S amplicon sequencing revealed Protists as the dominant group, followed by Animalia and Fungi. Within these groups, the most abundant phyla detected were Ciliophora (75 % on average), Rotifera (90 % on average), and Cryptomycota (60 % on average), respectively. Genera like Prorodon and Limnias stand out due to their high relative abundance within the Protist and Animalia group, respectively. A comparative analysis with routine microorganism composition assessment by microscopy revealed consistent taxonomic trends, affirming the feasibility of amplicon sequencing for routine microbial community surveillance. Furthermore, the incorporation of metabarcoding enhanced the depth of results. Prokaryotic composition analyzed by 16S amplicon sequencing revealed microorganisms previously described as relevant to the wastewater treatment process but highlighted dominant specific groups even when compared to other pulp facilities. Genera like Terrimonas, Defluviicoccus, and Methylocaldum appear as the most abundant among the identified genera. For proper reactor operation, microorganisms must aggregate around organic matter in flocs, which reflect reactor health and are composed of 5 % eukaryotes and 95 % prokaryotes. Hence, the characterization of both groups of microorganisms is crucial. Differences in communities between the wastewater treatment plant's aeration zones emphasize the importance of comprehensive community characterization. The study underscores metabarcoding's potential as a monitoring tool, for future linking of microbial communities with process patterns for improved environmental management in the pulp and paper industry and emphasizes the importance of establishing unique baselines for microbial communities in activated sludge systems.