Paper production consumes large amounts of water and energy. This can be reduced by using a closed water cycle and producing primary energy by anaerobic water treatment. This water treatment cycle consists of UASB reactors for anaerobic water treatment, a subsequent stripping of carbon dioxide and waste gases, a lime trap and the final treatment of waste air using a trickle bed reactor and a biofilter. Continuous operation revealed the formation of biofilms and odor as some challenges related to the microbial communities established in the systems.

The study aims at a better understanding of microbial functions and to identify key targets for process optimization.

Combined metagenomics and metaprotomics were applied to analyze the microbial communities. Extracted microbial proteins from water cycle were tryptically digested, transferred to nanoHPLC-MS/MS (TimsTOF Pro) and identified by search against a protein sequence database based on corresponding metagenome data.

The combined metagenomics and metaproteomics approach showed a complex microbial community which clearly differs between the process stages: (i) samples from paper production and contaminated water showed higher abundance of Bacillalles and Lactobacillales, (ii) anaerobic treatment in UASB reactors contained more Methanomicrobiales, Enterobacterales and Thiotrichales, (iii) aerobic treatment and cleaned water and finally (iv) exhausted air treatment showed higher abundance of Burkholderiales. These differences correlated well with the different process conditions.

Functional analysis showed hydrogenotrophic methanogenesis in the UASB reactors and acidogenesis from organics as well as methanogenesis from methylated amines in the contaminated water from paper production. Detecting methanogenesis from methylated amines was surprising but correlated with anaerobic conditions in these samples. The detection of methanogenic functions in the final aerobic cleaned water could be explained by a partial loss of methanogenic biomass from the UASB reactor.

The metaproteome analysis of microbial communities from closed water cycle of a paper mill provided insight into community structure and functions. Data can be used to further optimize the system.