Maria Riese

• Extracellular enzymes in microbial communities play a central role in nutrient cycling and the degradation of (pollutant) substances in various natural and anthropogenic systems. Bound in aquatic biofilms, sludge aggregates, or even unbound at their interfaces, they are of great importance for both the environment and human health. In particular, in wastewater treatment plants and inland waters, hydrolytic activities influence the wide-reaching efficiency of nutrient removal and self-purification, thus contributing significantly to overall water quality. The main goal of this dissertation project was to investigate the factors that influence enzymatic activity and the health of microbial communities in activated sludge and river systems, particularly in relation to anthropogenic influences and natural environmental conditions. The aim was to contribute to a better understanding of the sensitivity of our freshwater ecosystems and to support the long-term preservation of water quality and ecological stability. The development and optimization of appropriate methods, as well as their testing and applicability, were the focal points. For this purpose, a fluorometric microplate assay was developed and adapted to determine both extracellular enzyme activities (EEAs) in activated sludge samples and in intact biofilms. Its suitability for field studies was subsequently tested. Inhibition and activity of selected hydrolases under different conditions were investigated to better understand the mechanisms and potential environmental risks posed by anthropogenic influences and seasonal fluctuations of hydrochemical and climatic parameters. The first phase of the doctoral thesis involved studies on the inhibition of alkaline phosphatase in activated sludge by oxyanions. Using the fluorometric microplate assay, the inhibitory effect was sensitively detected over a pH range of 7.0 to 8.5. IC50- and IC20-concentrations were calculated from modeled dose-response functions. It was found that vanadate and tungstate caused strong inhibitory effects, while molybdate moderately inhibited the enzyme. An increasing pH led to a reduction in the inhibitory effect of tungstate and molybdate. The inhibition effects of vanadate were not significantly affected by the pH. In municipal wastewater, the concentrations of such metal ions are usually low, but industrial wastewater may have pollutant loads that can significantly impact the removal of phosphorus-containing compounds, and thus the efficiency of treatment plants. In the second phase, an attempt was made to further adapt the developed methodology to investigate EEA and kinetics in intact freshwater biofilms. Four different types of bead materials (lava, glass, sintered quartz, and ceramics) fitting into a 96-well microplate were tested as carriers for biofilms on both the laboratory and field scale. The analysis included a total of seven hydrolases as representatives of key nutrient cycles such as phosphorus, carbon, and nitrogen: phosphatases, glucosidases, peptidases (two different types), and sulfatase. Experiments with increasing substrate concentrations led to classical kinetic profiles according to the Michaelis-Menten mechanism. This allowed for the prediction of the biofilm enzymes’ response to different substrate concentrations. Parameters such as Vmax and Km could be derived from the modeled curves. Ceramic beads are particularly suitable for long-term studies due to their high stability, while sintered quartz beads should be preferred for the use in stagnant media (material loss under turbulent conditions). Lava and glass beads, on the other and, proved suboptimal for uniform biofilm development due to their surface properties. The potential use of this fast and sensitive test for ecotoxicological or even human-toxicological studies was demonstrated by the effects of caffeine on the activity of PDE. The result of this part of the research represents a powerful tool for assessing environmental pollution and monitoring water quality. The high application potential was clearly highlighted in the final phase of the project. The goal here was to deepen the understanding of interactions between seasonal factors, anthropogenic influences, and biofilm processes in rivers by investigating EEA and biofilm parameters such as biomass and relating them to hydrochemical and climatic factors. Ceramic beads were exposed both upstream and downstream of a wastewater treatment plant discharge and sampled over a period of seven months. EEAs and biomass varied depending on the season and location, with higher microbial activity observed upstream in winter. Winter conditions led to the dilution of most nutrients as well as in an increse of dissolved oxygen. Nutrient concentrations analyzed downstream were significantly higher in the summer. Accumulation of nutrient or pollutants during the summer months cannot be excluded, which may have led to a general reduction in enzyme activities. Potential causes could be inhibitory effects on the enzymes, or a reduced enzyme activity due to a sufficiently high nutrient supply. In general, the sampling site upstream showed a more pronounced seasonal dynamics, with a significant proportion of the variance in biological parameters (activity and biomass) attributable to seasonal factors. A secondary component, likely reflecting the impact of the treatment plant discharge, explained another portion of the data variance. Regardless of the season, high correlations between biological parameters were observed upstream, while downstream the data were more decorrelated. This could be because the biofilms, under chronic stress, respond less dynamically to seasonal fluctuations. This dissertation illustrates that in addition to anthropogenic stress factors, seasonal fluctuations of hydrochemical and climatic parameters should also be considered in "stress downstream the pipe" studies. The selected methods are recommended for explaining and considering the data variance, as they highlight the complex interplay between microbial enzymatic activity, environmental factors, and pollutants in the activated sludge of wastewater treatment plants and also in aquatic systems. The novel bead assay could pave the way for the future standardization of effect-oriented studies on intact aquatic biofilms.