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A continuously operated, thermophilic, municipal biogas plant was observed over 26 months (sampling twice per month) in regard to a number of physicochemical parameters and the biogas production. Biogas yields were put in correlation to parameters such as the volatile fatty acid concentration, the pH and the ammonium concentration. When the residing microbiota was classified via analysis of the 16S rRNA genes, most bacterial sequences matched with unidentified or uncultured bacteria from similar habitats. Of the archaeal sequences, 78.4% were identified as belonging to the genus Methanoculleus, which has not previously been reported for biogas plants, but is known to efficiently use H(2) and CO(2) produced by the degradation of fatty acids by syntrophic microorganisms. In order to further investigate the influence of varied amounts of ammonia (2-8 g/L) and volatile fatty acids on biogas production and composition (methane/CO(2)), laboratory scale satellite experiments were performed in parallel to the technical plant. Finally, ammonia stripping of the process water of the technical plant was accomplished, a measure through which the ammonia entering the biogas reactor via the mash could be nearly halved, which increased the energy output of the biogas plant by almost 20%.

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Funding information Deutsche Bundesstiftung Umwelt (DBU) Rationale: This study aims to develop a simplified denitrifier method for the δ 15 N and δ 18 O analysis of nitrate (NO 3 −) in natural water samples combining the method of Zhu et al (Sci Total Environ. 2018; 633: 1370-1378) and the original denitrifier method of Sigman et al (Anal Chem. 2001; 73: 4145-4153). Unlike in the aforementioned methods, the aerobic cultivation was performed without the addition or removal of nitrate in the liquid medium. We remove the nitrate contained in the nutrient medium as N 2 O in the gas phase by an additional purging step after incubation overnight before the water sample is injected. This eliminates the need for another preparation step, thus saving working time. Methods: The δ 15 N and δ 18 O values of dissolved NO 3 − were determined using a Delta V Plus isotope ratio mass spectrometer coupled to a GasBench II sample preparation device that included a denitrification kit. Results: After optimising the influencing factors (i.e., purging gas, purging time, and type of crimp seals), the method yielded high accuracy and precision (standard deviations were generally ≤0.7‰ for δ 18 O values and ≤0.3‰ for δ 15 N values), confirming the suitability of this procedure. Finally, the potential applicability of the method was demonstrated by measuring the isotopic composition of NO 3 − in natural water samples. Conclusions: The denitrifier method for converting NO 3 − into N 2 O for isotope analysis was optimised. This allowed the sample preparation time to be further reduced. The complete working time for sample preparation, including all steps, takes 10 min per vial if 60 vials are prepared in one run. The water samples are ready for isotope analysis on the fourth day after preparation has started. Isotope measurements can be performed up to 14 days after preparation. 1 | INTRODUCTION Understanding the geochemical processes that occur in the subsoil is necessary for risk assessments of groundwater and the protection of water resources. The identification of NO 3 − sources and the quantification of NO 3 − degradation in groundwater are particularly important. 1 As NO 3 − from different sources, such as fertiliser, wastewater, or animal waste, tends to exhibit characteristic isotopic compositions, its origin can be inferred by measuring the ratios of the heavy to light isotopes of nitrogen and oxygen. 2-4 In addition, biological processes can change these isotope ratios. Thus, isotope analysis facilitates the study of these processes 2 and is frequently used in the environmental sciences. 3 To determine the isotopic composition of NO 3 − in aqueous samples, it must first be