Monitoring of beta-proteobacterial ammonia oxidisers in waste water treatment plants differing in nitrification capacity

Nitrogen removal already is, or in a few years will be obligatory for both municipal and industrial wastewater treatment plants in Austria and the EC. Severe inhibition of the nitrification process is frequently observed in several cases when nitrification is studied in pilot- and full-scale plants making it difficult for the plant operator to meet the legal standards and the technical engineer to address the problem. Despite intensive research in the field of nitrification and nitrification inhibition, until now methodical shortcomings have prevented elementary investigations on the relation between microbial nitrification and basic abiotic conditions (e.g. wastewater composition, process parameters of wastewater treatment). The aim of this project is the application and development of new molecular technologies implying their molecular- genetic basics. This opens the possibility to monitor the microbial nitrification process on a molecular level using the respective gene encoding for AMO functioning as a key enzyme involved in microbial nitrification. These developed molecular approaches will be combined with already established methods in the investigation of wastewater treatment plants in order to achieve a more comprehensive insight into processes of microbial nitrification. The proposed study will allow to calculate explicit kinetic parameters for the microbial nitrification under specific environmental conditions and wastewater treatment processes. Finally this project will deliver strategies and suitable methods for elucidating the reasons of observed problems in nitrification in order to give engineers the clue for technical measures.

Studying the microbial process of nitrification in full-scale plants, severe inhibition of the nitrification process is observed especially at industrial plants treating process waters but also in municipal wastewater treatment plants. During incomplete nitrification the total nitrogen emission does not meet legal limits. According to the Austrian "Abwasseremissionsverordnung von 2001" for urban municipal wastewater (> 5.000 population equivalents) minimum reduction of 70% of total nitrogen has to be applied. For the upgrading and the design of new wastewater treatment plants (WWTPs) the monitoring of the bacterial community being responsible for the first step of the nitrification process is of great importance. Again and again nitrification breakdowns occurred at an activated sludge plant for biological pre-treatment of rendering plant effluents. As a consequence higher nitrogen emissions were observed. By molecular techniques only Nitrosospira sp. was detected in the activated sludge of this rendering plant with problems in nitrification. While reference plants without nitrification problems showed Nitrosomonas sp. as predominant ammonia oxidising bacteria (AOB). Further we could show that the nature of the process waters was responsible for the inhibiting effect. We confirmed our results based on molecular tools by a cultivation-based approach. During the time of incomplete nitrification, a new AOB species was enriched, isolated and identified. This species was named Nitrosospira sp. Nsp65. It forms an independent branch in the Nitrosospira lineage and currently represents the deepest branch within this evolutionary lineage. Summarizing, we were able to elaborate an applicable solution for the re-design and upgrading of the rendering plant. Also new, powerful molecular techniques and approaches were established for the investigation of the wastewater treatment processes. A simultaneous extraction protocol was established to obtain total RNA and genomic DNA from activated sludge. The gene encoding the active site of the ammonia monooxygenase (amoA) has been exploited as molecular marker for studying ammonia-oxidizing bacteria (AOB) diversity in the environment. We improved the detection of this functional marker gene, which facilitated further analyses.