Illuminating the Ecophysiology of Nitrotoga-like NOB

Nitrification is a two-step process catalyzed exclusively by microorganisms. First, ammonia is converted to nitrite by ammonia-oxidizing bacteria and archaea. Consecutively, nitrite is released and serves as substrate for nitrite- oxidizing bacteria (NOB). These convert the nitrite to nitrate, the end-product of nitrification. As nitrification is a key process of the biogeochemical nitrogen cycle, the nitrifying microorganisms are important drivers of N-cycling in nature and occur in virtually all habitats where their substrates ammonia, nitrite, and oxygen are available. In recent years, with the description of various Nitrospira lineages, Nitrotoga, and even phototrophic NOB, a surprising diversity of novel nitrite oxidizers has been discovered in natural and engineered ecosystems. This diversity sharply contrasts the still very limited insights into the biology of these microorganisms. The proposed project aims at bridging this gap for one of these novel groups, the genus Nitrotoga. A preliminary study using newly developed PCR primers and FISH probes already demonstrated that NOB closely related to "Candidatus Nitrotoga arctica" appear frequently in wastewater treatment plants (WWTPs) and can even constitute the sole known NOB in some technical systems. Based on these results as a starting point, this project will explore the abundance and physiological make-up of Nitrotoga-like bacteria. First, the already existing abundance data will be extended to cover not only engineered wastewater treatment systems, but also to get a glimpse at the distribution of Nitrotoga in selected terrestrial and freshwater habitats. Second, genomics will be employed to elucidate the genetic repertoire of two members of the genus Nitrotoga, one strain isolated from arctic permafrost soil and one from a WWTP. These genomes will allow alluring insights into the physiological potential of this novel genus and comparative analysis will further start to disclose the extent of change necessary to adapt from life in permafrost soils to activated sludge. Third, molecular tools like FISH-MAR will be used to directly prove the nitrite oxidizing lifestyle of Nitrotoga-like bacteria in an activated sludge sample, since the capability of purely chemolithoautotrophic growth with nitrite and CO 2 as sole energy and carbon source has so far only been observed for enrichment cultures. Further, the planned research will go beyond focus on one organism by monitoring in situ the competition of Nitrotoga and Nitrospira in activated sludge and by comparing the nitrite use efficiencies of Nitrotoga to those of Nitrobacter and Nitrospira - the most important and widespread known NOB in terrestrial and freshwater habitats and WWTPs. For this, incubations of NOB pure cultures will be performed in the presence of stable isotope-labeled substrates at different nitrite concentrations. Consecutively, a combination of whole-culture bulk measurements with single-cell NanoSIMS analyses will be employed to gain insights into nitrite use efficiencies of these NOB. In summary, the results obtained in this project will, on the one hand, identify factors supporting the growth and competitive success of Nitrotoga in natural and engineered systems, and, on the other hand, provide physiological parameters for Nitrotoga, Nitrospira, and Nitrobacter that are urgently needed for understanding and modeling nitrification. Such knowledge is becoming increasingly important for assessing human impact on the natural nitrogen cycle and will be relevant for research in Microbiology, Biogeochemistry, and Environmental Engineering.

Nitrification is a two-step process catalyzed exclusively by microorganisms. First, ammonia is converted to nitrite by ammonia-oxidizing bacteria and archaea. Consecutively, nitrite is released and serves as substrate for nitrite-oxidizing bacteria (NOB). These convert the nitrite to nitrate, the end-product of nitrification. As nitrification is a key process of the biogeochemical nitrogen cycle, the nitrifying microorganisms are important drivers of Ncycling in nature and occur in virtually all habitats where their substrates ammonia, nitrite, and oxygen are available. In recent years, with the description of various Nitrospira lineages, Nitrotoga, and even phototrophic NOB, a surprising diversity of novel nitrite oxidizers has been discovered in natural and engineered ecosystems. This diversity sharply contrasts the still very limited insights into the biology of these microorganisms. The proposed project aimed at bridging this gap for one of these novel groups, the genus Nitrotoga. To achieve this we firstly developed a set of molecular tools, so-called FISH probes and PCR primers, enabling us to detect NOB closely related to Nitrotoga arctica within their natural habitats. Applying these to samples from different wastewater treatment plants demonstrated that Nitrotoga-related NOB appear frequently in these systems and can even constitute the sole known NOB. Additionally, we could show that Nitrotoga-like NOB also are found in a range of soil and river sediment samples, indicating their environmental ubiquity. Secondly, we set out to prove the nitrite oxidizing lifestyle of Nitrotoga-like bacteria in activated sludge, since the capability to grow with nitrite and CO2 as sole energy and carbon source has so far only been observed for enrichment cultures. By combing microscopy with digital image analysis techniques we could demonstrate that Nitrotoga-like NOB always occur in close vicinity of ammonia-oxidizing bacteria in activated sludge flocs, strongly supporting their nitrite-oxidizing function. Finally, through incubation of live activated sludge samples in the presence of nitrite as energy source and radioactively labelled CO2 as carbon source, we could directly prove their role as previously overlooked NOB in wastewater treatment. These findings are of great value and interest to the scientific community and to wastewater engineers, as they demonstrate that previous research has missed a frequent key player in nitrification. Furthermore, the recognition of Nitrotoga will allow the design of more effective wastewater treatment plants especially in moderate and cold regions since this NOB thrives well at temperatures between 4 - 16C.