Microdiversity of Uncultured Nitrite-Oxidizing Bacteria

Nitrification is a key process of the biogeochemical nitrogen cycle and wastewater treatment. Our current knowledge on the involved organisms ammonia-oxidizing bacteria (AOB) and archaea, and nitrite-oxidizing bacteria (NOB) and their metabolic features, ecological niche differentiation, and biological interactions is very limited. In particular, almost nothing is known about the fine-scale population structure, genomic plasticity, and phenotypic traits within lineages of closely related (microdiverse) nitrifiers. Microdiversity has been identified in recent years as a key feature of many microbial communities in natural and engineered habitats. Its discovery has fueled research on microbial population genetics, speciation, and on ecological aspects such as the niche partitioning of coexisting functionally redundant populations and diversity-stability relationships. The lack of insight into nitrifier microdiversity strongly contrasts the ecological importance of these organisms, which catalyze essential processes in most natural ecosystems and wastewater treatment plants (WWTPs). This project will use a full-scale WWTP as model ecosystem to study the microdiversity of uncultured NOB, which coexist in the same activated sludge, in a concerted approach comprising in-depth comparative genomics, population genetics, and in situ single-cell physiology. It builds upon a novel method to identify, physically extract, and genome-sequence individual cell aggregates (microcolonies) of nitrifiers from activated sludge by Raman microspectroscopy, laser tweezing, and single-cell genomics. By using this recently established sorting/sequencing pipeline, ~50 "single- microcolony" metagenomes of coexisting NOB and ~50 such metagenomes of AOB are sequenced already before the start of the project, which will utilize this exceptionally large sequence dataset from nitrifiers. Population genetic analyses will be performed on the obtained NOB genomes to resolve the fine-scale population structure of microdiverse NOB. Genome-based hypotheses on (possibly novel) niche-defining metabolic features of NOB clades will be tested in situ with the same living NOB populations. We will apply a battery of single-cell tools such as FISH with a high phylogenetic resolution, isotope labeling, Raman microspectroscopy, microautoradiography, and NanoSIMS analyses. Symbiotic AOB-NOB interactions will be characterized by genomics, in situ experiments, and spatial co-aggregation analyses, and their specificity for microdiverse populations from both guilds will be determined. The genomes of microbial predators or other heterotrophic symbionts, which were attached to the extracted microcolonies, will be co-sequenced with the NOB genomes and will inform on possible interactions of microdiverse NOB with non-nitrifiers that can be tested by in situ experiments. The project will dramatically extend our knowledge of nitrifying microorganisms by providing for the first time a detailed picture of microdiverse NOB populations including their phylogenetic structure, genomic plasticity, and ecophysiological versatility. More generally, the project will make significant progress in microdiversity research by combining targeted "single-microcolony genomics" with in situ experiments to study the biology of multiple closely related and uncultured populations directly in a complex microbial ecosystem.

Nitrification, the oxidation of ammonia to nitrate, is an essential process of the nitrogen cycle in nature and in engineered systems, such as wastewater and drinking water treatment plants. However, nitrification also causes massive loss of nitrogen from fertilized agricultural soils. Nitrification is a two-step process: ammonia is oxidized by ammonia-oxidizing microorganisms to nitrite, which is further converted to nitrate by nitrite-oxidizing bacteria (NOB). Knowledge about the biology of NOB is limited, because most species cannot be cultured in the laboratory. In this project, microcolonies of NOB cells were sorted by micromanipulation from activated sludge and their metagenomes were sequenced in order to study the genomic microdiversity among closely related NOB. Surprisingly, the investigated wastewater treatment plant contained only a low diversity of NOB from the genus Nitrospira, which belonged to not more than three genomically distinct species. Intriguingly, however, a novel giant virus was identified in the metagenomic sequence dataset. This virus likely infects eukaryotic microorganisms and evolved from a much smaller virus by massive gain of host genes. Furthermore, the first pure culture from the NOB genus Nitrotoga was isolated from activated sludge. A detailed physiological and genomic analysis revealed that Nitrotoga evolved independently from other NOB and possess alternative metabolisms, such has hydrogen (H2) oxidation, which may support their survival under nitrite-depleted conditions. The most important result was the unexpected discovery that members of the genus Nitrospira are able to catalyze both ammonia and nitrite oxidation. These bacteria are called "complete ammonia oxidizers" ("comammox"). Their discovery contradicts a century-old textbook paradigm that two different microbes would be required for complete nitrification. The novel comammox bacteria turned out to occur in soils and freshwater ecosystems as well as in wastewater and drinking water treatment plants. Physiological analyses of the first comammox pure culture revealed an exceptionally high affinity of comammox for the substrate ammonia, which enables comammox to outcompete most other ammonia-oxidizing organisms in terrestrial and engineered habitats. In addition, comammox was found to grow slowly but produce more biomass than other ammonia oxidizers and NOB per amount of substrate utilized. Based on these first insights into the ecophysiology of comammox, strategies can be developed to foster comammox populations in engineered systems and soils, where complete nitrification by a single organism can provide significant advantages. Fluorescence in situ hybridization (FISH) with rRNA-targeted DNA probes is widely used to detect microorganisms directly in their habitats. Here, a new multicolor FISH technique was developed that allows for the simultaneous detection of more microbial populations than the standard FISH protocol. Together with newly designed FISH probes to detect ammonia-oxidizing bacteria, this method will facilitate future studies on nitrification in wastewater and other aquatic systems.