Illuminating the Ecology of Nitrite-Oxidizing Bacteria

Nitrite-oxidizing bacteria (NOB) are key players of the biogeochemical nitrogen cycle. By catalyzing the second step of nitrification, the oxidation of nitrite to nitrate, NOB are the biological source of nitrate, which is a major form of fixed nitrogen in the biosphere and an important nitrogen source for many microorganisms and plants. Moreover, the activity of NOB determines whether fixed nitrogen is retained in ecosystems as nitrate or is lost to the atmosphere via other processes that convert nitrite to gaseous N-compounds. Despite the huge ecological importance of NOB, current knowledge of their microbiology and ecology is severely limited. Past research on NOB, which are difficult to culture in the laboratory, mainly focused on few pure cultures or representatives in wastewater treatment plants. The NOB communities in natural ecosystems have been neglected and thus, their composition, population dynamics, and responses to shifts of environmental conditions and human impact remain enigmatic. This project will bridge this knowledge gap by studying the structure and function of NOB communities in representative pristine or human-affected terrestrial and aquatic ecosystems: soils from the Arctic and from tropical or temperate forests, agricultural soils, waters and sediments from freshwater streams and lakes, and moderately saline lakes in the East Austrian national park "Neusiedler See-Seewinkel". The saline lakes are characterized by highly variable environmental conditions, but host extremely productive microbial communities that have barely been studied. To analyze the phylogenetically diverse NOB independently from cultivation, a novel and highly specific marker gene will be used. Sequences of this gene, nxrB coding for the beta subunit of nitrite oxidoreductase, have become available only recently with the first sequenced genomes from all known lineages of NOB. As nxrB is a key functional gene of nitrite oxidation, it is present in all NOB, but occurs in distinct forms characteristic for each phylogenetic NOB clade. In a highly parallelized approach, the NOB community structures in plenty of environmental samples will be analyzed by cutting-edge next-generation sequencing of nxrB genes, and the in situ nitrite-oxidizing activities of the different populations will be studied by monitoring nxrB transcription. In a series of incubation experiments, the responses of NOB to changing environmental conditions and structure-function relationships of NOB communities will be investigated. NOB will also be enriched from selected samples, and the potential of these autotrophs to use various organic substrates will be examined by molecular single-cell methods. The project will provide for the first time a broad picture of environmental NOB populations and their ecology in diverse habitats. This knowledge is urgently needed to better understand the effects of natural environmental fluctuations on the nitrogen cycle and to assess the consequences of human impact, such as an increasing nitrogen deposition, for vulnerable ecosystems.

Nitrification, the oxidation of ammonia to nitrate, is a key process of the nitrogen cycle in nature. Moreover, it is important for biological wastewater treatment and drinking water treatment, but on the other hand causes massive loss of nitrogen from fertilized arable soils. Nitrification is a two-step process: ammonia is oxidized by ammonia-oxidizing microorganisms to nitrite, which is further transformed to nitrate by nitrite-oxidizing bacteria (NOB). Little is known about the biology of NOB, because most species cannot be cultured in the laboratory. In this project, new cultivation-independent molecular biological methods were developed to detect NOB directly in environmental samples. Their use led to the discovery of a hitherto unknown, very high diversity of NOB from the genus Nitrospira in soils from arctic, temperate, and tropical ecosystems. For example, up to 700 Nitrospira species occurred in a single soil sample, and more than 100 Nitrospira species were detected in a wastewater treatment plant. These coexisting Nitrospira populations preferred different nitrite concentrations, temperatures, pH values, and alternative substrates (such as formate). Thus, they occupy different ecological niches as basis of their coexistence in the same habitat. A combination of genome analyses with laboratory experiments revealed that some Nitrospira even grow independently of nitrification (without nitrite), utilizing molecular hydrogen (H2) or formate as the sole source of energy and CO2 as the source of carbon. However, the greatest surprise was the discovery that specific Nitrospira species oxidize both ammonia and nitrite, thus catalyzing the whole nitrification process on their own. These organisms are referred to as complete ammonia oxidizers or comammox. Their discovery disproved 100 years-old textbook knowledge that the two steps of nitrification would always be performed by different microbes. The newly discovered comammox bacteria are widespread in soils and freshwater, and they occur in wastewater and drinking water treatment plants. Their importance for the natural nitrogen cycle, and for nitrification in wastewater treatment, is being investigated worldwide in follow-up studies. Even in saline-alkaline lakes in the Austrian national park Neusiedler See-Seewinkel, NOB and comammox of the genus Nitrospira occur that are adapted to a high salinity and pH values up to 11. So far, scientists assumed that NOB would be specialized bacteria which oxidize nitrite but show no other activities. This project has completely changed this picture and demonstrated that NOB are flexible microorganisms. They are adapted to diverse environmental conditions and have unexpected ecological functions. This knowledge is important for a better understanding of the nitrogen cycle and can help optimize the efficiency of fertilization, wastewater treatment, and drinking water preparation.