C2

State of the art

Nitrification is an essential step in the nitrogen (N) cycle that catalyzes the conversion of ammonia to nitrate via the intermediate nitrite. This process is very important since the total ammonium concentration in water should not exceed 0.5 mg L^-1 to be suitable for drinking water (Deutsche Trinkwasserverordnung 2001). Nitrification is carried out by different guilds of microorganisms which possess a chemolithoautotrophic lifestyle or can switch between a chemolithoautotrophic and a mixotrophic lifestyle (e.g. Pester et al. 2011). Ammonia oxidation to nitrite, the first step in nitrification, can be carried out by ammonia oxidizing bacteria (AOB) or the recently discovered ammonia oxidizing archaea (AOA). Both are trophically linked to nitrite oxidizing bacteria, which convert the toxic nitrite to nitrate as the second step in nitrification (Jetten 2008). Just last year a completely new group of microorganisms was discovered that performs the whole process of nitrification in one step and was therefore called complete ammonia oxidizers (Comammox) (Daims et al. 2015; van Kessel et al. 2015). Since Comammox were just described in December 2015 and AOA were discovered only in 2005 in comparison to AOB (known since 100 years), the relative importance of these groups for nutrient flow is currently unclear. Even more, their response to an almost doubling of the nitrogen content in Lake Constance since the 1960’s (IGKB 2012) as one of the major changing baselines of this ecosystem is not understood at all.

Preparatory work

The leading PI, Michael Pester, studies nitrifying microorganisms since his early Post-doc time. During this time, he established molecular and bioinformatics tools to study ammonia oxidizing and nitrite oxidizing microorganisms using metagenomics approaches based on amplicon sequencing in various habitats such as soils and waste water treatment plants. In particular, he performed a biogeographical study on soil AOA using the functional marker gene amoA (coding for the alpha-subunit of the ammonia monooxygenase) (Pester et al. 2012) and established nxrB (coding for the beta-subunit of the nitrite oxidoreductase) as a functional and phylogenetic marker for nitrite oxidizing bacteria (Pester et al. 2014). Furthermore, he contributed to a study that disentangled niche partitioning between closely related nitrite-oxidizing Nitrospira in waste water treatment plants (Gruber-Dorninger et al. 2015). Bernhard Schink as the second PI has long-standing experience on the general microbial ecology of Lake Constance (e.g. Deutzmann et al. 2011, 2014).

Proposed project and role within the RTG

Nitrifying microorganisms bridge the nitrogen and carbon cycle by their chemolithoautotrophic or mixotrophic lifestyle in a unique way. Our hypothesis is that nitrifying microorganisms contribute to or likely dominate this “dark” primary production in lake ecosystems, which should gain importance the deeper and larger the water body is where no photosynthesis occurs. So far, the contribution of nitrifying microorganisms to ammonia turnover and CO₂ fixation into organic matter is heavily understudied in freshwater lakes. As a first step, we propose to study the contribution of dark primary production to overall primary production in Lake Constance and to disentangle, which particular nitrifying microbes contribute to this process. Particular focus will be laid on ammonia oxidation as the first and at the same time rate-limiting step in nitrification. The question whether AOA, AOB, or Comammox dominate ammonia oxidation is currently attracting a lot of attention but is not understood at all because of the recent discovery of the responsible microoganisms. The three different groups of microorganisms exploit homologous ammonia monooxygenases in order to activate ammonia and thus carry amo-genes in their genomes. Using amoA as a functional and phylogenetic marker gene, we will follow their abundance (based on amoA genes) and transcriptional activity (based on amoA transcripts) as connected to the annual succession of the lake plankton. This will be done in the photic as well as aphotic zone using amoA (gene) high-throughput amplicon sequencing. Based on the obtained results, we will perform a combined metagenomics and metatranscriptomics analysis of selected samples to gain deeper insights into the genetic make-up of the targeted microorganisms as well as their transcriptional activity. These molecular analyses will be flanked by activity measurements of the overall nitrification and “dark” primary production using tracer experiments based on ¹⁵N-ammonium and ¹³C-carbon dioxide, respectively. In a second step, the resilience of these microorganisms to shifting ecosystem baselines (e.g., increasing water temperatures and varying N:P ratios) and their reversibility after strong disturbance events will be studied in mesocosm experiments under controlled conditions.

The doctoral researcher in this project will strongly interact with the doctoral researchers in projects A2, C3, and C4