Nitrifiers and denitrifiers inhabiting Rothwald forest soil

The Rothwald forest is the biggest remnant of virgin forest within Central Europe. Microbiological studies have revealed that the microbial nitrogen (N) turnover is fast and that the concentration of mineral N is high in the Rothwald forest soil. Furthermore, a high capacity of this forest for N retention has been suggested. Microorganisms mediate most N cycling processes and play a key role in maintaining the N balance of ecosystems. The microbial oxidation of ammonia to nitrate during nitrification is of special ecological relevance, since it liberates nitrate as a potentially mobile form of N. Another important step is the reduction of nitrate to nitrite and then to nitric oxide, N2O and N2 during the denitrification process, because it results in gaseous N losses. Microbial community studies in soil have long been limited by the fact that the majority of bacteria are not accessible by cultivation. Therefore, the bacterial communities involved in N cycling processes in natural environments are largely unknown. Recent advances in molecular microbiology have made it possible to circumvent limitations related to bacterial isolation by use of cultivation-independent methods. The proposed project aims at obtaining a better understanding of the ammonia oxidizing and nitrate and nitrite reducing bacterial communities inhabiting Rothwald forest soil. The diversity of these bacterial groups will be assessed by T-RFLP-analysis of the functional genes amoA, narG, nirS and nirK, which encode key enzymes of the nitrification and denitrification processes. Furthermore, a microarray targetting the amoA gene will be developed, which will enable rapid analysis of ammonia oxidizing bacterial communities in soil. NarG, nirS and nirK clone libraries will be established and sequence analysis of nitrite and nitrate reductase genes will be performed. In order to assess the functional diversity of uncultured bacteria, a metagenomic library will be generated. The metagenome approach involves cloning of the metagenome, encompassing the collective genomes of microbes inhabiting a particular environment, into a BAC (bacterial artificial chromosome) vector. The resulting libraries will be screened for the presence of novel nitrite reductase (nir) genes. The suggested approach is highly appropriate to link structural and functional information of yet uncultured microorganisms. Results of this project will provide important data on the diversity and physiological capabilities of N cycling bacteria inhabiting a virgin forest soil.