Continuous Cultivation of Janssand Microbial Communities : Response to Varying Oxygen Concentrations and Temperatures

The Wadden Sea is the largest contiguous system of tidal sand and mud flats worldwide. It forms a variety of (micro-)habitats, which create ecological niches with an outstanding richness of thousands of species. The Wadden Sea is an area of intense biogeochemical cycling and mineralization processes such as the nitrogen cycle, which comprises the transformation of nitrogen containing molecules. Denitrification, the stepwise reduction of nitrate (NO3) to dinitrogen (N2), is such a transformation process usually employed in the reduction of nitrate concentrations of waste-water and a source of nitrous oxide (N2O), a long living, potent greenhouse gas. This thesis addresses the effect of temperature and dynamic oxic/anoxic conditions on marine denitrifying bacteria. The experimental approach consisted of the long term continuous cultivation of microbial communities sampled from tidal Wadden Sea sediments. Continuous cultivation enabled the natural selection of simplified microbial communities under defined, stable and environmentally relevant conditions. Compared to more complex communities, these simple communities were traceable with genomics, transcriptomics and proteomics approaches in order to identify the involved organisms and determine their metabolic traits. Furthermore, fluorescence in situ hybridization (FISH ) approaches, determination of metabolites in culture liquid and off gas, and stoichiometric modelling completed the picture. In part one, the development of the experimental setup for continuous cultivation is described. This chapter shows that with the presented setup it was possible to select a microbial community comprised of representatives of populations that were important in situ. One of the selected populations represented a clade of potential sulfur oxidizing Gammaproteobacteria that were highly abundant in situ. Another population was a member of the uncultured BD1-5/SN-2 division that was shown to translate the stop codon UGA as the amino acid glycine. Its enrichment also enabled microscopy images of a bacterium belonging to this enigmatic clade. In part two, the developed experimental setup was used to study the outcome of natural selection under tidal (oxic/anoxic) conditions. It was shown that the resulting community facilitated the parallel occurrence of thermodynamically unsorted redox processes: fermentation, sulfate reduction, denitrification and aerobic respiration. Oxygen sensitive enzymes were protected by a combination of cellular aggregation and active oxygen consumption by cells performing anaerobic metabolism. In part three, the effect of temperature on natural selection of denitrifying communities was investigated. The results were consistent with those of chapter 2, and showed co-selection of fermentative and denitrifying bacteria. However, whereas the fermentative/denitrifying consortia selected at 25 degree celsius denitrified effectively, this was not the case at 10 degree celsius, leading to a reduced denitrification activity at this temperature. Temperature was shown to have a strong selective effect on the denitrifying populations. Although the observations could not be explained conclusively, we speculate that at 10 degree celsius, cells were selected that performed denitrification and fermentation in parallel, whereas at 25 degree celsius division of labor was more pronounced. Thus, at 10 degree celsius fermentation may have outcompeted denitrification at the single cell level while at 25 degree celsius fermentative and denitrifying microbes were able to co-exist. Overall, the work of this thesis pioneers a new approach to the microbial ecology of denitrification in the context of other redox processes. The research provided new insight about how denitrification interacts with other redox processes and opened the way for direct, hypothesis based assessment of these interactions in nature