Autotrophy in Groundwater Ecosystems

The major role in global net CO2 fixation plays photosynthesis of green plants, algae and cyanobacteria, but other microorganisms are also important concerning autotrophy; i.e. autotrophic microorganisms can be found in most bacterial groups (Eubacteria) and there are even numerous representatives within the Archaea. CO2 fixation is not only one of the world’s most important biogeochemical processes and responsible for the buildup of organic compounds which are needed for biological functions (e.g. cell growth or nutrition of heterotrophic organisms); ultimately all ecosystems are based on inputs of carbon and energy provided by autotrophic organisms which can be found in almost all environments. While the importance of CO2 fixation on the surface is known, there is almost no information about autotrophic processes in the subsurface. The widespread opinion is that subsurface communities are dominated by heterotrophic microorganisms, but it is unlikely that all subsurface biomass depends on the limited amounts of organic carbon imported from the surface or on pollution dumping. Groundwater systems comply with all requirements for autotrophic growth processes (electron donors e.g. H2, S2O3 2- and electron acceptors e.g. NO3-, O2 are available as well as plenty of inorganic carbon), so autotrophic microorganisms could significantly contribute to the carbon flux in at least some of those systems. In summary, the existence and the role of chemolithoautotrophic CO2 fixation in the terrestrial subsurface is hardly known. To date, five CO2 fixation pathways are described, i.e. the Calvin-Benson-Bassham cycle (Calvin cycle), the reductive tricarboxylic acid cycle, the reductive acetyl CoA pathway, the 3-hydroxypropionate cycle and the 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation pathway, with the Calvin cycle being the most intensively studied and probably the most abundant one. A sixth fixation pathway was just recently discovered. Objective of this thesis was to prove the CO2 fixation potential within the microbial communities in different groundwater ecosystems by means of functional gene analysis (cbbL, cbbM and acl genes) and to link this potential with in situ autotrophic activities as evaluated by different isotope and fatty acid approaches (FISH-MAR and PLFA analysis). Furthermore enrichment cultures under obligate chemolithoautotrophic conditions were started to get an idea about the diversity of those communities. The detection of the cbb genes in a contaminated and a pristine aquifer proved the occurrence of CO2 fixation potential being present in the bacterial communities of those ecosystems. Concerning the tar-oil contaminated aquifer, the majority of all retrieved cbb sequences was closely related to the cbbL and cbbM sequences belonging to the genus Thiobacillus, indicating that this genus might be of importance in groundwater ecosystems. This hypothesis is further supported by the results retrieved in the investigation at the organically poor site, the Testfield Scheyern. Here, most cbbM sequences detected were also closely related to the cbb sequences of Thiobacillus ssp. The successful labelling of bacterial cells deriving from the tar-oil contaminated aquifer via fluorescent in situ hybridization (FISH) indicated considerable bacterial activity in this aquifer, but the detection of radiolabeled cells failed. 13C-labelled CaCO3 was exposed together with sterile sediment in the same aquifer. Cell counts suggested a successful colonization of the exposed sediments, but PFLA concentration was low. However, the incorporation of 13C-carbon into two of the detected fatty acids was a direct hint for bacterial CO2-uptake. Successful enrichment cultures out of both investigated aquifers proved the actual occurrence of autotrophs in those ecosystems. In total four new chemolithoautotrophic bacterial strains could be isolated, one of them, belonging to the genus Thiobacillus, was further characterized. It was an obligate chemolithoautotrophic strain, using the Calvin cycle for CO2 fixation. It was described as a new species, Thiobacillus thiophilus D24TN sp. nov.