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..