Gerhard Weidler

Communities of Archaea and Bacteria in a Subsurface Radioactive Thermal Spring in the Austrian Central Alps, and Evidence of Ammonia-Oxidizing Crenarchaeota

ABSTRACT Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the “Franz-Josef-Quelle” in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated Euryarchaeota. These findings support recent reports that Crenarchaeota are not restricted to high-temperature environments. Most of the bacterial sequences were related to the Proteobacteria (α, β, γ, and δ), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (δ-Proteobacteria) and three others grouped with Nitrospira. Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were obtained which were related to crenarchaeotic amoA genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.

Crenarchaeota and Their Role in the Nitrogen Cycle in a Subsurface Radioactive Thermal Spring in the Austrian Central Alps

ABSTRACT Previous results from a 16S rRNA gene library analysis showed high diversity within the prokaryotic community of a subterranean radioactive thermal spring, the “Franz-Josef-Quelle” (FJQ) in Bad Gastein, Austria, as well as evidence for ammonia oxidation by crenarchaeota. This study reports further characterization of the community by denaturing gradient gel electrophoresis (DGGE) analysis, fluorescence in situ hybridization (FISH), and semiquantitative nitrification measurements. DGGE bands from three types of samples (filtered water, biofilms on glass slides, and naturally grown biofilms), including samples collected at two distinct times (January 2005 and July 2006), were analyzed. The archaeal community consisted mainly of Crenarchaeota of the soil-subsurface-freshwater group (group 1.1b) and showed a higher diversity than in the previous 16S rRNA gene library analysis, as was also found for crenarchaeal amoA genes. No bacterial amoA genes were detected. FISH analysis of biofilms indicated the presence of archaeal cells with an abundance of 5.3% (±4.5%) in the total 4′,6-diamidino-2-phenylindole (DAPI)-stained community. Microcosm experiments of several weeks in duration showed a decline of ammonium that correlated with an increase of nitrite, the presence of crenarchaeal amoA genes, and the absence of bacterial amoA genes. The data suggested that only ammonia-oxidizing archaea (AOA) perform the first step of nitrification in this 45°C environment. The crenarchaeal amoA gene sequences grouped within a novel cluster of amoA sequences from the database, originating from geothermally influenced environments, for which we propose the designation “thermal spring” cluster and which may be older than most AOA from soils on earth.

Astrobiology with haloarchaea from Permo-Triassic rock salt

Several viable halophilic archaebacteria were isolated previously from rock salt of Permo-Triassic age in an Austrian salt mine; one of these strains was the first to be recognized as a novel species from subterranean halite and was designated Halococcus salifodinae . The halophilic microorganisms have apparently survived in the salt sediments over extremely long periods of time. Halobacteria could therefore be suitable model organisms for exploring the possibility of long-term survival of microbes on other planets, in particular, since extraterrestrial halite has been detected in meteorites and is assumed to be present in the subsurface ocean on Europa. Our efforts are directed at the identification of the microbial content of ancient rock salt and the development of procedures for the investigation of the halobacterial response to extreme environmental conditions. Using modified culture media, further halophilic strains were isolated from freshly blasted rock salt and bore cores; in addition, growth of several haloarchaea was substantially improved. Molecular methods indicated the presence of at least 12 different 16S rRNA gene species in a sample of Alpine rock salt, but these strains have not been cultured yet. The exploration of Mars is a target of space missions in the 21st century; therefore, testing the survival of haloarchaea under conditions comparable to present-day Mars, using a simulation chamber, was begun. Preliminary results with Halococcus and Halobacterium species suggested at least tenfold higher survival rates when cells were kept in liquid brines than under dry conditions; staining of cells with the LIVE–DEAD kit, which discriminates between damaged and intact membranes, corroborated these data.

Thaumarchaeal ammonium oxidation and evidence for a nitrogen cycle in a subsurface radioactive thermal spring in the Austrian Central Alps

Previous studies had suggested the presence of ammonium oxidizing Thaumarchaeota as well as nitrite oxidizing Bacteria in the subsurface spring called Franz Josef Quelle (FJQ), a slightly radioactive thermal mineral spring with a temperature of 43.6–47°C near the alpine village of Bad Gastein, Austria. The microbiological consortium of the FJQ was investigated for its utilization of nitrogen compounds and the putative presence of a subsurface nitrogen cycle. Microcosm experiments made with samples from the spring water, containing planktonic microorganisms, or from biofilms, were used in this study. Three slightly different media, enriched with vitamins and trace elements, and two incubation temperatures (30 and 40°C, respectively) were employed. Under aerobic conditions, high rates of conversion of ammonium to nitrite, as well as nitrite to nitrate were measured. Under oxygen-limited conditions nitrate was converted to gaseous compounds. Stable isotope probing with 15NH4Cl or (15NH4)2SO4as sole energy sources revealed incorporation of 15N into community DNA. Genomic DNA as well as RNA were extracted from all microcosms. The following genes or fragments of genes were successfully amplified, cloned and sequenced by standard PCR from DNA extracts: Ammonia monooxygenase subunit A (amoA), nitrite oxidoreductase subunits A and B (nxrA and nxrB), nitrate reductase (narG), nitrite reductase (nirS), nitric oxide reductases (cnorB and qnorB), nitrous oxide reductase (nosZ). Reverse transcription of extracted total RNA and real-time PCR suggested the expression of each of those genes. Nitrogen fixation (as probed with nifH and nifD) was not detected. However, a geological origin of NH+4 in the water of the FJQ cannot be excluded, considering the silicate, granite and gneiss containing environment. The data suggested the operation of a nitrogen cycle in the subsurface environment of the FJQ.

Prokaryotic Communities Below Planetary Surfaces and Their Involvement in the Nitrogen Cycle

Nitrogen is essential to the chemistry of all living organisms. While NASA’s plans for the search for life in the universe focus mainly on the detection of water. Capone et al. (2006) suggested to look for the presence of nitrogen, since its detection would be an important clue for potential life. Even the absence of extinct life on Mars could possibly be declared, if only abiotic nitrate deposits were found there (Capone et al., 2006). Microorganisms are the principal participants in the terrestrial global nitrogen cycle, and several surprising discoveries during the last years have changed the understanding of their involvement (Ward et al., 2007). For instance, ammonia oxidation had long been thought to be performed only by chemolithoautotrophic bacteria, but our studies (see below) as well as those of others suggested strongly the occurrence of possibly widespread archaeal ammonia oxidation. The first hints came from metagenomic studies of Crenarchaeota (Schleper et al., 2005); subsequently, marine and freshwater environments, various soils, hot springs, and wastewaters were found to contain ammonia-oxidizing archaea (You et al., 2009). Here, a little explored subsurface ecosystem will be considered in more detail with respect to a potential nitrogen cycle.

VIABLE HALOBACTERIA FROM ANCIENT OCEANS—AND IN OUTER SPACE?

About 250 million years ago the continents were close together and formed Pangaea, a supercontinent, which persisted for about 100 million years and then fragmented. The landmasses at that time were located predominantly in the southern hemisphere. The climate was arid and dry; the average temperature is thought to have been several degrees higher than at present. This was one of the time periods in the history of the Earth, when huge salt sediments formed. A total of about 1.3 million cubic kilometers of salt were deposited during the late Permian and early Triassic period alone (Zharkov 1981). The thickness of the salt sediments can reach 1000 to 2000 meters. When Pangaea broke up, land masses were drifting in latitudinal and Northern direction. Mountain ranges such as the Alps, the Carpathians and the Himalayas were pushed up due to the forces of plate tectonics. The salt deposits in Austria originated in the Alpine basin, which extended from Innsbruck to Vienna. Some salt mines in the Alps are still in operation, and these were the sources of our samples. In the Alpine basin and in the Central European basin (Zechstein sea), no more salt sedimentation took place after the Triassic period; however, in other locations, e.g. in Poland, significant salt deposits were still formed until about 20 million years ago. Dating of the salt deposits by sulfur-isotope analysis (ratios of 32S/34S as measured by mass spectrometry), in connection with information from stratigraphy, indicated a Permo-Triassic age for the Alpine and Zechstein deposits, which was independently confirmed by the identification of pollen grains from extinct plants in the sediments (Klaus 1974).