Karstic caves represent one of the most important subterranean carbon storages on Earth and provide windows into the subsurface. The recent discovery of the Herrenberg Cave, Germany, gave us the opportunity to investigate the diversity and potential role of bacteria in carbonate mineral formation. Calcite was the only mineral observed by Raman spectroscopy to precipitate as stalactites from seepage water. Bacterial cells were found on the surface and interior of stalactites by confocal laser scanning microscopy. Proteobacteria dominated the microbial communities inhabiting stalactites, representing more than 70% of total 16S rRNA gene clones. Proteobacteria formed 22 to 34% of the detected communities in fluvial sediments, and a large fraction of these bacteria were also metabolically active. A total of 9 isolates, belonging to the genera Arthrobacter, Flavobacterium, Pseudomonas, Rhodococcus, Serratia, and Stenotrophomonas, grew on alkaline carbonate-precipitating medium. Two cultures with the most intense precipitate formation, Arthrobacter sulfonivorans and Rhodococcus globerulus, grew as aggregates, produced extracellular polymeric substances (EPS), and formed mixtures of calcite, vaterite, and monohydrocalcite. R. globerulus formed idiomorphous crystals with rhombohedral morphology, whereas A. sulfonivorans formed xenomorphous globular crystals, evidence for taxon-specific crystal morphologies. The results of this study highlighted the importance of combining various techniques in order to understand the geomicrobiology of karstic caves, but further studies are needed to determine whether the mineralogical biosignatures found in nutrient-rich media can also be found in oligotrophic caves.

The traditional view of the dependency of subsurface environments on surface-derived allochthonous carbon inputs is challenged by increasing evidence for the role of lithoautotrophy in aquifer carbon flow. We linked information on autotrophy (Calvin-Benson-Bassham cycle) with that from total microbial community analysis in groundwater at two superimposed-upper and lower-limestone groundwater reservoirs (aquifers). Quantitative PCR revealed that up to 17% of the microbial population had the genetic potential to fix CO 2 via the Calvin cycle, with abundances of cbbM and cbbL genes, encoding RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) forms I and II, ranging from 1.14 ؋ 10 3 to 6 ؋ 10 6 genes liter ؊1 over a 2-year period. The structure of the active microbial communities based on 16S rRNA transcripts differed between the two aquifers, with a larger fraction of heterotrophic, facultative anaerobic, soil-related groups in the oxygen-deficient upper aquifer. Most identified CO 2 -assimilating phylogenetic groups appeared to be involved in the oxidation of sulfur or nitrogen compounds and harbored both RubisCO forms I and II, allowing efficient CO 2 fixation in environments with strong oxygen and CO 2 fluctuations. The genera Sulfuricella and Nitrosomonas were represented by read fractions of up to 78 and 33%, respectively, within the cbbM and cbbL transcript pool and accounted for 5.6 and 3.8% of 16S rRNA sequence reads, respectively, in the lower aquifer. Our results indicate that a large fraction of bacteria in pristine limestone aquifers has the genetic potential for autotrophic CO 2 fixation, with energy most likely provided by the oxidation of reduced sulfur and nitrogen compounds. Due to the lack of light-driven primary production, groundwater ecosystems were originally believed to be controlled by surface-derived allochthonous organic matter input (1-3) and to be dominated by heterotrophic prokaryotes adapted to nutrient limitation. However, there is increasing evidence of the important role of lithoautotrophy for carbon flow in aquifers (4-7). A large proportion of drinking water originates from groundwater resources (8), with karstic aquifers providing ϳ25% of the drinking water sources on a global scale (9). Despite the crucial role of microbial activity in shaping groundwater geochemistry (10-12), the links between microbial diversity and function in groundwater ecosystems, especially with regard to chemolithoautotrophy, are still poorly understood (7). Recent studies suggest that microbial CO 2 assimilation in aquifers could be fueled by energy conserved by nitrification, oxidation of ferrous iron and reduced sulfur compounds (6, 7), or oxidation of H 2 or methane (13, 14). Oxidation of electron donors present as solid minerals such as pyrite can even yield highly reactive dissolved ions that might affect other minerals and dissolved ions in the aquifer, leading to changes to the makeup of rocks and groundwater.Today, there are six known autotrophic CO 2 fixation pathways (reviewed in refere...

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Bacterial communities associated with decomposing rhizomes of Phragmites australis were investigated in Lake Ferto (Neusiedlersee, Hungary). Alkaliphilic and alkalitolerant strains were isolated on cellulose-containing alkaline medium spread with dilutions of scrapings taken from the surface of the decaying plant material. Fifty-one strains were grouped by numerical analysis based on physiological tests and BIOLOG sole carbon source utilization data. The strains identified by 16S rDNA sequence comparisons included members of low G+C Gram positives (Marinibacillus marinus, Bacillus cereus, and Exiguobacterium aurantiacum), high G+C Gram positives (Nesterenkonia halobia and Dietzia natronolimnea), alpha-proteobacteria (Pannonibacter phragmitetus), and gamma-proteobacteria (Pseudomonas pseudoalcaligenes and Halomonas venusta). Most of the strains were characterized by aerobic chemoorganotrophic respiratory metabolism and utilized several different carbon sources, although no direct cellulolytic activity was observed. Results of the pH and salt tolerance tests revealed optimuma in most cases at pH 11 and at the presence of 2.5-5% NaCl. These bacteria probably occupy niches in the aerobic, alkaline, water-influenced environments on the decomposing reed surfaces.

Raman gas spectrometry is introduced as a unique tool for the investigation of the respiratory activity that is indicative for growth of bacteria involved in biomineralization. Growth of these bacteria cannot be monitored using conventional turbidity-based optical density measurements due to concomitant mineral formation in the medium. The respiratory activity of carbonate-precipitating Arthrobacter sulfonivorans , isolated from the recently discovered Herrenberg Cave, was investigated during its lifecycle by means of innovative cavity-enhanced Raman gas analysis. This method allowed rapid and nonconsumptive online quantification of CO2 and O2 in situ in the headspace of the bacterial culture. Carbon dioxide production rates of A. sulfonivorans showed two maxima due to its pleomorphic growth lifecycle. In contrast, only one maximum was observed in control organism Pseudomonas fluorescens with a one-stage lifecycle. Further insight into the biomineralization process over time was provided by a combination of Raman macro- and microspectroscopy. With the help of this spatially resolved chemical imaging of the different types of calcium carbonate minerals, it was elucidated that the surface of the A. sulfonivorans bacterial cells served as nuclei for biomineralization of initially spherical vaterite precipitates. These vaterite biominerals continued growing as chemically stable rock-forming calcite crystals with rough edges. Thus, the utilization of innovative Raman multigas spectroscopy, combined with Raman mineral analysis, provided novel insights into microbial-mediated biomineralization and, therefore, provides a powerful methodology in the field of environmental sciences.

Seasonal studies of surface sediment bacterial communities, from two basins with differing trophic states within Lake Balaton (Hungary), were carried out using molecular (denaturing gradient gel electrophoresis, DGGE) and cultivation-based techniques. The presence of polyphosphate accumulates was tested using Neisser staining, and phosphatase activity was investigated on organic phosphorus (P) compound. Aerobic viable cell counts were significantly higher in the eutrophic than mesotrophic basin in each season. The lowest viable counts were observed in the autumn and the highest in spring and summer month in both basins. The DGGE fingerprints of the samples reflected that the composition of sediment bacterial communities in the two basins were distinct in spring and summer, and similar in autumn, but similarly diverse in all seasons. On the basis of partial 16S rDNA sequences, the 216 strains were affiliated with six major bacterial lineages: Firmicutes; Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Common species characterized from both basins constituted up to 66% of all identified phylotypes. Strains related to Bacillus sp. were dominant in all but one sample. Isolates affiliated with Aeromonas sp. prevailed in the sample taken from the mesotrophic basin in spring. The majority of the strains showed excess poly-P accumulation. Association of Neisser staining and phosphatase activity test results suggested that excess poly-P accumulation serves as P storage for sediment bacteria. Our study implied the importance of Firmicutes, Actinobacteria, Alphaproteobacteria, and Aeromonas species in benthic bacterial P retention.

The community structure of sulfate-reducing bacteria (SRB) associated with reed (Phragmites australis) rhizosphere in Lake Velencei (Hungary) was investigated by using cultivation-based and molecular methods. The cultivation methods were restricted to recover lactate-utilizing species with the exclusion of Desulfobacter and some Desulfobacterium species presumably not being dominant members of the examined community. The most-probable-number (MPN) estimations of lactate-utilizing SRB showed that the cell counts in reed rhizosphere were at least one order of magnitude higher than that in the bulk sediment. The number of endospores was low compared to the total SRB counts. From the highest positive dilution of MPN series, 47 strains were isolated and grouped by restriction fragment length polymorphism (RFLP) analysis of the amplified 16S ribosomal RNA (rRNA) and dsrAB (dissimilatory sulfite reductase) genes. Contrary to the physiological diversity of the isolates, the combined results of RFLP analysis revealed higher diversity at species as well as at subspecies level. Based on the partial 16S rRNA sequences, the representative strains were closely affiliated with the genera Desulfovibrio and Desulfotomaculum. The partial dsrAB sequences of the clones, recovered after isolation and PCR amplification of the community DNA, were related to hitherto uncultured species of the genera Desulfovibrio and Desulfobulbus. Nevertheless, the representative of the second largest clone group was shown to be closely affiliated with the sequenced dsrAB gene of a strain isolated from the same environment and identified as Desulfovibrio alcoholivorans. Another clone sequence was closely related to a possible novel species also isolated within the scope of this work.

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