Dagmar Kock

Quantitative Microbial Community Analysis of Three Different Sulfidic Mine Tailing Dumps Generating Acid Mine Drainage

ABSTRACT The microbial communities of three different sulfidic and acidic mine waste tailing dumps located in Botswana, Germany, and Sweden were quantitatively analyzed using quantitative real-time PCR (Q-PCR), fluorescence in situ hybridization (FISH), catalyzed reporter deposition-FISH (CARD-FISH), Sybr green II direct counting, and the most probable number (MPN) cultivation technique. Depth profiles of cell numbers showed that the compositions of the microbial communities are greatly different at the three sites and also strongly varied between zones of oxidized and unoxidized tailings. Maximum cell numbers of up to 109 cells g−1 dry weight were determined in the pyrite or pyrrhotite oxidation zones, whereas cell numbers in unoxidized tailings were significantly lower. Bacteria dominated over Archaea and Eukarya at all tailing sites. The acidophilic Fe(II)- and/or sulfur-oxidizing Acidithiobacillus spp. dominated over the acidophilic Fe(II)-oxidizing Leptospirillum spp. among the Bacteria at two sites. The two genera were equally abundant at the third site. The acidophilic Fe(II)- and sulfur-oxidizing Sulfobacillus spp. were generally less abundant. The acidophilic Fe(III)-reducing Acidiphilium spp. could be found at only one site. The neutrophilic Fe(III)-reducing Geobacteraceae as well as the dsrA gene of sulfate reducers were quantifiable at all three sites. FISH analysis provided reliable data only for tailing zones with high microbial activity, whereas CARD-FISH, Q-PCR, Sybr green II staining, and MPN were suitable methods for a quantitative microbial community analysis of tailings in general.

Quantification of Microbial Communities in Subsurface Marine Sediments of the Black Sea and off Namibia.

Organic-rich subsurface marine sediments were taken by gravity coring up to a depth of 10 m below seafloor at six stations from the anoxic Black Sea and the Benguela upwelling system off Namibia during the research cruises Meteor 72-5 and 76-1, respectively. The quantitative microbial community composition at various sediment depths was analyzed using total cell counting, catalyzed reporter deposition – fluorescence in situ hybridization (CARD–FISH) and quantitative real-time PCR (Q-PCR). Total cell counts decreased with depths from 109 to 1010 cells/mL at the sediment surface to 107–109 cells/mL below one meter depth. Based on CARD–FISH and Q-PCR analyses overall similar proportions of Bacteria and Archaea were found. The down-core distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes (16S and 18S rRNA) as well as functional genes involved in different biogeochemical processes was quantified using Q-PCR. Crenarchaeota and the bacterial candidate division JS-1 as well as the classes Anaerolineae and Caldilineae of the phylum Chloroflexi were highly abundant. Less abundant but detectable in most of the samples were Eukarya as well as the metal and sulfate-reducing Geobacteraceae (only in the Benguela upwelling influenced sediments). The functional genes cbbL, encoding for the large subunit of RuBisCO, the genes dsrA and aprA, indicative of sulfate-reducers as well as the mcrA gene of methanogens were detected in the Benguela upwelling and Black Sea sediments. Overall, the high organic carbon content of the sediments goes along with high cell counts and high gene copy numbers, as well as an equal abundance of Bacteria and Archaea.

Quantification of Microorganisms Involved in Cemented Layer Formation in Sulfidic Mine Waste Tailings (Freiberg, Saxony, Germany)

Cemented layers predominantly consisting of gels/poorly crystalline mineral phases have been formed as a consequence of mineral weathering in sulfidic tailings near Freiberg, Saxony, Germany. These layers function as natural attenuation barrier for toxic compounds and reduce oxidation and erosion processes of tailings surfaces. Quantitative molecular biological and cultivation methods were applied to investigate the role of microorganisms for mineral weathering and cemented layer formation. High resolution depth profiles of numbers of microorganisms showed maximal cell numbers in the oxidation zone where cemented layers had been formed. Highest total cell numbers of >109 cells g-1 dry weight (dw) were detected by SybrGreen direct counting. Using quantitative real-time PCR (Q-PCR) between 107 and 109 Bacteria g-1 dw and up to 108 Archaea g-1 dw were determined. As well high numbers of cultivable and living Bacteria could be detected by MPN (most probable number) for Fe(II)- and S-oxidizers and CARD-FISH (catalyzed reporter deposition - fluorescence in situ hybridization). Overall, the high numbers of microorganisms determined with various quantification techniques argue for a significant role of microorganisms in cemented layer formation due to microbial mineral weathering. It is hypothesized that EPS (extracellular polymeric substances) mediate the formation of secondary mineral phases.

Geomicrobiology of Sulfidic Mine Dumps: A Short Review

The geomicrobiology of sulfidic mine dumps is reviewed. More than 30 microbiological studies of sulfidic mine dumps have been published. Mainly culturing approaches such as most probable number (MPN) or agar plates were used to study the microbial communities. More recently, molecular biological techniques such as FISH, CARD-FISH, Q-PCR, T-RFLP, DGGE, or cloning have been applied to quantify microorganisms and to investigate the microbial diversity. Aerobic Fe(II)- and sulfur compound oxidizing microorganisms oxidize pyrite, pyrrhotite and other metal sulfides and play an important role in the formation of acid mine drainage (AMD). Anaerobic microorganisms such as Fe(III)-reducing microorganisms dissolve Fe(III)(hydr)oxides and may thereby release adsorbed or precipitated metals. Sulfate-reducing microorganisms precipitate and immobilize metals. In addition to the microbial communities several biogeochemical processes have been analyzed in mine dumps. Pyrite or pyrrhotite oxidation rates have been measured by different techniques: Column experiments, humidity cells, microcalorimetry, or oxygen consumption measurements. Analyses of stable isotopes of iron, oxygen and sulfur have yielded valuable information on biogeochemical reactions. The microbiology and the biogeochemical processes in sulfidic mine dumps have to be understood for control and prevention of AMD generation and to provide different possibilities for remediation concepts. Today, remediation measures, e.g. under water storage of the waste or covering of the dumps, focus on the inhibition of pyrite oxidation to keep the toxic compounds inside the mine waste dumps. As an alternative to the inhibition of pyrite oxidation, metals which also have economic value could be extracted from mine dumps by the application of different metal extraction technologies including bioleaching.

Reservoir and Cap Rock Monitoring

One aim of the CLEAN project was to develop and test monitoring methods for the reservoir cap rock and the reservoir itself. It is shown here that advanced injection and production profile evaluation can be achieved using a combination of pressure, temperature and spinner flow meter data. Using distributed temperature sensing, temperature profiles in gas-filled wells can be acquired, as the sensor cable can be stationary during the measurement allowing for simultaneous thermal equilibration along the entire logged profile. A first field test of the developed hybrid wireline logging system was successfully performed under static conditions and the feasibility of warm-back monitoring was shown based on the results of numerical simulations for a possible CO2 injection scenario. A combined approach using the developed hybrid system for enhanced production logging during injection followed by warm-back monitoring within a subsequent shut-in period would allow for accurate determination of the spatial extent and injectivity of individual CO2 injection intervals.