Evelyn Krawczyk-Bärsch

Chlorite dissolution in the acid pH-range: A combined microscopic and macroscopic approach

Abstract The dissolution of chlorite with intermediate Fe-content was studied macroscopically via mixed flow experiments as well as microscopically via atomic force microscopy (AFM). BET surface area normalized steady state dissolution rates at 25 °C for pH 2 to 5 vary between 10−12 and 10−13 mol/m2.s. The order of the dissolution reaction with respect to protons was calculated to be about 0.29. For pH 2 to 4, chlorite was found to dissolve non-stoichiometrically, with a preferred release of the octahedrally coordinated cations. The additional release of octahedrally coordinated cations may be due to the transformation of chlorite to interstratified chlorite/vermiculite from the grain edges inward. In-situ atomic force microscopy performed on the basal surfaces of a chlorite sample, which has been preconditioned at pH 2 for several months, indicated a defect controlled dissolution mechanism. Molecular steps with height differences which correspond to the different subunits of chlorite, e.g. TOT sheet and brucite like layer, originated at surface defects such or compositional inhomogenities or cracks, which may be due to the deformation history of the chlorite sample. In contrast to other sheet silicates, at pH 2 nanoscale etch pits occur on the chlorite basal surfaces within flat terraces terminated by a TOT-sheet as well as within the brucite like layer. The chlorite basal surface dissolves layer by layer, because most of the surface defects are only expressed through single TOT or brucite-like layers. The defect controlled dissolution mechanism favours dissolution of molecular steps on the basal surfaces compared to dissolution of the grain edges. At pH 2 the dissolution of the chlorite basal surface is dominated by the retreat of 14 A steps, representing one chlorite unit cell. The macroscopic and microscopic chlorite dissolution rates can be linked via the reactive surface area as identified by AFM. The reactive surface area with respect to dissolution consists of only 0.2% of the BET-surface area. A dissolution rate of 2.5 × 10−9 mol/m2s was calculated from macroscopic and microscopic dissolution experiments at pH 2, when normalized to the reactive surface area.

Combined use of flow cytometry and microscopy to study the interactions between the gram-negative betaproteobacterium Acidovorax facilis and uranium(VI)

The former uranium mine Königstein (Saxony, Germany) is currently in the process of remediation by means of controlled underground flooding. Nevertheless, the flooding water has to be cleaned up by a conventional wastewater treatment plant. In this study, the uranium(VI) removal and tolerance mechanisms of the gram-negative betaproteobacterium Acidovorax facilis were investigated by a multidisciplinary approach combining wet chemistry, flow cytometry, and microscopy. The kinetics of uranium removal and the corresponding mechanisms were investigated. The results showed a biphasic process of uranium removal characterized by a first phase where 95% of uranium was removed within the first 8h followed by a second phase that reached equilibrium after 24h. The bacterial cells displayed a total uranium removal capacity of 130mgU/g dry biomass. The removal of uranium was also temperature-dependent, indicating that metabolic activity heavily influenced bacterial interactions with uranium. TEM analyses showed biosorption on the cell surface and intracellular accumulation of uranium. Uranium tolerance tests showed that A. facilis was able to withstand concentrations up to 0.1mM. This work demonstrates that A. facilis is a suitable candidate for in situ bioremediation of flooding water in Königstein as well as for other contaminated waste waters.

The influence of biofilms on the migration of uranium in acid mine drainage (AMD) waters.

The uranium mine in Königstein (Germany) is currently in the process of being flooded. Huge mass of Ferrovum myxofaciens dominated biofilms are growing in the acid mine drainage (AMD) water as macroscopic streamers and as stalactite-like snottites hanging from the ceiling of the galleries. Microsensor measurements were performed in the AMD water as well as in the biofilms from the drainage channel on-site and in the laboratory. The analytical data of the AMD water was used for the thermodynamic calculation of the predominance fields of the aquatic uranium sulfate (UO(2)SO(4)) and UO(2)(++) speciation as well as of the solid uranium species Uranophane [Ca(UO(2))(2)(SiO(3)OH)(2)∙5H(2)O] and Coffinite [U(SiO(4))(1-x)(OH)(4x)], which are defined in the stability field of pH>4.8 and Eh<960 mV and pH>0 and Eh<300 mV, respectively. The plotting of the measured redox potential and pH of the AMD water and the biofilm into the calculated pH-Eh diagram showed that an aqueous uranium(VI) sulfate complex exists under the ambient conditions. According to thermodynamic calculations a retention of uranium from the AMD water by forming solid uranium(VI) or uranium(IV) species will be inhibited until the pH will increase to >4.8. Even analysis by Energy-filtered Transmission Electron Microscopy (EF-TEM) and electron energy loss spectroscopy (EELS) within the biofilms did not provide any microscopic or spectroscopic evidence for the presence of uranium immobilization. In laboratory experiments the first phase of the flooding process was simulated by increasing the pH of the AMD water. The results of the experiments indicated that the F. myxofaciens dominated biofilms may have a substantial impact on the migration of uranium. The AMD water remained acid although it was permanently neutralized with the consequence that the retention of uranium from the aqueous solution by the formation of solid uranium species will be inhibited.

Eukaryotic life in biofilms formed in a uranium mine

The underground uranium mine Königstein (Saxony, Germany), currently in the process of remediation, represents an underground acid mine drainage (AMD) environment, that is, low pH conditions and high concentrations of heavy metals including uranium, in which eye‐catching biofilm formations were observed. During active uranium mining from 1984 to 1990, technical leaching with sulphuric acid was applied underground on‐site resulting in a change of the underground mine environment and initiated the formation of AMD and also the growth of AMD‐related copious biofilms. Biofilms grow underground in the mine galleries in a depth of 250 m (50 m above sea level) either as stalactite‐like slime communities or as acid streamers in the drainage channels. The eukaryotic diversity of these biofilms was analyzed by microscopic investigations and by molecular methods, that is, 18S rDNA PCR, cloning, and sequencing. The biofilm communities of the Königstein environment showed a low eukaryotic biodiversity and consisted of a variety of groups belonging to nine major taxa: ciliates, flagellates, amoebae, heterolobosea, fungi, apicomplexa, stramenopiles, rotifers and arthropoda, and a large number of uncultured eukaryotes, denoted as acidotolerant eukaryotic cluster (AEC). In Königstein, the flagellates Bodo saltans, the stramenopiles Diplophrys archeri, and the phylum of rotifers, class Bdelloidea, were detected for the first time in an AMD environment characterized by high concentrations of uranium. This study shows that not only bacteria and archaea may live in radioactive contaminated environments, but also species of eukaryotes, clearly indicating their potential influence on carbon cycling and metal immobilization within AMD‐affected environment.

A spectroscopic study on U(VI) biomineralization in cultivated Pseudomonas fluorescens biofilms isolated from granitic aquifers.

The interaction between the Pseudomonas fluorescens biofilm and U(VI) were studied using extended X-ray absorption fine structure spectroscopy (EXAFS), and time-resolved laser fluorescence spectroscopy (TRLFS). In EXAFS studies, the formation of a stable uranyl phosphate mineral, similar to autunite (Ca[UO2]2[PO4]2•2–6H2O) or meta-autunite (Ca[UO2]2[PO4]2•10–12H2O) was observed. This is the first time such a biomineralization process has been observed in P. fluorescens. Biomineralization occurs due to phosphate release from the cellular polyphosphate, likely as a cell’s response to the added uranium. It differs significantly from the biosorption process occurring in the planktonic cells of the same strain. TRLFS studies of the uranium-contaminated nutrient medium identified aqueous Ca2UO2(CO3)3 and UO2(CO3)34− species, which in contrast to the biomineralization in the P. fluorescens biofilm, may contribute to the transport and migration of U(VI). The obtained results reveal that biofilms of P. fluorescens may play an important role in predicting the transport behavior of uranium in the environment. They will also contribute to the improvement of remediation methods in uranium-contaminated sites.

The Effect of Chlorite Dissolution on the Sorption Behavior of U(VI)

During the dissolution of chlorite the sorption of uranyl(VI) can be described by two different sorption sites. The first site is attributed to a surface site on the chlorite which is occupied after 138 hours of our mixed-flow experiment. Due to the fact that uranyl(VI) is continuously sorbed we conclude that as a second site the sorption sites of the newly forming ferrihydrite colloids are responsible for the additional sorption beyond the available sorption sites on chlorite.

Metabolism-dependent bioaccumulation of uranium by Rhodosporidium toruloides isolated from the flooding water of a former uranium mine

Remediation of former uranium mining sites represents one of the biggest challenges worldwide that have to be solved in this century. During the last years, the search of alternative strategies involving environmentally sustainable treatments has started. Bioremediation, the use of microorganisms to clean up polluted sites in the environment, is considered one the best alternative. By means of culture-dependent methods, we isolated an indigenous yeast strain, KS5 (Rhodosporidium toruloides), directly from the flooding water of a former uranium mining site and investigated its interactions with uranium. Our results highlight distinct adaptive mechanisms towards high uranium concentrations on the one hand, and complex interaction mechanisms on the other. The cells of the strain KS5 exhibit high a uranium tolerance, being able to grow at 6 mM, and also a high ability to accumulate this radionuclide (350 mg uranium/g dry biomass, 48 h). The removal of uranium by KS5 displays a temperature- and cell viability-dependent process, indicating that metabolic activity could be involved. By STEM (scanning transmission electron microscopy) investigations, we observed that uranium was removed by two mechanisms, active bioaccumulation and inactive biosorption. This study highlights the potential of KS5 as a representative of indigenous species within the flooding water of a former uranium mine, which may play a key role in bioremediation of uranium contaminated sites.

Multidisciplinary characterization of U(VI) sequestration by Acidovorax facilis for bioremediation purposes

The contamination of the environment by U may affect plant life and consequently may have an impact on animal and human health. The present work describes U(VI) sequestration by Acidovorax facilis using a multidisciplinary approach combining wet chemistry, transmission electron microscopy, and spectroscopy methods (e.g. cryo-time resolved laser-induced fluorescence spectroscopy, extended X-ray absorption fine structure spectroscopy, and in-situ attenuated total reflection Fourier transform infrared spectroscopy). This bacterial strain is widely distributed in nature including U-contaminated sites. In kinetic batch experiments cells of A. facilis were contacted for 5 min to 48 h with 0.1 mM U(VI). The results show that the local coordination of U species associated with the cells depends upon time contact. U is bound mainly to phosphate groups of lipopolysaccharide (LPS) at the outer membrane within the first hour. And, that both, phosphoryl and carboxyl functionality groups of LPS and peptidoglycan of A. facilis cells may effectuate the removal of high U amounts from solution at 24-48 h of incubation. It is clearly demonstrated that A. facilis may play an important role in predicting the transport behaviour of U in the environment and that the results will contribute to the improvement of bioremediation methods of U-contaminated sites.