Papers by Author: Wolfgang Sand

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Authors: Bianca M. Florian, Nanni Noël, Soeren Bellenberg, J. Huergo, Thore Rohwerder, Wolfgang Sand
Abstract: The aim of the study was to quantify and to visualize colonization of metal sulfides by pure and mixed cultures. Strains of the genera Acidithiobacillus and Leptospirillum were tested. Sessile and planktonic cells were visualized by fluorescence microscopy using 4',6-diamidino-2-phenylindole (DAPI) and FISH. Additionally, atomic force microscopy was used for the investigations on cell morphology, spatial arrangement of cells on metal sulfides and mineral surface topography. It was shown that the morphology of sessile cells was totally different as compared with planktonic ones. Interactions of different species resulted in increased production of extracellular polymeric substances (EPS) or caused negligible-attaching bacteria to be incorporated into a biofilm by the good attaching ones. Consequently, biofilm formation was furthered.
Authors: Christian Thyssen, David Holuscha, Jens Kuhn, Friederike Walter, Wolfram Fürbeth, Wolfgang Sand
Abstract: Bioleaching and biocorrosion are based on similar biochemical processes. Microbe-surface interaction, biofilm formation and concomitant extracellular polymeric substance (EPS) production gained increasing interest in the past decades. Nowadays it is generally accepted that biofilm formation and an accompanying formation of manganese oxides by manganese oxidizing bacteria such as Leptothrix spp. account for one type of pitting corrosion of stainless steel (SS). However, little is known about biofilm formation, EPS composition of manganese oxidizing microorganisms and their influence on microbiologically influenced corrosion. Consequently, we studied biofilm formation of Leptothrix discophora, the biooxidation of manganese in biofilms on floating filters as well as biofilm formation on stainless steel and the involved corrosion processes. Cells were visualized by epifluorescence (EFM) or confocal laser scanning –microscopy (CLSM). Additionally, the influence of biofilm formation and biooxidation of manganese by L. discophora on the open circuit potential (OCP) and pitting potential (Epit) of stainless steel was measured using a 3 electrode setup. L. discophora grew well in biofilms on floating filters and on SS coupons and incorporated in both conditions Mn2+ in the form of MnO2 from the bulk phase into the biofilm. OCP measurements of actively manganese-oxidizing biofilms on stainless steel showed a significant ennoblement of ≥200 mV.
Authors: Qian Li, Rui Yong Zhang, Beate A. Krok, Mario Vera, Wolfgang Sand
Abstract: In this study, initial attachment to and biofilm formation of Sulfobacillus thermosulfidooxidans DSM 9293T on pyrite in the presence of Leptospirillum ferriphilum DSM 14647T were investigated. Interactions of S. thermosulfidooxidansT and L. ferriphilumT were studied by means of monitoring attachment behavior and biofilm formation on pyrite. Our preliminary results showed that 1): Pre-established biofilms of L. ferriphilumT had effects on attachment of S. thermosulfidooxidansT to pyrite; 2): physical contact between cells of L. ferriphilumT and S. thermosulfidooxidansT on pyrite were visible 3): Pyrite leaching by cells of S. thermosulfidooxidansT was inhibited by the presence of inactive cells of L. ferriphilumT.
Authors: Lina María Ruíz, Wolfgang Sand, Carlos A. Jerez, Nicolas Guiliani
Abstract: Acidithiobacillus ferrooxidans, an acidophilic, chemolithotrophic, γ-proteobacterium, is involved in the bioleaching of metal sulfides. For this process, bacterial attachment to mineral surface and biofilm development play a pivotal role. Generally, biofilm formation and production of exopolysaccharides is regulated by the second messenger cyclic diguanylic acid (c-di-GMP) whose cellular level depends on the synthesis and degradation activities of diguanylate cyclase (DGCs, with GGDEF domain) and phosphodiesterase (PDE, with EAL or HD-GYP domains), respectively. The analysis of the genomic sequence of A. ferrooxidans ATCC 23270 allowed us to identify 5 putative orfs encoding DGC and/or PDE-like proteins. Four of them encode for bifunctional putative proteins with GGDEF and EAL domains and are named AFE_0053, AFE_1360, AFE_1373 and AFE_1379. The fifth one named AFE_1852 has an EAL domain. The putative proteins also include PAS and GAF domains involved in signal transduction. These features suggest an involvement in signalling transduction through the metabolism of c-di-GMP. The amino acid sequences of these putative proteins were aligned with known DGCs and PDEs. Alignments indicate that AFE_1360 and AFE_1373 share more consensus sequences with active PDEs, whereas AFE_0053 and AFE_1379 do with active DGCs. On the other hand, in AFE_1852 some conserved residues of known active PDEs are changed. RT-PCR-experiments revealed that the genes that encode for these putative DGCs and/or PDEs are expressed by growth on two different substrates. These preliminary results suggest that A. ferrooxidans possesses a c-di-GMP pathway that should be involved in biofilm formation, as it occurs in many bacteria.
Authors: Mario A. Vera, Thore Rohwerder, Soeren Bellenberg, Wolfgang Sand, Y. Denis, Violaine Bonnefoy
Abstract: Bioleaching is the extraction of metals, such as copper or gold, from ore by microorganisms. Bacterial attachment increases leaching activities due to the formation of a "reaction space" between the metal sulfide surface and the cell. This process depends on abiotic characteristics such as purity and degree of crystallization of the metal sulfide, as well as biotic ones such as the capacity of the bacteria for detecting favourable attachment sites and synthesizing a suitable cell envelope (EPS), for adhesion. Planktonic and sessile cells should differ significantly in their metabolic activities and therefore in their gene expression patterns. To help to understand At. ferrooxidans biofilm formation, microarray transcript profiling was carried out to compare planktonic and sessile cells. The high contents of EPS and ferric iron of the biofilms are interfering with RNA extraction, causing inhibition of DNAse, reverse transcriptase and/or polymerase activities required to get labelled target cDNA. In order to have sufficient high quality RNA suitable for transcriptomic analysis, we have optimized the biofilm formation of At. ferrooxidans on pyrite (FeS2) and the RNA extraction from the sessile cell population. DNA microarrays have been hybridized with labelled cDNAs from sessile and planktonic cells and preliminary data suggest that some genes are differently expressed between these two subpopulations. The understanding of these differences may help us to shift populations of leaching bacteria from the planktonic state towards the sessile state in order to influence bioleaching.
Authors: Stefanie Mangold, Kerstin Harneit, Wolfgang Sand
Abstract: Leaching bacteria attach to their substrates, i.e. mineral sulfides, and form monolayered biofilms. In this study the biofilm formation of Acidithiobacillus ferrooxidans A2 on pyrite was examined using atomic force and epifluorescence microscopy (AFM and EFM, respectively). A novel system by JPK instruments, the BioMaterial WorkstationTM, allows the investigation of the same location on an opaque sample with AFM and EFM. Until recently this was only possible for translucent samples. Sessile bacteria on pyrite coupons were stained with 4’,6-diamidino-2- phenylindol (DAPI) and visualized by EFM as well as AFM. The best imaging conditions for AFM were assessed. Scans of bacteria attached to pyrite were performed in contact mode in air as well as in tapping mode in fluid. Imaging in fluid was more challenging than imaging in air as bacteria tend to detach from their substratum. To avoid the dislocation of microorganisms by the AFM probe the sample was dried in air for 1 h prior to scanning in fluid. Scanning in air was performed with the whole range of cantilever spring constants tested (k = 0.03 N/m to k = 0.65 N/m) while, for scanning in fluid, best results were achieved using stiffer cantilevers (k = 0.65 N/m).
Authors: Thore Rohwerder, Wolfgang Sand
Abstract: Both, the employment of leaching bacteria for metal winning as well as the mitigation of bioleaching processes at AMD/ARD sites, require reliable monitoring methods for assessing bacterial activities. Therefore, we have developed a robust and rapid test system combining two sensitive analytical techniques: quantification of heat evolution by microcalorimetry and determination of all relevant inorganic sulfur species by chromatographic methods (IC and HPLC). Generally, only about 1 g of sample is sufficient for a complete analysis. The combined test has been applied to various leaching biotopes such as bioreactors, columns, heaps and natural sites. The bacterial activity of diverse sulfidic materials such as lignite and coal wastes, pure metal sulfides and complex ores has been investigated. In our labs, microcalorimetry can be performed in the range of 5 to 80 °C, covering most of the temperature spectrum of leaching bacteria. Hence, the heat evolution values of samples as a direct measure for calculating leaching rates can be obtained at nearly all relevant in situ temperatures. The combination with sulfur species determination results in additional information on leaching mechanisms (thiosulfate or polysulfide pathway) and general leaching performance (e. g. accumulation of sulfur intermediates).
Authors: Soeren Bellenberg, Bianca M. Florian, Mario A. Vera, Thore Rohwerder, Wolfgang Sand
Abstract: The aim of this study was to investigate interspecies interaction by quantifying pyrite dissolution and to visualize the colonization of metal sulfides in pure and mixed cultures of leaching bacteria. Strains, such as chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 23270 (type strain) and chemoorganoheterotrophic Acidiphilium cryptum JF-5, were used. Sessile, pyrite-detached and planktonic cells were visualized by epifluorescence microscopy using DAPI staining and FISH. Additionally, atomic force microscopy was used for investigations on cell morphology, bacterial distribution on pyrite and mineral surface topography. In pure At. ferrooxidans cultures and in mixed cultures with Ap. cryptum JF-5, it could be shown that the bacterial morphology of sessile cells differed significantly from those of planktonic cells by decreased cell sizes and enhanced production of extracellular polymeric substances in case of sessile cells. Interspecies interaction in mixed cultures resulted in increased pyrite leaching and production of extracellular polymeric substances and consequently, enhanced biofilm formation.
Authors: Laura Castro, Mario Vera, Jesús Angel Muñoz, María Luisa Blázquez, Felisa González, Wolfgang Sand, Antonio Ballester
Abstract: Aeromonas hydrophila is a facultative anaerobe which, under conditions of oxygen depletion, is able to respire iron (III). Scanning electron microscopy (SEM) and conducting-probe atomic force microscopy (AFM) revealed the presence of filaments between cells and cell-substrate and their conductive nature. These results indicate that the pili of A. hydrophila might serve as biological nanowires, transferring electrons from the cell to the surface of Fe (III) oxides. Conductive pili could also play a role in bacterial interactions and in inter/intra species signalling, and could lead to biotechnological approaches for novel materials.
Authors: Thore Rohwerder, Claudia Janosch, Wolfgang Sand
Abstract: The alpha-proteobacterial genus Acidiphilium consists of several acidophilic species, generally known as a part of the mesophilc microbial flora of leaching biotopes. All of them can grow chemoorganotrophically on carbon sources like sugars and many express additional photosynthetic pigments. Thus far, only Ap. acidophilum is known to be capable of chemolithotrophic growth on elemental sulfur oxidation. The oxidation potential of inorganic sulfur species by the other strictly heterotrophic species has not yet been thoroughly investigated. Here, we demonstrate the unequivocal evidence of inorganic sulfur compound oxidation by strains of Ap. cryptum and other Acidiphilium species. Evolutionary and biochemical aspects of this new feature among the heterotrophic Acidiphilium spp. are discussed. This finding will possibly help to solve the long-standing question about the biochemical nature of elemental sulfur oxidation in mesophilic leaching bacteria.
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