Advanced Materials Research Vols. 20-21

Abstract: Microbiologically influenced corrosion (MIC) is a serious problem that continues to plague many industrial systems. In this study, a method to prevent MIC by the use of an azole-type organic compound on the metal substrates was studied. Inhibition of MIC of mild steel and stainless steel 316 by 2-Methylbenzimidazole (MBI) in seawater with sulphate-reducing bacteria (SRB) was investigated using electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). MBI was shown to be an effective inhibitor in controlling MIC by two strains of sulphate-reducing bacteria: Desulfovibrio desulfuricans, and a local marine isolate. EIS analysis shows an increase in charge transfer resistance for both mild steel and stainless steel 316 after the addition of MBI in the aqueous solution. AFM analyses show a decrease in the surface roughness and pit depth after the addition of MBI. Of the two bacterial strains, it is found that MBI is more effective in the inhibition of corrosion by D. desulfuricans. At a concentration of 1mM, MBI shows a higher MIC inhibition effect on stainless steel 316 (corrosion inhibition 99.5%) than on the mild steel (corrosion inhibition 59.4%). These results indicate that MBI shows potential application in the inhibition of MIC of metal substrates.

Abstract: Extracellular polymeric substances (EPS) of Acidithiobacillus ferrooxidans and other leaching microorganisms mediate the attachment of cells to pyrite and other minerals. They also play a pivotal role in indirect leaching of base and precious metals via the contact mechanism. The aim of this study is to get more insight on the influence of the growth substrates iron(II) ions, pyrite, chalcopyrite and elemental sulfur on EPS formation, attachment and biofilm formation. The synthesis of EPS by cells of A. ferrooxidans strain A2 is strongely influenced by the growth substrate or attachment substratum of the cells. Cells grown with soluble iron(II) ions generally generate less EPS than cells grown with solid pyrite, chalcopyrite or elemental sulfur. Planktonic cells grown in the presence of solid substrate produce two to four times more EPS than iron(II) ion grown cells. With sessile cells, this factor is further increased to 50 to 240 depending on the specific substrate. The EPS of all the differently grown planktonic and sessile cells of A. ferrooxidans strain A2 contained neutral sugars, fatty acids, uronic acids, proteins and metal ions. The composition of these compounds varied with the growth substrate and type (planktonic or sessile). The attachment behavior of cells of A. ferrooxidans strain A2 also differed with the substrate of the pre-culture. Cells grown on iron(II) ions, pyrite or chalcopyrite attached rapidly to pyrite and chalcopyrite, while attachment to elemental sulfur was poor. On the contrary, sulfur grown cells attached well to elemental sulfur but weakly to pyrite and chalcopyrite. Attachment of EPS-free cells to all substrates was also diminished. Cells of A. ferrooxidans strain A2 cover mineral surfaces with a dense biofilm after a few days of cultivation, as visualized by fluorescence microscopy and AFM. Large amounts of EPS are formed, which eventually cover the cells and the mineral surface. Even after a few weeks of cultivation the biofilm remained monolayered on all substrates.

Abstract: Attachment to metal sulfide surfaces is a prerequisite of bioleaching activity via the contact mode [Rohwerder et al.: Appl. Microbiol. Biotechnol. Vol. 63 (2003), p. 239]. Until now, the mechanisms and possible variations of this attachment process among leaching bacteria is not well understood. Therefore, we are studying the diversity in the attachment behaviour of different strains of the genus Acidithiobacillus, which is one of the main bacterial group involved in the bioleaching of metal sulfides. Among the species At. ferrooxidans, cells of the type strain (ATCC 23270) were found to attach the least. Whereas bacterial cells of strains A2 and R1 attached the most. Other strains that were investigated in this study included At. ferrooxidans D-26 and R7. Highly likely, these strains exhibit different attachment behaviour due to specific variations in their extracellular polymeric substance (EPS) composition as loss of part of the EPS has previously been observed to have a negative effect on attachment and bioleaching activities. Differences in several EPS parameters such as the amount of complexed Fe(III) ions and the sugar composition are possible reasons for the observed strain-specific attachment behaviour. In addition to attachment assays and microscopic studies (AFM and epifluorescence), leaching activities of the strains were comparatively investigated by short-term as well as growth experiments.

Abstract: Systems Microbiology is a new way to approach research in microbiology. The idea is to treat the microorganism or community as a whole, integrating fundamental biological knowledge with OMICS research (genomics, proteomics, transcriptomics, metabolomics) and bioinformatics to obtain a global picture of how a microbial cell operates in the community. The oxidative reactions resulting in the extraction of dissolved metal values from ores is the outcome of a consortium of different microorganisms. Therefore, this bioleaching community is particularly amenable for the application of Systems Microbiology. As more genomic sequences of different biomining microorganisms become available, it will be possible to define the molecular adaptations of bacteria to their environment, the interactions between the members of the community and to predict favorable or negative changes to efficiently control metal solubilization. Some key phenomena to understand the process of biomining are biochemistry of iron and sulfur compound oxidation, bacteria-mineral interactions (chemotaxis, cell-cell communication, adhesion, biofilm formation) and several adaptive responses allowing the microorganisms to survive in a bioleaching environment. These variables should be considered in an integrative way from now on. Together with recently developed molecular methods to monitor the behavior and evolution of microbial participants during bioleaching operations, Systems Microbiology will offer a comprehensive view of the bioleaching community. The power of the OMICS approaches will be briefly reviewed. It is expected they will provide not only exciting new findings but also will allow predictions on how to keep the microbial consortium healthy and therefore efficient during the entire process of bioleaching.

Abstract: Oxidation of ferrous iron by moderately thermophilic species of the genus Ferroplasma is of considerable potential value in commercial bioleaching operations. A collection of strains was enriched and isolated from a number of natural or industrial acidic sites at 45 and 55°C. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that all strains were members of the Order Thermoplasmatales. Stains BH8 and BH12 showed 98% sequence similarity with Ferroplasma acidiphilum strain YT and DR1 respectively. Strains BH7 and BH10 were closely related to the moderately thermophilic species Ferroplasma cupricumulans. Strain BH9, a moderately thermophilic organotroph, was unrelated to previously described species and probably represents a novel genus. Strains BH8 and BH12 showed some unique physiological differences to the type species F. acidiphilum. Unlike F. acidiphilum, both strains were moderately thermophilic with a temperature range for strains BH8 and BH12 of 24 to 61°C and 27 to 49°C respectively. Strain BH12 grew organotrophically on Darland’s Medium containing glucose and yeast extract and chemomixotrophically on Darland’s Medium supplemented with ferrous sulphate. Moderately thermophilic species of Ferroplasma and Ferroplasma-like genera appear widely distributed geographically and possess considerable physiological and phylogenetic diversity that may benefit industrial bioleaching processes.

Abstract: Members of Leptospirillum genus have emerged not only as one of the most representative bacteria in the Río Tinto ecosystem, but also in other acidic environments (AMD), and in biohydrometallurgical operations. The main objective of this work was to study the role of chemolithoautotrophic bacteria of the genus Leptospirillum in the Río Tinto iron cycle (an extreme acidic environment, characterized by its constant low pH) to better understand and control industrial biohydrometallurgical processes. Different strains of Leptospirillum were isolated from the Río Tinto basin and physiologically and genetically characterized. Certain metabolic capabilities, such as pyrite leaching, iron oxidation and nitrogen fixation, were determined for each strain. Complementary molecular ecology techniques (FISH, CARD-FISH and cloning) were used to study the microbial diversity and the distribution of leptospirilli along the iron gradient in the different phases of Río Tinto: water column, anaerobic sediments and biofilms.

Abstract: Microbial diversity associated with uranium mine areas of Jaduguda, India has been investigated using a culture independent molecular approach. Soil samples collected from existing and proposed mine sites were analyzed for physicochemical parameters. Community DNA was extracted from five samples. Small subunit rRNA gene (16S rRNA) was PCR amplified using bacterial primers. The diversity of the total bacterial community was described at molecular level by amplified ribosomal DNA restriction analysis (ARDRA). Dominant bacterial groups (represents by OTUs) selected by ARDRA were identified by sequencing the 16S rRNA genes. From the bacterial rDNA clone library around 230 clones were used for further analysis. The unique OTUs and number of clones representing such OTUs were determined. Dominant OTUs were sequenced and identified. These phylotypes spanned a wide range within the bacterial domain occupying Proteobacteria, Acidobacteria, Bacteroidetes, Firmicutes, Cyanobacteria as major phyla. About 46 % of clones sequenced from various sites were identified as Proteobacteria. The present findings on microbial diversity at the molecular level are the first of its kind for uranium mine sites of India. Around 20 % of the clone sequences showed little affiliation with known taxa and probably represent new organisms adapted to this habitat.

Abstract: Acidithiobacillus caldus and Leptospirillum ferriphilum dominate the microbial consortium used in continuous-flow, stirred tank processes used to treat gold-bearing arsenopyrite concentrates in South Africa. These microorganisms were adapted to high concentrations of arsenic over several years and both types of microorganisms were found to contain two sets of arsenic resistance genes. One set was present on all isolates of a species irrespective of whether they were highly arsenic resistant or not, while a second, transposon-located set was present in only those strains that had been adapted to high concentrations of arsenic. We isolated a Leptospirillum ferrooxidans from the microbial consortium present in arsenopyrite treatment tanks from Tamboraque (near Lima, Peru) that had not been inoculated with an arsenic-adapted consortium from South Africa. This allowed us to determine whether these microorganisms had acquired similar arsenic resistance mechanisms as contained on the transposons in the highly arsenic resistant South African cultures. Several isolates of both Leptospirillum ferriphilum and L. ferrooxidans from Europe as well as a "Leptospirillum ferrodiazotrophum" were also screened to detect whether they contained similar arsenic resistance transposons even though they had not been selected for enhanced arsenic resistance. Transposons containing arsenic resistance genes that were identical or closely related to those from South Africa were found in both L. ferrooxidans and L. ferriphilum isolates from South America and Europe. The widespread occurrence of arsenic resistance transposons suggests that it should be possible to select for highly arsenic resistant biomining microorganisms from many different sources and therefore, unnecessary to acquire pre-adapted arsenic resistant consortia.

Abstract: An arsenic resistant ferrous iron oxidizing bacterium Acidithiobacillus ferrooxidans (GenBank no. EF010878) was isolated from reactor leachate. The reactor leachate showed extreme environmental parameters. Ferrous iron concentrations of more than 60 g/L were found to be inhibitory in the presence and absence of arsenite. Ks values of 12.5 and 8.0 g/L ferrous sulphate and Vmax of 0.124 and 0.117 g/L/h/0.8 mg of protein were found in the presence and absence of arsenite respectively. At 14.9 g/L of arsenite and arsenate the culture showed 26.8 and 59.7 % ferrous iron oxidizing activity respectively. Amongst the metals studied, copper was found to be more toxic as compared to nickel and zinc. In the presence of 3.51 g/L nickel or 4.68 g/L zinc, about 30 % biooxidation activity was registered. In the pyrite oxidation study 87, 67 and 64 % of pyrite oxidation was found and 2.02, 3.19 and 5.96 g/L total iron was solubilized with 5, 10 and 20 g/L of pyrite respectively. The isolate was also able to oxidize refractory arsenopyrite gold ore and 0.531 g/L of arsenic was solubilized along with 0.872 g/L of soluble total iron. During this period the numbers of planktonic bacteria increased from 2.4 x 106 to 1.0 x 108 cells/mL.