Advanced Materials Research Vol. 825

Abstract: Empirical field tests in which phosphate mining wastes (NPR) were added to various sulphidic mining wastes resulted in neutral drainage. A biofilm was documented on waste rock covering the pyrite. This was likely the cause of the improved effluents. NPR was added to sterile columns containing pyritic lignite and spiked with granulated pyrite. The biological columns were inoculated with 10⁸ cells/g of lignite-cultured microbes from Rio Agrio, Argentina. The microbes were maintained at 30°C using pyrite as sole energy source. The microbial populations were monitored with MPN before NPR addition and 2, 4 and 10 weeks later. After 45 days strong bioleaching was evident, as measured by electrical conductivity. Concurrently, a shift in the microbial populations took place. In the presence of NPR about 90% of populations consisted of heterotrophic neutrophiles covering the pyrite surfaces with a 10 μm thick biofilm, whereas without NPR, 99% of the populations were iron oxidizing acidophiles in mono-layered biofilms. These results are relevant to controlling processes in heap leaching and acid rock drainage.

Abstract: In a field study, biogeochemical processes in a large lignite coal spoil area with moderate AMD generation were investigated. Underneath this area, large amounts of groundwater are impacted by degradation and transformation processes of coal remainders in the former open pit mining area. An investigation was performed to find out the sources for the ground and surface water contaminations of larger areas. Samples were taken from different places and different depths of the coal spoil area and were investigated for different metabolic groups of microorganisms. As a result, fungi are able to degrade humic matter in coal spoil heaps in a first step to oligomers. Other microorganisms do a further degradation of first intermediates in a commensalic community. Streptomycetes do a cleavage of lignocelluloses, strepto- and other actinomycetes also degrade cellobiose and xylose related parts of the humic coal spoil matter. The different members of the microbial community exist in different “floors” of the spoil area: fungi and most Actinomycetes prefer the oxic zone, whereas degraders of aromatic and heterocyclic compounds can also exist in the capillary and ground water zones; here more frequently Arthrobacter, Pseudomonas, Rhodococcus and Mycobacterium strains were detected. Ferric iron formed in biooxidation of pyrite seems to play an important role as a catalyst for oxic as well as anaerobic degradation of complex organic matter in the underground. A complex linkage between microbial Fe-, S-, C- and N-cycles was figured out on this site that induces a high and long-term impact on ground water contamination in this area.

Abstract: The distribution and diversity of bacterial community in Zijinshan commercial non-aeration copper bioheapleaching system operated at pH 0.8 for three years were investigated. The 24 meters high heap was cut off by mechanical digger. On the trapezoidal cross-section of the heap, 9 ore samples were taken from different vertical and horizontal locations and investigated by 16S rRNA gene clone library. Another 3 liquid samples from raffinate solution pond, spray solution pond and pregnant solution pond were also applied to 16S rRNA gene clone library analysis. The retrieved 1166 clone sequences from 12 samples were mainly related to genus Acidithiobacillus (42.36%), genus Leptospirillum (37.73%) and genus Sulfobacillus (6.52%). Relative high amount of heterotrophic bacteria were distributed at the ore surface in the internal part of the heap and in the liquid samples respectively. The retrieved heterotrophic bacterial sequences were mainly related to genus Acidiphilium (accounting 11.11% to 32.00% percent in the liquid samples), genus Acidovorax (accounting 12.37% in A1 sample), genus Pelomonas (accounting 4.17% to 10.31% in several ore samples) and genus Aquabacterium (accounting 10.31% in C2 sample). Bacterial diversity in the heap was increased from the surfcae layer to the interior of the heap. The proportion of genus Leptospirillum horizontally increased from the inner to the outer part while vertically decreased from lower depth (2-3 years leaching time) to higher depth(3-6 month leaching time), and reverse correlation of genus Acidithiobacillus was found in the heap. Our finding indicated that heterotrophic bacteria may play very important roles in the commercial bioheapleaching system, and revealed high distribution of genus Leptospirillum in the outer part of this non-aerated heap.

Abstract: New acidophilic iron-oxidising bacteria like Ferrovum spp. and Gallionella relatives have recently been detected in several mine waters by culture-independent methods. To get information about their physiological characteristics, isolation of respective species is required. In former isolation studies, using a previously described iron-containing medium (iFeo) combined with the overlay-plate technique, mainly Acidithiobacillus ferrooxidans or A. ferrivorans had been obtained, although F. myxofaciens dominated the microbial community. Therefore, a new medium, called APPW was designed based on the water chemistry of a pilot plant for iron oxidation. The growth of iron-oxidising bacteria on overlay plates with iFeo and APPW media, respectively, was compared. Additionally, the iron, phosphate, and ammonium concentrations of APPW were separately adapted to iFeo. Colonies grown were analysed by PCR with primers specific for Ferrovum spp., Gallionella relatives, Acidithiobacillus spp., and Acidiphilium spp. While on all media most colonies obtained were contaminated by heterotrophic Acidiphilium spp., on iFeo Acidithiobacillus, as expected, turned out to be the dominating genus among the iron oxidisers. Interestingly on APPW and APPW-NH₄ Ferrovum dominated among the iron oxidisers. While APPW-Fe, as compared to APPW, seemed to inhibit the Gallionella relatives, APPW-PO₄ resulted in an overall lower number of colonies, but a higher percentage of this taxon.

Abstract: Using indigenous microorganisms in biomining processes assures few ecological distortions and less time consumption for adaptation. However, there is a debate as to whether an indigenous microbial community is more efficient than one composed of microbial strains selected for specific physiological traits. In order to provide thoughtful opinions in such a debate, a cross-comparison of six strains isolated from two different regions: Chambishi Copper Mine (Zambia) and Dexing Copper Mine (China), was conducted. Physiological properties and leaching performances of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, and Leptospirillum ferriphilum in pure and mixed culture were studied. The results showed that strains within species presented similar traits while copper resistance was not species-specific. In terms of leaching efficiency, native strains always achieved higher cell density and greater iron and copper extraction rates than the exogenous. In addition, microbial community analysis revealed the different mixed culture shared almost the same profile, and At. ferrooxidans strains always outcompeted the other strains.

Abstract: The aim of this study was to enrich phosphorus solubilizing microorganisms from high-phosphorus iron ores, apatite ores and phosphogypsum waste. Phosphorus solubilizing microorganisms can be utilized in dephosphorization of high-phosphorus iron ores and in phosphorus leaching from fluorapatite ores. Low grade fluorapatite ore (3.6% P, pH 6.8), fluorapatite concentrate (13% P, pH 8.3), phosphogypsum waste (0.7% P, pH 2.3), iron ore 1 (0.19% P, pH 7.6) and iron ore 2 (0.18% P, pH 7.6) were used as potential sources of phosphorus solubilizing microorganisms. The samples were cultured in NBRIP media at pH 5 and 8 with either glucose or sucrose as a carbon source, and in modified 9K media at pH 1.5 and 2.5 for 3 weeks. Phosphorus solubilizing bacteria were enriched only from the fluorapatite concentrate at the pH of 8. The four obtained heterotrophic isolates were identified by 16S rRNA gene sequencing, and were shown to be closest related to Burkholderia fungorum. These results indicate that the diversity of culturable phosphorus solubilizing bacteria present in apatite and iron ores is relatively low. The isolated Burkholderia strain showed phosphorus solubilizing potential.

Abstract: The extremely thermophilic archaea have become a research hotspot in the recent years because of their extreme living conditions, physicochemical characteristics such as the oxidation of sulphur, metal sulphide ore and excellent leaching capability of metal sulphides. A novel thermoacidophilic archaea (ALE1 strain) of the genus Acidianus, Candidatus Acidianus copahuensis, was isolated from the Copahue Volcano area, in Neuquén, Argentina. This strain was able to metabolize different sulphur compounds under aerobic and anaerobic conditions. ALE1 strain was inoculated in M88, a selective medium for thermoacidophilic archaea recommended by DSMZ. Elemental sulphur (S^o) and potassium tetrathionate (T) were used alternatively as the energy source, while oxygen and iron (III) were the electron acceptors in the aerobic and anaerobic tests respectively. All systems were incubated at 70oC in shake flasks. The pH, Eh, [H⁺] and cell concentration were measured. The results from the aerobic test confirm that ALE1 strain was able to oxidize (S^o) and (T) decreasing the pH significantly, reaching the values 1 and 1.5, respectively. A light yellow precipitate was formed only in the inoculated systems in presence of (T). It was characterized by SEM and FTIR techniques showing that only sulphur atoms are bonded together in some way not confirmed yet. In the anaerobic tests, it was found that ALE1 was able to oxidize sulphur and tetrathionate ion transferring electrons to iron (III) acceptor. The Eh decreased in all inoculated systems from nearly 500mV to 300mV approximately. This work shows that ALE1 strain is helping to keep active the sulphur cycle in the Copahue volcanic environment; however the challenge is to elucidate the metabolism involved.

Abstract: In this work we have studied the attachment and colonization of pyrite surfaces by the recently isolated thermophilic archaeon Candidatus Acidianus copahuensis (isolated from the geothermal Caviahue-Copahue system, Argentina). Cells pregrown with sulfur, iron (II) or pyrite were tested. In order to characterize the EPS glycoconjugates of this strain, fluorescent lectins were used. Concanavalin A (ConA) gave the best signal and was selected for further studies. Coupons and grains of pyrite were treated with DAPI (to stain attached cells) and TRITC-ConA (to stain polysaccharides from EPS). Pyrite surfaces were imaged by epifluorescence (EFM) and confocal laser microscopy (CLSM). Initial cell attachment to pyrite grains was estimated to be 26%, 35% and 43% when cells were pregrown with sulfur, iron (II) and pyrite as electron donors, respectively. It was observed that the cell adhesion correlated with an increase of EPS production; both processes were favoured when cells were pregrown with pyrite or iron (II). Also the effect of phosphate (Pi) starvation in the attachment of Acidianus was studied in similar tests using a base medium without Pi. An increase of cell attachment under Pi starvation conditions was detectable.

Abstract: Mesophilic iron and sulfur-oxidizing acidophiles are commonly used for the extraction of base metals from low-grade sulfide ores in some copper Chilean mines. However, relatively little is known about their activities in cold environments. Some natural ecosystems present in the Andes Mountains, such as the Chilean Altiplano, meet environmental conditions for the growth of psychrotolerant leaching microorganisms. In this work, we obtained enrichment cultures of iron- and sulfur-oxidizing microorganisms from an acid river in the Chilean Altiplano. Molecular identification was performed using PCR products of bacterial 16S rRNA clone libraries, and the sequences analysis revealed the presence of a microorganism related to the recently described psychrotolerant Acidithiobacillus ferrivorans. The Acidithiobacillus strain was able to grow at temperatures ranging between 4 and 30°C, and pH values ranged between 1.7 and 2.5. According to the energy sources, this microorganism was able to grow using ferric iron, sulfur, thiosulfate and tetrathionate. Optimal growth was observed in presence of ferric ion, where the culture reached a potential redox value of 600 mV and a cellular number of 3×10⁷ cells/mL. Molecular analysis of variants of gene encoding for rusticyanin showed that rusB gene was amplified from A. ferrivorans strain and no PCR product was obtained for the rusA gene. Our description is consistent with data previously reported for A. ferrivorans strains. Finally, results of this study highlight the importance and potential of novel native bacterial species proficient in mineral oxidation under low-temperature conditions.

Abstract: A novel industrial-scale bioreactor was implemented by Alcoa of Australia (Alcoa) at its Kwinana alumina refinery (Western Australia) for the degradation of oxalate, an organic byproduct of the Bayer alumina refining process. At the Kwinana refinery oxalate is removed from the Bayer Liquor via a separate side-stream as it increases the operating costs associated with the process and, at sufficiently high levels, may adversely affect the quality and yield of the final alumina product. The bioreactor process provides a more economic and environmentally friendly method for the treatment of removed oxalate compared with chemical conversion or storage of the solid by-product. In previous studies, the microbial community composition of the bioreactor was investigated and was found to be largely dominated by microorganisms of the α-, β- and γ-Proteobacteria subgroups. During the present study, two bacteria that had the ability to use oxalate as a sole source of carbon and energy were isolated from samples obtained from the bioreactor. Phylogenetic and physiological analyses indicated that the two isolates were probably strains of a novel species of a novel genus within the β-Proteobacteria subgroup. Isolation and characterisation of the microbial communities within the bioreactor system has the potential to improve process operation, which may have a positive impact on the biological oxalate destruction process and the footprint of alumina production.