Advanced Materials Research Vols. 71-73

Abstract: 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.

Abstract: The composition of microbial communities in five acid mine water samples were studied, using culture-independent 16S rDNA based cloning and restriction fragment length polymorphism (RFLP) analysis. Phylogenetic analysis revealed that the bacteria in these five samples fell into 4 major groups: Proteobacteria, Nitrospira, Firmicutes and Bacteroidetes. Proteobacteria organisms such as A. ferrooxidans appeared in samples SX3, K1 and K2, but was scarce in samples SX1 and SX2; Nitrospira organisms Leptospirillum ferrooxidans, Leptospirillum ferriphilum and Leptospirillum group III, were prevalent in samples SX1, K1 and K2, but with fewer in samples SX2 and SX3. Archaea were only detected in samples K1 and K2 from the Tong shankou copper mine. Thermoplasma and Ferroplasma lineages were detected abundantly in these two samples. Meanwhile, the results of Principal Component Analysis (PCA) based on the percentages of OTUs and data of biogeochemical parameters, revealed that biogeochemical properties affected the diversity of microbial communities in mine water. The pH, temperature and different concentrations of elements such as S, Ni, Co and Cu seemed to be key factors resulting in the diverse distribution of microbes.

Abstract: More than 100 cultures of acidophilic Fe(II)- and/or sulfur-oxidizing microorganisms from mine waste dumps in 10 different countries all over the world have been maintained in liquid media in the BGR-strain collection for many years. Our 16S rDNA analysis showed that most of the cultivated Fe(II)-oxidizers belong to four genera: Acidithiobacillus, Acidimicrobium, “Ferrimicrobium” and Leptospirillum. All analyzed Acidithiobacillus strains were identified as At. ferrooxidans. The Leptospirillum strains were affiliated with L. ferriphilum or L. ferrooxidans. The Gram-positive strains related to Acidimicrobium or ”Ferrimicrobium” were phylogenetically more diverse than the strains of the genera Acidithiobacillus and Leptospirillum and fell into three separate clusters. While several strains could be identified as syngeneic (16S rDNA) with “Ferrimicrobium acidiphilum”, two other 16S rDNA clusters were distantly related and might represent new species or even new genera. In addition, one new Sulfobacillus strain and one new Alicyclobacillus strain could be identified. Furthermore several strains related to Acidiphilium acidophilum have been detected and form one 16S rDNA cluster.

Abstract: The distribution and diversity of acidophilic microbe at the ore surface of Zijinshan commercial low-grade copper bioleaching heap operated at pH 0.8 were investigated. Samples taken from -1m, -2 m, -3m from the top of heap surface were investigated by 16S rRNA gene clone library. Phylogenetic analyses of 16S rRNA fragments revealed that the retrieved bacterial sequences mainly related to genus Acidithiobacillus (64.6%), genus Leptospirillum (27.3%), genus Sulfobacillus (3.1%) and genus Ferrimicrobium(1.6%). For archaea, only Ferroplasma acidiphilum was detected. Bacterial diversity in the heap was increased from surface layer to underground. The proportion of genus Leptospirillum was sharply reduced (from 48.5% to 5%) from higher depth to lower depth and reverse correlation of increased A.ferrooxidans (from 0.9% to 15%) and S. thermotolerans (from undetectable to 7%) were found in the heap. Sulfur oxidizers including A.albertensis, A.caldus and A.thiooxidans also vertically increased from higher depth to lower depth (from 50.5% to 80%). These results indicated that genus Acidithiobacillus especially sulfur oxidizers A.albertensis, A.caldus and A.thiooxidans may play very important roles in the commercial low-grade copper bioleaching heap.

Abstract: The Escondida mine, located in northern Chile, is the largest copper producing mine in the world. It has an abundant low-grade (ca. 0.5% Cu) sulfide copper ore reservoir, which is processed in large heap bioreactors at the mine. To understand better how microorganisms adapt to heap leaching environments, we have isolated and identified acidophiles from pregnant leach solution (PLS) from the heaps. Six bacteria and one archaeon were isolated directly on solid overlay media, and identified by phylogenetic analyses of their 16S rRNA genes as strains of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillum ferriphilum, Acidiphilium cryptum and Ferroplasma acidiphilum. The sequences of the 16S rRNA genes from isolated strains showed high similarity with those detected previously by culture-independent analyses performed on samples from a pilot plant for this process. Of the three known species of Leptospirillum, only L. ferriphilum has been detected in Escondida PLS. Tolerance of the Escondida isolate (coded IESL-25) to copper and some other transition metals such as zinc, nickel and silver was compared with several other strains of both L. ferriphilum and Leptospirillum ferrooxidans. It was noted that all L. ferriphilum strains (including IESL-25) displayed far greater tolerance to both copper and silver than strains of L. ferrooxidans, though tolerance to zinc and nickel was similar among isolates of both species. Micro-representational-difference analysis (MRDA) was used to study the genetic response of L. ferriphilum IESL-25 to high copper concentration. Gene sequences obtained by MRDA were analyzed using available genomic information for L. ferriphilum and one copper-induced gene identified appears to be involved in lipopolysaccharide biosynthesis.

Abstract: In order to explore new options to optimize the low-grade copper ore bioleaching process, it is important to understand the kinetics of microbial oxidation at industrial level. This work studies the changes of iron and sulfur oxidation rates of microbial communities in solution from an industrial low grade copper bioleaching heap process at Escondida Mine in Chile. Pregnant leach solution (PLS) samples were analyzed periodically to determine physico-chemical parameters. The total numbers of the different microorganism species in industrial samples were determined by Real Time PCR. In addition, Most Probable Number assays (MPN) were performed for iron and sulfur oxidizing microorganisms. Kinetics incubation tests of PLS in the presence of iron or sulfur were performed to study the iron and sulfur oxidation, in total, 102 oxidation profile tests were obtained. Based on the oxidation profiles obtained, the tests were divided into four groups, labeled as fast, normal, stepped shape, and incomplete. The grouping system was established by considering oxidation time and rates, during the initial oxidation stages and accounted for any lag phase. A data mining technique, called decision trees was used to analyze the data and to generate rules that represented patterns in the data. Strong correlations were found between the predominant microorganisms and the behavior of the oxidation tests. Preliminary results indicate that the magnitude order of MPN of the iron oxidizing microorganisms is an important factor in the microbial oxidizing activity, followed by the predominant specie within the microbial population, PLS temperature and Eh.

Abstract: The study of microbial populations of biohydrometallurgic processes is generally more focused on the solutions involved than on the minerals. This is mainly due to complexity and costs associated with taking mineral samples during the leaching cycle. The present study compares the microorganisms in the solutions and the minerals in 1 m test columns and in samples taken from two strips of an industrial heap from the Escondida Copper Mine in Chile. The column test was carried out in six 1 m columns that were operated with similar parameters than the industrial heap. The industrial heap is divided into strips, and samples were collected from Strip 1 (after the end of the operation) and from Strip 9 (during the irrigation period). To analyze the bacterial population Real-Time PCR and CARD-FISH techniques were used. From the results of the column leaching tests a slightly greater cellular density was observed for the mineral than the solution, when the total bacterial count was considered. In both types of samples the predominant microorganisms were Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans D2 and Leptospirillum ferriphilum. In the samples from the industrial heap A. ferrooxidans predominated at the start of operation and A.thiooxidans at the end of the leach cycle.

Abstract: Bioleaching test work was performed in continuously operated multi-stage reactor systems at 70°C using a thermophilic culture treating an Aguablanca Ni-Cu concentrate from Spain and a blend of Cu concentrates from Bor, Serbia. The copper in both these concentrates occurs as chalcopyrite and therefore the use of thermophiles was applied, which resulted in copper recoveries of over 95%. Qualitative assessment of the microbial community in the bioreactors was performed by terminal restriction enzyme fragment length polymorphism (T-RFLP) and clone library analysis of the 16S rRNA genes amplified by PCR. T-RFLP analysis revealed that only archaea were present, and that the communities in both the Aguablanca and Bor systems consisted of two different microorganisms. A 16S rRNA gene clone library using DNA from the Aguablanca system was constructed and screened. Again, two archaea were detected in similar relative abundance in the population as found by T-RFLP analyses. The sequences of these two cloned genes revealed that the dominant archaeon (up to 98% of the total archaea detected) was Acidianus brierleyi, and the other was Metallosphaera sedula. Quantitative assessment of the microbial community was performed by Q-PCR and confirmed the dominance of archaea in the system with Acidianus being the dominant strain (98-99% of the total population) and a minor part of the population (1-2%) consisted of Metallosphaera. Additionally, very small amounts of Sulfolobus spp. were detected. This study, along with other recent studies on the diversity of thermoacidophiles involved in the solubilization of copper from chalcopyrite concentrates, revealed that a wider variety of thermoacidophiles are involved in bioprocessing of metal sulfides, and showed that A. brierleyi should be considered an important biomining acidophile.

Abstract: Terminal Restriction Fragment Length Polymorphism (T-RFLP) was used to determine the diversity of the bacterial and archaeal populations in a bioleaching column charged with a low-grade chalcopyrite ore and operated at 50 oC. Differing populations were identified in the leachate and the column solids but there was not discernible effect in respect of location in the column.

Abstract: Thermotolerant “Thiobacillus prosperus”-like bacteria were enriched from warm, acidic sediments of the island of Milos in the Aegean Sea. Analysis of 16S rRNA gene sequences indicated at least two thermotolerant species, with at least one of them present in similar niches at Vulcano, Italy. Iron solubilization in a pyrite-enrichment culture at 47°C was most rapid in the presence of NaCl at 30 g.l 1. One of the novel species (strain M7) grew in pure culture on pyrite with NaCl at 50 g.l-1, but iron solubilization was most rapid with 20 g NaCl.l 1 at just below 50°C.