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The huge industrial data recorded by several years in copper bioleaching operations represents an opportunity for the technology improvement. A systematic approach is being developed to get insights from the field data from an industrial process and to deliver the obtained knowledge with the aim to serve as the foundation for optimal industrial decision making even in presence of inherent process variations. The development of this Decision Support System (DSS) considers a Q-PCR array, a database for data logging and storage, the application of suitable statistical and computational tools for data analyses and knowledge acquiring and finally the creation of a system of knowledge translation to transform it into action (operational suggestions). The user can accurately retrieve data and design similar matches to the historic operation to get e.g. the expected metallurgical performance of a strip based on its mineralogical parameters. In addition, the user can get computed information and recommendations that should be analyzed. We will discuss the process followed to construct the base of knowledge of the DSS.

Acidithiobacillus thiooxidans is one of the most stable components in the consortium of microorganisms inhabiting the copper bioleaching heap of Minera Escondida Limitada. CalvinBenson-Bassham (CBB) is the CO2 fixation pathway most used by aerobic bacteria, and is the exclusive pathway used by A. thiooxidans. In this work, the population dynamics and the transcriptomic dynamics of five key genes involved in the A. thiooxidans CBB pathway were studied in bioleaching column and pure culture tests at different concentrations of CO2 availability. Association between CO2 level, population dynamics and relative expression of CBB-genes was discovered. Differences between CBB pathways from closely related species were reported. The transcription profile modelling could provide useful knowledge for improving industrial bioleaching operations.

Flotation tailings are hazardous due to physical and chemical instability and are currently considered one of the main pollution concerns of mining industry. Most of these tailings are fine-grained residues with low copper content (0.1-0.8%) and include toxic compounds, such as heavy metals, arsenic, and flotation reagents, among others. Another hazard is the generation of acid mine drainage (AMD) due to biological oxidation of tailings. On the other hand, low-grade ore is challenging the mining industry to commercially produce copper.The main objective of this work was to assess the use of flotation tailings and low-grade ore to produce a biologically-produced copper sulfide, as a product for further pyrometallurgical or fine chemistry processing. Low-grade ore and flotation tailings were first agglomerated at different proportions and leaching tests were performed in 1m columns, using either industrial or synthetic raffinate solution. Then, the PLS solution obtained from the column tests was subjected to selective precipitation of copper by using biogenic hydrogen sulfide (H2S) generated by a mixed culture of neutrophilic sulfate reducing bacteria (SRB), enriched from sediments of Caracarani river (located at 4,600 m over sea level in northern Chile).Over 91% of the copper soluble present in the PLS was recovered as copper sulfide in 28 days, while ferrous iron, other metals and arsenic remained in solution. Analysis performed by XRD confirmed the presence of copper in the precipitate (58% as covellite). This work demonstrates the possibility to apply bioleaching and bio-precipitation technologies to treat tailings and low-grade copper ore to produce a high quality copper sulfide.

Bioleaching processes are usually open systems where introduced and native microorganisms survive to changes in pH, temperature, salt and metal concentration, among others. Spatial and temporal description of the microbial community could be relevant for better comprehension of copper extraction process and help in the development of operative procedures to improve the metal extraction. We performed metagenomics and high-throughput sequencing of 16S rRNA genes analyses on samples from Escondida mine bioleaching heap and laboratory columns tests. Archaeal community structure in samples was assessed using three pairs of Archaea-specific primers, and results were highly depending on the primers pairs used. Similarly, three pairs of Bacteria-specific primers were used to assess the bacterial community. Moreover, according to the metagenomics analysis, At. thiooxidans, F. acidarmanus, Leptospirillum spp., Acidiphilium sp. JA12-A1, Acidiphilium spp., At. ferrivorans, and Leptospirillum ferriphilum were the most representative microorganisms. The repercussion of the different methodologies and outputs in the characterization of the bioleaching microbial community is discussed. A better understanding of the microbial community in bioleaching processes could improve the analysis regarding environmental changes in the heap process, its metallurgical performance and, can be used to assist in the decision-making process.

In bioleaching, the chemiolithotrophic community plays an important role as oxidizers of sulfur compounds and ferrous iron. Ferrous iron oxiding microorganisms are key players in the process, as ferric iron is absolutely required to solubilize metal sulfide ores. Members of the Sulfobacillus genus (able to oxidize ferrous iron) were predominant (22 - 95%) in a chalcopyrite bioleaching columns test. In order to obtain new insight about the mechanism of iron oxidation in Sulfobacillus we investigated the presence and expression of genes potentially related to iron oxidation by Sulfobacillus, especially the group of the so-called blue-copper proteins rusticyanin (rus) and sulfocyanin (soxE) in the course of the experiment. The physicochemical parameters and the population dynamics were monitored periodically in the columns and the metatranscriptome was analyzed by using pyro-sequencing. The average temperature inside the column ranged from 22 to 57 °C and the Fe(II) oxidation rate at 45 °C varied between 8 and 42 mg L-1h-1 along 300 days of operation. The metatranscriptomic analysis reveals an over-expression of 9-13 folds of the putative rus and soxE genes in four strains of Sulfobacillus spp. when the Sulfobacillus proportion in the column was >80% and the Fe(II) oxidation rate measured at 45 °C reached 10 mg L-1h-1. Some cytochromes from the electron transport chain were also over-expressed, on a range of 7 - 10 folds under those operational conditions. These results support the hypothetical participation of blue-copper proteins in the iron oxidation pathway of Sulfobacilli. Culture assays and more specific expression analysis are necessary in order to confirm this hypothesis. In addition, we attempt to establish the relationship between rusticyanin and sulfocyanin genes and perform a protein sequence analysis that allows us to infer the actual function of these proteins in Sulfobacillus species.

Recently, a preliminary model has been proposed for relating the microbial succession of bioleaching heaps with the activity of different CO2 fixation pathways. In order to confirm this hypothesis and to understand the impact of the carbon metabolism in the metallurgical performance, the expression levels of carbon fixation pathways were investigated in a chalcopyrite bioleaching column test by transcriptomic analysis. The community structure, the physicochemical conditions and the metallurgical parameters were also analyzed. Gene expression profiles obtained by microarrays confirmed the temporal distribution of microorganisms as a function of the temperature and the different pathways for CO2 fixation. These results revealed the impact of the different CO2 fixation pathways in the composition of the microbial assemblage as the bioleaching proceeds.

The structure of the microbial community inhabiting the copper bioleaching heap at Escondida mine has been systematically monitored since the operation was started up (2006), using biomolecular and microbiological analyses. Recent molecular analyses showed thatL. ferriphilumwas one of the most abundant organisms in the process during year 2012. In order to study the biological dynamics of carbon and nitrogen in the process, the expression levels of thirteen genes ofL. ferriphilumwere analyzed by RT-qPCR. The 16S rRNA andalaSgenes were used as reference, and two relative quantification methods (ΔΔct and and Pffafl) were applied to estimate the relative expression levels of metabolic genes. On the day 110 of operation, a significant increment in the expression level of one gene involved in the cycle of tricarboxilic acids (2-oxoglutarate-acceptor oxidoreductase,oorA) was detected. By other hand, the expression level of two genes involved in carbohydrate metabolism (glgP, Glycogen phosphorilase, andglgA, Glycogen synthase) gradually increased, as the operation time progressed. The expression levels of genes involved in the fixation and assimilation of nitrogen increased at later stages of the process. A significant increase of the expression level of the gene annotated for Nitrogenase iron protein (nifH) was detected on the day 185 of operation. The opposite trend was observed for the gene annotated as Ammonium transporter protein (amt), as an elevated expression level was observed in earlier stages to suddenly decrease on the day 185 of operation, suggesting a change of the nitrogen source. In agreement with molecular quantitative analyses, this work confirmed thatL. ferriphilumwas an active member of the community during the period studied. This work gives new insights into biological dynamics of carbon and nitrogen, and suggests the potential guidelines to enhance the efficiency of biological components in industrial heap bioleaching processes.

Heap bioleaching is an established technology for recovering copper from low-grade sulphide ores. Recently, genetics-based approaches have been employed to characterize mineral-processing bacteria. In these approaches, data analysis is a key issue. Consequently, it is of fundamental importance to provide adequate mathematical models and statistical tools to draw reliable conclusions. The present work relates to current studies of the consortium of organisms inhabiting the bioleaching heap of the Escondida mine in Northern Chile. These studies aim to describe and understand the relationship between the dynamics of the community and the performance of the industrial process. Here, we consider a series of quantitative real-time polymerase chain reaction (PCR) experiments performed to quantify six different microorganisms at various stages of the bioleaching cycle. Establishing the reliability of the data obtained by real-time PCR requires the estimation of the error variance at several different levels. The results obtained show that the sampling component of the error variance is the dominant source of variability for most microorganisms. An estimate for the proportional reduction in residual standard deviation from the use of extraction and real-time PCR triplicates was found to range from 3% to 27% for the different organisms. This result suggests that triplicate assays would produce only a modest reduction in error variance compared to more frequent sampling from the heap.

The design of the industrial copper bioleaching plant at Escondida mine involves the recycling of the solutions in the system, with a consequent increase of ionic solutes in the raffinate solution and Pregnant Leach Solution (PLS). It is known that elevated concentrations of solutes in these solutions have a negative effect on the microbial population leading to decrease the dissolution of copper containing ore. To study the effect of the ionic load on the microbial oxidizing activity, a regression model was built using the systematic data obtained for the concentrations of sulfate, aluminum, chloride and manganese in solutions, during five years of operation. Then, this model was used to support a design of experiment (DOE). The DOE revealed an important effect on the microbial activity produced by sulfate and/or aluminum, decreasing the activity to minimum values, but the effect of chloride and manganese were not significant on the microbial oxidizing activity. This work highlights the importance of determining operational limits for the ionic load of biomining solutions in order to prevent future inhibitions of the microbial oxidizing-activity.

The material treated in the industrial heap bioleaching plant at Escondida Mine includes sulfide, oxide and mixtures of run of mine ore (ROM) with 5% of total copper average grade. The design of the heap considers seven lifts of eighteen meters height each. Nowadays, the heap is being operated with ore loaded in the second and third lifts. The amount of acid required for the process can be increased by increasing the height of the heap and also by the characteristics of the ore. This effect can be attributed to the increase of contact time between ore and solution. The goal of this work was to assess the effect of using raffinate solution with elevated concentration of acid on a microbial culture obtained from process solutions. Reactor tests of culture grown in industrial raffinate solution were performed and DNA and RNA were extracted for qPCR and RT-qPCR analyses, respectively. A design of experiment (DOE) was considered to determine the number of replicates in order to assess the effect of two concentrations of sulfate (80 and 100 g/L) and two incubation temperatures (30 and 45 °C). In addition, analysis of the Most Probable Number (MPN) of iron and sulfur oxidizing organisms and oxidation tests were used to determine the microbial activity in the tests. The results obtained from the DOE showed that acid, temperature and the interaction of temperature and energy source had a significant effect on the microbial activity. Both iron and sulfur oxidizing activities decreased when acid was added at elevated concentration. By other hand, molecular analyses showed differences in levels of specific concentrations of microorganisms and the expression of 16S rRNA gene of the different species thriving in the culture tests.

Acidithiobacillus ferrooxidans strain D2 was isolated from a copper bioleaching operation in Atacama Desert, Chile. Copper is widely used as cofactor in proteins but high concentrations of copper are toxic. Cells require certain mechanisms to maintain the copper homeostasis and avoid toxic effects of high intracellular concentration. The molecular response of A. ferrooxidans strain D2 grown in the presence/absence of copper was examined using a A. ferrooxidans whole-genome DNA microarrays. Roughly 23% of 3,147 genes represented on the microarray were differentially expressed; about 9% of them were upregulated in the presence of copper. Among the upregulated genes, those encoding for the copper efflux protein (CusA) and for the copper-translocating P-type ATPase (CopA) were upregulated. The expression of genes encoding proteins related to iron transport was repressed. Similarly, genes related with assimilative metabolism of sulfur (L-cysteine biosynthesis) cysB, cysJ, cysI, CysD-2 and cysN were upregulated. Our results show that when A. ferrooxidans strain D2 was challenged with high copper concentrations, genes related to copper stress response were upregulated as well as others that have not been reported to be related to that mechanism. In addition, some genes related to other metabolic pathways were repressed, probably because of the energy cost of the stress response.

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.

Microbial heap bioleaching is being used as an industrial process to recover copper from low grade ores. It is known that a consortium of different microorganisms participates in this process. Therefore identification and quantification of communities inhabiting heap bioleaching operations is a key step for understanding the dynamics and role of these microorganisms in the process. A quantitative real-time PCR approach was used to investigate the microbial dynamics in this process. To study the microbial population inhabiting a low-grade copper sulphide ore bioleaching industrial heap process at Escondida Mine in Chile, 16S rRNA genetic libraries were constructed using bacterial and archaeal universal primers. Phylogenetic analyses of sequences retrieved from genetic libraries showed that the community is mainly composed by microoganisms related to Acidithiobacillus ferrooxidans (2 strains), Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and the archaea Ferroplasma. Specific primers for real-time PCR determination were designed and tested to amplify each of the sequences obtained by cloning. Standard curves for real time PCR were performed using plasmid DNA from selected clones. This methodology is actually being used to monitor relevant microorganisms inhabiting this low-grade copper sulphide ore bioleaching industrial heap.

During the last decades, microbial bioleaching of metallic sulphide ores has become a very important process in the mining industry. The study of biodiversity in those systems has revealed the occurrence of several acidophilic organisms, forming a dynamic community including Bacteria and Archaea. Despite the importance of the heap bioleaching process, the metabolic behaviour of a microbial community in bioleaching systems remains unknown. The role played by members of the community inhabiting a bioheap environment has been estimated by the phenotypic characteristics of pure cultures, such as iron oxidation, sulphur oxidation, resistance to high ion concentration and metal tolerance, among others. The global genomic expression of the community inhabiting an industrial bioheap of low-grade copper sulphide ore at Escondida mine in Chile was investigated by random arbitrary primed polymerase chain reaction (RAP-PCR). Three random primers were used and twenty differentially expressed bands were cloned and sequenced. Several sequences were related to 16S rRNA of members of the microbial community; two sequences were related to the α subunit of pyruvate dehydrogenase from Acidithiobacillus ferrooxidans. The expression levels of the pyruvate dehydrogenase gene in samples from an industrial bioleaching operation at different stages were checked by quantitative real-time PCR.