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Mining contributes to water pollution through the generation and uncontrolled release of sulfuric acid and metalliferous drainage (AMD). While sulfate reducing bacteria have been used for the bioremediation of AMD, one of the main limitations to using sulfate-reducing bioreactors is that most species are highly sensitive to acidity; therefore it is necessary to avoid direct contact between acidic mine waters and the bacteria, which is normally achieved by using “off-line” systems. There have been few successful applications using acidophilic, sulfate-reducing bacterial populations, which tolerate low pH and high metal concentrations and allow direct metal sulfide precipitation to occur within a single bioreactor unit. Here, we describe the performance of a low pH sulfidogenic bioreactor inoculated with an indigenous microbial community to treat mine-impacted water. The inoculum was obtained from anaerobic sediments collected from an acidic river located in northern Chile. The sulfidogenic bioreactor system (2.3 L) was operated as a continuous flow mode unit for 99 days at 30 °C and fed with synthetic water based on the chemical composition of the acidic river, characterized by extremely low pH (2.1) and, zinc and iron as main transition metals (0.5 and 2 mM, respectively). The bioreactor pH was set to 4.5 initially and was increased in stages to pH 6.0 during the experiment. The results show that zinc concentrations in liquors draining the bioreactor were below the detection level in most of the samples analyzed. In addition, by progressively increasing the glycerol concentration, it was possible to increase the removal of iron (70% of the total present), though more acetic acid (from 1 to 5 mM) was generated. Analysis of the microbial populations showed that they changed with varying operation parameters, and a known acetogenic sulfidogen (Desulfoporosinus acididurans) became more dominant over time. The modular unit used for treating mine-impacted water demonstrated robust activity with low complexity engineering.

The use of information available in the organizations to understand what good performance looks like has been proposed for improving the decreased productivity in mine sector. Detailed monitoring has been performed at the heap bioleaching process in Minera Escondida since the start of the industrial operation in 2006. The huge industrial data recorded represents an opportunity to raise knowledge about complex bioleaching processes for improving the technology. A systematic approach using machine learning tools for the analysis of High Dimensional Feature Space is now being developed to deliver experience-based learning with the aim to serve as the foundation for optimal production planning and operational decision making, in the presence of inherent process variations. The construction of a Decision Support System (DSS) is reported, which considers a Real Time PCR array, a database for data logging and storage, the application of suitable statistical and computational tools for knowledge acquiring and finally the creation of a system of knowledge translation to transform it into action by applying recommendations that come to terms with operational limitations. The user can accurately retrieve data and design similar matches to the historic operation to get, for instance the expected metallurgical performance (such as copper recovery, acid consumption and bacterial activity) and recommendations. The process followed to construct the base of knowledge of the DSS is discussed.

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.

Microorganisms, which play a key role during mineral dissolution in bioleaching processes, require O 2 to promote the oxidation processes of sulfide mineral dissolution and CO 2 or more complex molecules as carbon source for cell growth. Preliminary models have been proposed for relating the microbial succession in bioleaching heaps with the activity of different CO 2 fixation pathways. The increase of internal heap temperature during bioleaching improves the rate of chalcopyrite dissolution. However, it also negatively affects CO 2 and O 2 availability, consequently the microbial community changes and only the best adapted microorganisms are capable of growing under the limiting conditions. In this work we attempt to elucidate how microbiological succession proceeds in a semi-industrial bioleaching column test system, with emphasis in determining the identity of the microorganisms participating and the metabolic dynamics of carbon fixation pathways. In silico reconstruction of the carbon fixation metabolisms based on available genomes and the gene expression studies using microarray and RNA-seq were performed. The physicochemical conditions and the metallurgical parameters were also included in the analysis. Although the investigation is based on a non-homogeneous system - which leads to some seemingly contradictory data - the results showed a clear change in the structure of the microbial community as well as in the expression of pathways for CO 2 fixation, as the column test progressed. These changes were directly related to two factors, the temperature inside column and the CO 2 availability. The gene expression analyses confirmed the temporal distribution of microorganisms as a function of the temperature and the different pathways for CO 2 fixation. The evidences obtained here support the fact that low CO 2 availability becomes an important asset to the microbial population survival and growth promotion in the bioleaching environments. The determination of the expression level of the key genes is a good opportunity to improve a sound understanding of the high temperature bio-assisted heap leaching of chalcopyrite and to optimize the technology.

The major industrial heap bioleaching processes are located in desert regions (mainly Chile and Australia) where fresh water is scarce and the use of resources with low water activity becomes an attractive alternative. However, in spite of the importance of the microbial populations involved in these processes, little is known about their response or adaptation to osmotic stress. In order to investigate the response to osmotic stress in these microorganisms, six species of acidophilic bacteria were grown at elevated osmotic strength in liquid media, and the compatible solutes synthesised were identified using ion chromatography and MALDI-TOF mass spectrometry. Trehalose was identified as one of, or the sole, compatible solute in all species and strains, apart from Acidithiobacillus thiooxidans where glucose and proline levels increased at elevated osmotic potentials. Several other potential compatible solutes were tentatively identified by MALDITOF analysis. The same compatible solutes were produced by these bacteria regardless of the salt used to produce the osmotic stress. The results correlate with data from sequenced genomes which confirm that many chemolithotrophic and heterotrophic acidophiles possess genes for trehalose synthesis. This is the first report to identify and quantify compatible solutes in acidophilic bacteria that have important roles in biomining technologies.

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.

CLST is an extremely acidophilic gamma-proteobacteria that was isolated from the Gorbea salt flat, an acidic hypersaline environment in northern Chile. This kind of environment is considered a terrestrial analog of ancient Martian terrains and a source of new material for biotechnological applications. plays a key role in industrial bioleaching; it has the capacity of generating and maintaining acidic conditions by producing sulfuric acid and it can also remove sulfur layers from the surface of minerals, which are detrimental for their dissolution. CLST is a strain of able to tolerate moderate chloride concentrations (up to 15 g L−1 Cl−), a feature that is quite unusual in extreme acidophilic microorganisms. Basic microbiological features and genomic properties of this biotechnologically relevant strain are described in this work. The 3,974,949 bp draft genome is arranged into 40 scaffolds of 389 contigs containing 3866 protein-coding genes and 75 RNAs encoding genes. This is the first draft genome of a halotolerant strain. The release of the genome sequence of this strain improves representation of these extreme acidophilic Gram negative bacteria in public databases and strengthens the framework for further investigation of the physiological diversity and ecological function of populations.

article i nfo Available online 5 October 2014 A comprehensive monitoring program has been performed at an industrial bioleaching heap at the Escondida mine in Chile since 2006, in order to study possible changes to the indigenous microbial population. In the initial stage of thebioleachingheapoperation,themicrobialpopulation wasdominatedby mesophilic microorganisms. When the height of the heap increased, the microbial population changed from predominantly mesophilic to thermotolerant and moderate thermophilic microorganisms. The results of molecular analyses indicated that under these conditions Leptospirillum ferriphilum and Sulfobacillus thermosulfidooxidans are the most abundant microorganisms. Those results obtained show that the highest ferrous iron oxidation activity at the third lift occurs at the same operational temperature window as that of the thermotolerant and moderate thermophiles. An increase in the expression of genes (including rfaF1) associated with cell envelope biogenesis was indicative of the active growth of L. ferriphilum. We conclude that the increasing heap height and the consequent increase in temperature within the heap triggered a change in the microbial community from mesophilic to moderate thermophilic microorganisms. Further, this shift had a relevant impact on the metallurgical performance of the industrial bioheap process at Escondida mine.

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 industrial run of mine (ROM) bioleaching heap of Escondida mine is monitored monthly from Pregnant Leach Solution (PLS) to assess concentration of microorganisms, microbial activity and physicochemical parameters generating a huge amount of information. To obtain a better description of the iron microbial activity and the dissolution rate of sulfide ores occurring in the leaching cycle, iterative process based on “Knowledge Discovery in Databases” (KDD) was used. A data mining technique, called “hierarchical clustering”, was employed with mineralogical characteristics of the ores loaded in the heap as input. Three different groups of strips were distinguished by this model. Chalcocite and chalcopyrite contents were the most relevant parameters selected by “decision tree” technique. The results showed that there was a good coincidence between the three groups defined and the metallurgical performance in terms of copper recovery. Moreover, another decision tree analysis was performed including the three mineralogical groups, the gathered data about physicochemical parameters and the microbial community composition and function from the PLS. The PLS temperature and the MPN were the most relevant factors selected to explain the differences between the operation of the defined groups of strips. The group with the lowest chalcocite and highest chalcopyrite content, reached the highest PLS temperature and the highest MPN of ferrous iron oxidizing microorganisms and also the fastest kinetics of copper recovery. The estimated temperature inside the heap following conservatively the available models for heaps loaded with agglomerated material, together with the sustainable occurrence of a moderate thermophile population and the confirmation of some chalcopyrite dissolution by analysis of tail allowed us to account the chalcopyrite as a microbial substrate in that industrial scale operation. Those results confirmed the relationship between the microbial community function and the copper release and the hypothesis that the substrate availability is an important parameter to describe the microbial activity. Nevertheless, the model is not yet able to explain the low bacterial growth in the group with the highest chalcocite content that is considered a good microbial substrate. Then it is needed to perform another cycle of the KDD process to complete the model. This kind of information should be considered in defining models to describe and to predict copper recovery in ROM industrial bioleaching processes.

Indexación: Web of Science; PubMed Leptospirillum ferriphilum Sp-Cl is a Gram negative, thermotolerant, curved, rod- shaped bacterium, isolated from an industrial bioleaching operation in northern Chile, where chalcocite is the major copper mineral and copper hydroxychloride atacamite is present in variable proportions in the ore. This strain has unique features as compared to the other members of the species, namely resistance to elevated concentrations of chloride, sulfate and metals. Basic microbiological features and genomic properties of this biotechnologically relevant strain are described in this work. The 2,475,669 bp draft genome is arranged into 74 scaffolds of 74 contigs. A total of 48 RNA genes and 2,834 protein coding genes were predicted from its annotation; 55 % of these were assigned a putative function. Release of the genome sequence of this strain will provide further understanding of the mechanisms used by acidophilic bacteria to endure high osmotic stress and high chloride levels and of the role of chloride-tolerant iron-oxidizers in industrial bioleaching operations. https://standardsingenomics.biomedcentral.com/articles/10.1186/s40793-016-0142-1

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.

During the past few decades the microbial bioleaching of metallic sulphide ores has become a very important process utilized by the mining industry. The biodiversity studies of industrial systems have revealed the occurrence of acidophilic microorganisms, forming dynamic communities that include bacteria and archaea. A combination of molecular and cultivation-based approaches has been used during the leaching cycle to understand the key microorganisms present and their function in the Escondida. Sulphide Leach Plant. The global genomic expression in solution samples has been analyzed by random arbitrary primed polymerase chain reaction (RAP-PCR), to pinpoint those microorganisms with active metabolism in the communities present in regions of the heap leach operation of different ages. Four random primers were used and one hundred and twenty RAP-PCR products were cloned, sequenced and compared to databases using the BLAST search algorithm. Approximately 48% of the RAP-PCR products were 16S and 23S rRNA genes, and many of these results are consistent with community analysis carried out on the same heap using 16S rRNA gene libraries and real-time PCR. Interestingly, different microorganisms to those previously detected by 16S rRNA PCR-DGGE, represented by sequences distantly related to the archaea Picrophilus torridus, and Thermoplasma acidophilum sequences, were detected using RNA analysis. Eight different RAP-PCR products shared similarity with functional genes, and sequence analysis revealed the expression of genes associated with transcription (RNA polymerase) and translation (elongation factor and ribosomal protein) components, metabolic function (2-methylisocitrate synthase and pyruvate dehydrogenase), and electron transport (cytochrome). Other genes such as mercuric reductase and ABC transporter ATP-binding proteins were also detected. However, 30% of RAP-PCR products showed no similarity with any sequences currently in the database. This report represents the first transcriptomic study of the microbial community inhabiting an industrial bioleaching system. The data suggest that the functional spectrum of expressed genes was consistent with genomic and proteomic reports from pure cultures and environmental AMD samples containing microorganisms involved in bioleaching processes. Both studies revealed the expression of genes mainly related to energetic metabolisms and stress responses. (C) 2008 Published by Elsevier B.V.

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.

A comprehensive monitoring program by culturing and molecular techniques, physicochemical analysis plus the record of the operational conditions have been performed at an industrial bioleaching process for run-of-mine (ROM) material at Escondida mine since 2006. The data collected have been systematized in a data base to allow further analysis for advancing the understanding of commercial bioheap applications. The microbial succession during a heap bioleaching cycle has been formerly described by means of the analysis of the data obtained from the first lift of the industrial process. Data visualization techniques were used to analyze the information and to find patterns in the data. When only the first and second floor of the heap was operative, the mesophile population, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, was predominant in the microbial community. After the start of the third lift operation, a relevant change in the abundance of the moderate thermophile population was evident. Leptospirillum ferriphilum (optimum temperature 35-39°C) and Sulfobacillus thermosulfidooxidans (optimum temperature 45-50°C) became the most abundant microorganism. In addition, the specific iron oxidation activity showed by the moderate thermophilic dominated community is higher than the mesophilic dominated one. A patched distribution of Sulfolobus allowed us to infer the temporal occurrence of small niches with temperature over 50°C inside the heap. The temperature turned out to be a key factor driving the dynamic of the microbial community mainly after the start of the third lift. That shift had a relevant impact on the metallurgical performance of the industrial process.

13 pages, 5 figures, 3 tables, Athalassohaline lakes are inland saline aquatic environments with ionic proportions quite different from the dissolved salts in seawater. Prokaryotes inhabiting athalassohaline environments are poorly known and very few of such places have been surveyed for microbial diversity studies around the world. We analyzed the planktonic bacterial and archaeal assemblages inhabiting several of these evaporitic basins in a remote and vast area in northern Chile by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene fragments. Most systems were springs and athalassohaline ponds in different saltflats of the Atacama Desert region, including Salar de Llamará (in the Central Depression), Salar de Atacama (in the Pre-Andean Depression) and Salar de Ascotán (in the Altiplano). Overall, we analyzed more than 25 samples from 19 different environments with strong gradients of altitude, qualitative ionic compositions and UV influence. Between 4 and 25 well-defined DGGE bands were detected for Bacteria in each sample, whereas Archaea ranged between 1 and 5. Predominant DGGE bands (defined by intensity and frequency of appearance) were excised from the gel and sequenced. Bacterial assemblages were dominated by the Cytophaga-Flavobacterium- Bacteroides (CFB) phylum and a few Proteobacteria. There was a tendency for increasing contribution of CFB with higher salinities and altitude. Thus, CFB accounted for the major fraction of band intensity in the Ascotán samples and for lower percentages in Atacama and Llamará. When the distribution of particular CFB sequences was examined, there were several relatives of Psychroflexus torquis substituting each other as salinity changed in Ascotán. Another set of CFB sequences, very distantly related to Cytophaga marinovorus, was abundant in both Llamará and Atacama at salinities lower than 7%. Archaeal assemblages were dominated by uncultured haloarchaea distantly related to cultured strains mostly obtained from thalassohaline environments. Most of the archaeal sequences did not have a close match with environmental 16S rRNA genes deposited in the database either. Therefore, athalassohaline environments are excellent sources of new microorganisms different from their counterparts in thalassohaline sites and useful tools to relate microbial genetic diversity and environmental characteristics such as changes in salinity (both qualitative and quantitative) and altitude. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved, Sampling and measurements carried out in Chile were funded by projects FONDEF D97F1078 and D99I1026. Measurements carried out in Barcelona were funded by project MicroDIFF (REN2001-2120/MAR) from the Spanish Ministerio de Ciencia y Tecnología. Exchange agreements between Chilean CONICYT (#1999 2 02 136) and the Generalitat de Catalunya (ACI99-27) and CSIC (2001CL0020) provided funds for travels between Spain and Chile. [...] EOC was supported by the Programa Ramón y Cajal from the Spanish Ministerio de Ciencia y Tecnología

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.

14.1 IntroductionA series of astrobiological high-altitude expeditions to the South AmericanAndean Mountains were initiated in 2002 to explore the highest perenniallakes on Earth, including several volcanic crater lakes at or above 6000 min elevation. During the next five years, they will provide the first integratedlong-term astrobiological characterization and monitoring of lacustrineenvironments and their biology at such an altitude. These extreme lakesare natural laboratories that provide the field data, currently missingabove 4000 m, to complete our understanding of terrestrial lakes and biota.Research is being performed on the effects of UV in low-altitude lakesand models of UV flux over time have been developed (Cockell, 2000). Thelakes showing a high content of dissolved organic material (DOM) shieldorganisms from UV effects (McKenzie et al., 1999; Rae et al., 2000). DOMacts as a natural sunscreen by influencing water transparency, and thereforeis a determinant of photic zone depth (Reche et al., 2000). In sparselyvegetated alpine areas, lakes tend to be clearer and offer less protectionfrom UV to organisms living in the water. Transparent water, combinedwith high UV irradiance may maximize the penetration and effect of UVradiation as shown for organisms in alpine lakes (e.g., Vincent et al., 1984;Vinebrook and Leavitt, 1996). Shallow-water benthic communities in theselakes are particularly sensitive to UV radiation. Periphyton, which definescommunities of microorganisms in bodies of water, can live on varioussusbtrates. While on rocks, they include immobile species that cannot seeklow UV refuges unlike sediment-dwelling periphyton (Happey-Wood, 1988;Vincent et al., 1993) or alpine phytoflagellates (Rott, 1988) which bothundergo vertical migration. Inhibition of algal photosynthesis by UV radia-tion has been documented in the laboratory (Ha ¬der, 1993) and it has beenshown that phytoplankton production is reduced by formation of nucleic acidlesions (Karentz et al., 1991) or production of peroxides and free oxygenradicals (Cooper et al., 1989). Most of the experiments that have demon-strated in situ suppression of algal growth by UV radiation have eitherused artificially enhanced UV irradiance (Worrest et al., 1978) or shallowsystems (

Se estudiaron las comunidades estratificadas de microorganismos fotosintéticos que se encuentran en el Salar de Llamará ubicado en el desierto de Atacama, norte de Chile, mediante métodos microscópicos y espectrofotométricos. El espesor de la zona fótica de los tapetes descritos varió entre 8 y 30 mm lo cual podría atribuirse a la granulometría y la composición mineralógica de los sedimentos. Se diferencian tres tipos de tapetes. El primero con una única capa pigmentada de color verde; el segundo con capas de colores verde y naranja y un tercero en el que se observa, además de las capas verde y naranja, una de color púrpura. En uno de los sitios muestreados no se encontraron capas pigmentadas. Debajo de la zona pigmentada el sedimento es de color blanco, excepto en uno de los sectores donde se observó una coloración negra atribuible a sulfuro de hierro. Los microorganismos predominantes de la capa naranja fueron diatomeas y cianobacterias unicelulares principalmente de los grupos Cyanothece y Synechococcus. Las cianobacterias filamentosas Microccoleus sp. y Oscillatoria sp. fueron las más abundantes en la capa verde. No se observaron diatomeas en los sitios estudiados donde la salinidad del agua intersticial osciló entre 12 y 33 %. En la capa verde de estos sitios predominaron las cianobacterias cocoides, de los grupos Synechococcus, Cyanothece y Gloeocapsa y del género Gloeobacter, sobre las cianobacterias filamentosas. La capa púrpura estuvo compuesta principalmente por bacterias fototróficas anoxigénicas similares a células de los géneros Chromatium y Thiocapsa. Los espectros de absorción revelaron que la clorofila a es el pigmento más abundante en la mayoría de las muestras analizadas. Los valores integrados de clorofila a y bacterioclorofila a alcanzaron 230 y 144 mg m-2 en el espesor de la zona pigmentada, respectivamente. También se detectaron abundantes microorganismos no fotosintéticos en los tapetes incluyendo cocos y bacilos no identificados. En todos los tapetes muestreados en el Salar se encontraron bacterias reductoras de sulfato.