Pedro A. Galleguillos

Monitoring of Microbial Community Inhabiting a Low-Grade Copper Sulphide Ore by Quantitative Real-Time PCR Analysis of 16S rRNA Genes

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.

Microbial Diversity and Genetic Response to Stress Conditions of Extremophilic Bacteria Isolated from the Escondida Copper Mine

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.

Temporal Dynamics of Genes Involved in Metabolic Pathways of C and N of L. ferriphilum, in the Industrial Bioleaching Process of Escondida Mine, Chile

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 that L. ferriphilum was 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 of L. ferriphilum were analyzed by RT-qPCR. The 16S rRNA and alaS genes 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, and glgA, 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 that L. ferriphilum was 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.

Characterization of Oxidizing Activity of a Microbial Community in an Industrial Bioleaching Heap

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.

Analysis of gene expression in response to copper stress in Acidithiobacillus ferrooxidans strain D2, isolated from a copper bioleaching operation

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.

Effect of Increased Acid Concentration on the Microbial Population Inhabiting an Industrial Heap Bioleaching Plant

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.

From Knowledge to Best Practices in Bioleaching

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.

Microbial Survey on Industrial Bioleaching Heap by High-Throughput 16S Sequencing and Metagenomics Analysis

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.

Estimation of Ionic Load Effect on the Oxidizing Activity of the Microbial Population in the Heap Bioleaching Process at Escondida Mine

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.

From Mesophilic to Moderate Thermophilic Populations in an Industrial Heap Bioleaching Process

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.