Mark Dopson

William Nyasha Mavengere

Sabrina Hedrich
email: sabrina.hedrich@bgr.de

Anne-Gwenaëlle Guézennec

Mickaël Charron

Axel Schippers

Catherine Joulian

Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate

Keywords: quantitative real-time PCR, bioleaching, community monitoring, Acidithiobacillus, Leptospirillum, Sulfobacillus

Monitoring of the microbial community in bioleaching processes is essential in order to control process parameters and enhance the leaching efficiency. Suitable methods are, however, limited as they are usually not adapted to bioleaching samples and often no taxon-specific assays are available in the literature for these types of consortia. Therefore, our study focused on the development of novel quantitative real-time PCR (qPCR) assays for the quantification of Acidithiobacillus caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, and Sulfobacillus benefaciens and comparison of the results with data from other common molecular monitoring methods in order to evaluate their accuracy and specificity. Stirred tank bioreactors for the leaching of copper concentrate, housing a consortium of acidophilic, moderately thermophilic bacteria, relevant in several bioleaching operations, served as a model system. The microbial community analysis via qPCR allowed a precise monitoring of the evolution of total biomass as well as abundance of specific species. Data achieved by the standard fingerprinting methods, terminal restriction fragment length polymorphism (T-RFLP) and capillary electrophoresis single strand conformation polymorphism (CE-SSCP) on the same samples followed the same trend as qPCR data. The main added value of qPCR was, however, to provide quantitative data for each species whereas only relative abundance could be deduced from T-RFLP and CE-SSCP profiles. Additional value was obtained by applying two further quantitative methods which do not require nucleic acid extraction, total cell counting after SYBR Green staining and metal sulfide oxidation activity measurements via microcalorimetry. Overall, these complementary methods allow for an efficient quantitative microbial community monitoring in various bioleaching operations.

INTRODUCTION

Bioleaching, the extraction of metals by means of microorganisms, is nowadays a well-established process and an economic alternative to conventional roasting or pressure oxidation techniques for sulfidic low-grade ores. This environmental-friendly and low cost technology becomes especially important in the current context, where mineral resources are becoming more complex and of lower grade. In order to enhance the application spectrum and improve the bioleaching performance, fundamental research is required to understand the process and optimize operating parameters.

Bioleaching performance is mainly driven by the colonization of the ore by adapted microbial species and their subsequent activity which is influenced by process parameters such as, e.g., agitation/aeration rates, nutrient medium composition and carbon dioxide enrichment. Accurate monitoring of the microbial bioleaching community is therefore essential for process control and to enhance the leaching efficiency, as well as to understand the microbiological aspects of the process as a key factor to design and operate a bioleaching system.

In recent years, rapid advances especially in tools for molecular microbial ecology have emerged and various techniques to monitor the microbial community in bioleaching processes are available (e.g., [START_REF] Johnson | Techniques for detecting and identifying acidophilic mineral-oxidizing microorganisms[END_REF]. They comprise culture-dependent (plating, MPN counts) and biomolecular approaches such as the genetic fingerprinting techniques, terminal restriction fragment length polymorphism (T-RLFP; [START_REF] Wakeman | Microbiological and geochemical dynamics in simulated-heap leaching of a polymetallic sulfide ore[END_REF], capillary electrophoresis single-strand conformation polymorphism (CE-SSCP; [START_REF] Foucher | Evolution of the bacterial population during the batch bioleaching of a cobaltiferous pyrite in a suspended-solids bubble column and comparison with a mechanically agitated reactor[END_REF], denaturating gradient gel electrophoresis (DGGE; [START_REF] Demergasso | Molecular characterization of microbial populations in a low-grade copper ore bioleaching test heap[END_REF][START_REF] Halinen | Heap bioleaching of a complex sulfide ore. Part I: effect of pH on metal extraction and microbial composition in pH controlled columns[END_REF], microscopic methods like fluorescence in situ hybridization (FISH and CARD-FISH, Schippers et al., 2008), microarray approaches (e.g., [START_REF] Garrido | An oligonucleotide prokaryotic acidophile microarray (PAM): its validation and its use to monitor seasonal variations in extreme acidic environments with total environmental RNA[END_REF][START_REF] Remonsellez | Dynamic of active microorganisms inhabiting a bioleaching industrial heap of low-grade copper sulfide ore monitored by realtime PCR and oligonucleotide prokaryotic acidophile microarray[END_REF], quantitative real-time PCR (qPCR; [START_REF] Liu | Rapid specific detection and quantification of bacteria and archaea involved in mineral sulfide bioleaching using real-time PCR[END_REF][START_REF] Zhang | Application of real-time PCR to monitor population dynamics of defined mixed cultures of moderate thermophiles involved in bioleaching of chalcopyrite[END_REF], and next generation sequencing techniques (e.g., [START_REF] Cardenas | Genomic and metagenomic challenges and opportunities for bioleaching: a minireview[END_REF].

Depending on the process and nature of the samples, suitable methods are, however, limited as parameters such as low sample amount (combined with low cell numbers of, e.g., autotrophic leaching organisms), the presence of particles, low pH and high concentrations of metals often negatively influence adequate implementation of the analysis.

When applying molecular analysis, efficient nucleic acid extraction is key to subsequent quantification of all microorganisms in the sample. Also low pH and high metal content can inhibit nucleic acid extraction and downstream processing such as polymerase chain reaction (PCR) and often pre-treatment of the sample is required. Furthermore, detachment of cells from particles and disruption of biofilms are critical when extracting nucleic acids from bioleaching samples (e.g., [START_REF] Zammit | The recovery of nucleic acid from biomining and acid mine drainage microorganisms[END_REF].

Species-specific (semi-)quantification can be achieved by various molecular methods, e.g., T-RFLP, CE-SSCP, or qPCR. While T-RFLP and CE-SSCP represent semi-quantitative methods based on PCR, qPCR is currently the most common method for quantitative microbial community monitoring. Even so qPCR is widely used and numerous assays have been described in the literature for the quantification of total bacteria, archaea, and special groups of microorganisms, there is only a limited number of assays published for bioleaching organisms (e.g., [START_REF] Liu | Rapid specific detection and quantification of bacteria and archaea involved in mineral sulfide bioleaching using real-time PCR[END_REF][START_REF] Remonsellez | Dynamic of active microorganisms inhabiting a bioleaching industrial heap of low-grade copper sulfide ore monitored by realtime PCR and oligonucleotide prokaryotic acidophile microarray[END_REF][START_REF] Zhang | Application of real-time PCR to monitor population dynamics of defined mixed cultures of moderate thermophiles involved in bioleaching of chalcopyrite[END_REF]. When searching for appropriate assays to monitor a defined bioleaching community it was found that most of these assays do not target the desired species or are not specific enough for quantification on species-level.

Quantification of cell abundances can also be achieved by microscopy-based approaches such as fluorescence in situ hybridization (FISH), catalyzed-reporter-deposition FISH (CARD-FISH), and total cell counts by SYBR Green staining. These methods often suffer from issues such as low cell numbers, cell attachment to particles and auto-fluorescence of these particles. These methods are greatly affected by the acidic pH and elevated metal concentrations which influence the binding properties of the DNA stain and lower the fluorescence signal intensity. Therefore, it is often difficult to stain the cells properly and to differentiate between living organisms and particles.

This study aims to develop and evaluate a selection of molecular methods to monitor the microbial community in bioleaching operations in order to define specific, quick, and reliable methods to be applied in further monitoring studies. In particular, our investigations focus on the quantification of microorganisms at species level via qPCR and comparison with T-RFLP and CE-SSCP data. Further focus is on the application and improvement of total cell counting assays for the bioleaching samples. In addition, microcalorimetric bioleaching activity measurements [START_REF] Rohwerder | Determination of reaction energy values for biological pyrite oxidation by calorimetry[END_REF] are provided for comparison. The model system in our study is a bioreactor set up for the bioleaching of copper concentrate which has been applied in previous studies before and houses a specially adapted microbial consortium proven very efficient for the bioleaching of this material [START_REF] Spolaore | Relationship between bioleaching performance, bacterial community structure and mineralogy in the bioleaching of a copper concentrate in stirred-tank reactors[END_REF].

MATERIALS AND METHODS

Microorganisms, Media, and Growth Conditions

The bioleaching culture used was the KCC consortium successfully applied in previous Cu concentrate bioleaching experiments containing Leptospirillum ferriphilum, Sulfobacillus benefaciens, Sulfobacillus thermosulfidooxidans, and Acidithiobacillus caldus [START_REF] Spolaore | Relationship between bioleaching performance, bacterial community structure and mineralogy in the bioleaching of a copper concentrate in stirred-tank reactors[END_REF]. The consortium was routinely kept on 3% Cu concentrate in basal salt medium pH 1.8 [START_REF] Wakeman | Microbiological and geochemical dynamics in simulated-heap leaching of a polymetallic sulfide ore[END_REF] at 42 • C, which served as inoculum for this study.

Pure cultures of each strain were grown in shake flasks containing basal salts pH 1.8-2.0 supplemented with 10 mM ferrous sulfate (L. ferriphilum) or 1% sulfur (sulfobacilli and A. caldus). The ferrous sulfate solution was filter-sterilized (0.1 µm) and the sulfur used was sterilized by tyndallization.

Batch Bioleaching Reactor

Batch bioleaching reactors operated with the described acidophilic consortium served as model system for microbial monitoring studies. In order to adapt the microorganisms to the mineral and higher solid loads and to achieve maximum metal bioleaching, the experiments followed a 5-step adaptation protocol:

(1) cultivation in shake flasks with 3% solid load for about 2 weeks; (2) transfer of this culture to a 2 L bioreactor with 5% solid load for one week; (3) transfer to a 2 L bioreactor with 10% solid load; (4) repeat step 3;

(5) final bioleaching step in three parallel 2 L bioreactors with 10% solid load.

The experiments of step 3-5 were terminated when copper dissolution reached a plateau approximately between day 6 and 8. At this point the cultures were immediately transferred to the next step to start a new bioreactor run.

Bioleaching experiments were conducted in 2 L temperaturecontrolled (42 • C), stirred batch bioreactors (Electrolab, UK). The reactors were fully baffled and agitation was performed using a dual impeller system consisting of a standard 6-blade Rushton turbine in combination with a 6-blade 45 • axial flow impeller with speed set at 400 rpm. Tests were carried out at 5-10% (w/v) solid load and an initial acidification of the pulp with sulfuric acid to about pH 2.0 (acid consumption of 182.3 g H 2 SO 4 /kg). Inoculation was performed by adding 200 mL of the above described culture at 0.5 days after initial acidification of the material. The bioreactors were sparged with air at 120 L/h. Each experiment was carried out in triplicate and copper dissolution and kinetics during bioleaching was followed. The material used was a copper concentrate originating from a black shale ore and was supplied by the company KGHM (Poland). The concentrate had a particle size of <90 µm and had the following main characteristics: 13.3% Cu, 9.4% Fe, 16.5% S, 1.1% total inorganic carbon and 9.1% total organic carbon.

Chemical Analysis

Daily pH (Blueline 18 pH electrode, Schott, Germany) and redox (BlueLine 31 Rx electrode, Schott, Germany) measurements on bioreactor runs were carried out directly in the pulp.

The measured redox values were corrected to the standard hydrogen electrode and reported as E h . Ferrous and ferric iron concentrations were measured in 0.45 µm-filtered samples using Ferrozine [START_REF] Lovley | Rapid assay for microbially reduced ferric iron in aquatic sediments[END_REF]. Dissolved metals were regularly determined in filtered, acidified samples using ICP-OES or atomic absorption spectroscopy (Varian SpectrAA-300). At the end of bioleaching step 5, the residue was completely harvested from the three bioreactors, washed with acidified (pH 1.5) water and dried before performing digestion with nitric acid following analysis of the metals by ICP-OES or atomic absorption spectroscopy.

Nucleic Acid Extraction and Quantification

For DNA extraction, 2-5 mL of homogeneous slurry samples were regularly taken from the bioreactors and centrifuged for 20 min at 13,000 × g, and the pellet was washed twice with 10 mM Tris buffer, pH 8. DNA extraction was achieved by using the FastDNA Spin Kit for Soil (MP Biomedicals) according to a modified protocol [START_REF] Webster | Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale[END_REF]. Concentration of DNA extracts, as well as of PCR products and plasmids, was determined by measuring absorbance at 260 nm in a FoodALYT photometer (Omnilab) equipped with a Hellma Tray cell (Hellma Anlytics) or in a Quantus fluorometer (Promega).

Quantitative Real-Time PCR

Primer Design

For the design of specific primers, full length 16S rRNA gene sequences of the target strains (NR_044352, AF356829, NR_040945, Z29975) and also 40 other sequences from various strains of each species and related organisms were downloaded from NCBI and aligned using Clustal X in Mega 6.0 [START_REF] Tamura | MEGA6: molecular evolutionary genetics analysis version 6.0[END_REF]. Primers were selected by manual search for variable regions in the 16S rRNA gene sequence of each species which was most likely different from the other species in the alignment. Selected primers (Table 1) were finally checked via in silico PCR using AmplifX (version 1.7.0; [START_REF] Jullien | AmplifX 1.7.0[END_REF].

qPCR Quantification and Thermocycling

Quantitative real-time-PCR was applied in two laboratories (BGR, Hannover, Germany and BRGM, Orleans, France) to quantify total bacteria [START_REF] Nadkarni | Determination of bacterial load by real-time PCR using a broad range (universal) probe and primer set[END_REF] and specific bioleaching species (Table 1) by targeting their 16S rRNA genes. Extracted DNA was amplified by qPCR using the devices StepOne TM (Applied Biosystems) or CFX Connect (BioRad). Master mixes from the companies Applied Biosystems TM (for SYBR green assays at BGR), Quanta Biosciences Inc. (TaqMan R assays at BGR), or BioRad (SYBR assays at BRGM) were used. Reactions were performed in a total volume of 10 µL containing 1X master mix, 0.5 µM each primer and different concentrations of DNA. Each DNA extract was measured in duplicate or A temperature gradient was applied to determine the optimal annealing temperature for each primer pair. Final cycling conditions were an initial denaturation at 95 • C for 10 min, 30 or 40 cycles of denaturation at 95 • C for 15 s and annealing/elongation for 30 s at 60 • C. Melt curves were constructed after each qPCR run with the following parameters: one cycle of 95 • C for 15 s and 60 • C for 1 min followed by temperature ramping up to 95 • C in increments of 0.3 • C.

Assay Validation and Specificity Test

The specificity of each primer pair for the target species was evaluated from the efficiency and melt curves of the qPCR assays. The amplification products were furthermore analyzed on an agarose gel to confirm the absence of unspecific products. Species specificity of primers was checked by using genomic and plasmid DNA of S. benefaciens, S. thermsosulfidooxidans, S. acidophilus, L. ferriphilum, L. ferrooxidans, A. caldus, A. thiooxidans, A. ferridurans, A. ferrivorans, A. ferriphilus, and A. ferrooxidans as template with each primer set to ensure that there was no crossreactivity. If no signal and amplification product was detected in the qPCR curves and on the agarose gel for the other species the assay was classified as specific for the target species. The assays were then tested on various bioreactor samples which had previously been analyzed with other quantitative methods.

T-RFLP Monitoring

Amplification of a 900 bp fragment of the 16S rRNA gene for T-RFLP analysis from DNA extracts was achieved using DreamTaq PCR Master Mix (Thermo Fisher) and a Cy5-labeled 8F forward primer [START_REF] Frank | Molecular phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases[END_REF] and 907R [START_REF] Muyzer | Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments[END_REF] as described previously [START_REF] Okibe | Enumeration and characterization of acidophilic microorganisms isolated from a pilot plant stirred tank bioleaching operation[END_REF]. Up to 4 µL of PCR product were digested in a 10 µL reaction with 1 U of the restriction endonuclease HaeIII (Thermo Scientific) and 1 µL appropriate buffer. The reactions were incubated at 37 • C for 2-3 h. Terminal restriction fragments (T-RFs) were analyzed on a capillary sequencer (Beckman Coulter, GenomeLab GeXP Genetic Analysis System) using a 600 bp size standard and identified by reference to the databank of acidophilic microorganisms held at BGR (including the T-RFs of the species within the bioleaching consortium). Relative abundances of T-RFs were calculated on the basis of peak areas.

CE-SSCP Monitoring

Amplification of a 200 bp fragment of the 16S rRNA gene for CE-SSCP analysis from DNA extracts was achieved using GoTaq polymerase PCR mix (Promega) and the w49 forward primer and the 5 -end FAM-labeled w34 primer [START_REF] Foucher | Evolution of the bacterial population during the batch bioleaching of a cobaltiferous pyrite in a suspended-solids bubble column and comparison with a mechanically agitated reactor[END_REF]. One microliter of 10-to 50-fold diluted PCR product and 0.2 µL of Genescan-600 LIZ internal standard (Applied Biosystems) were heat denatured in deionized formamide (Applied Biosystems), and immediately cooled on ice. Fragment analyses were performed on an ABI Prism 310 genetic analyser using the non-denaturing CAP polymer (Applied Biosystems). Raw data analyses and assignment of peak position were done with the software GeneScan (Applied Biosystems) and relative abundances were calculated on the basis of peak areas.

SYBR Green Staining of Slurry Samples from Bioleaching Reactors

Slurry samples from the bioreactors were either processed directly or fixed in 4% formaldehyde and stored at 4 • C until further processing. SYBR Green staining for total cell numbers was carried out according to [START_REF] Lunau | An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy[END_REF] following homogenization of the samples by ultrasonic treatment (20 s, 20 cycles, 20% intensity). After appropriate dilution, the sample was applied onto a membrane filter (Whatman Nucleopore, d = 25 mm, 0.2 µm pore size). To enhance the visibility of the cells and avoid interactions with the metals and particles the following treatments were tested:

(i) Washing with acidified (pH 1.8) basal salts medium to remove metals and remaining iron to avoid precipitation of ferric iron during TE buffer treatment (higher pH) (ii) Treatment with 0.05% Triton X to detach cells and allow homogenous distribution of the sample on the filter (iii) Treatment with 0.1% Tween 20 to detach cells and allow homogenous distribution of the sample on the filter Each pre-treatment step was followed by rinsing with TE buffer in order to reach the appropriate neutral pH again for SYBR Green staining. Afterward the filter was put onto a microscopic slide and covered with 20 µL staining solution (6% SYBR Green, 7% Mowiol, 1% ascorbic acid) before counting cells under the microscope.

All treatment methods were performed in triplicate on at least three independent bioleaching samples to validate the method. Cell numbers were determined for each sample by counting across the whole filter area and at least 50 fields of view.

Microcalorimetry

Microcalorimetric measurements were carried out at the beginning and end of each experiment in order to determine the activity of the cultures on the concentrate [START_REF] Schippers | Bioleaching: analysis of microbial communities dissolving metal sulfides[END_REF]. Therefore 1 mL of the pulp was put into a 4 mL glass ampoule and the supernatant was removed after 5 min of settling. The ampoule was sealed and the heat output measured in a TAM III microcalorimeter (TA Instruments) at 42 • C. Samples were measured in triplicate for about 12 h. The weight of the residue before the experiment and the dry weight afterward were determined in order to calculate the heat output per gram solids. Chemical control experiments were conducted with the same set up.

Statistical Analysis

Statistical analyses were run to compare for one species the ratio determined by qPCR and the ratio determined by CE-SSCP or T-RFLP. Differences were determined with a nonparametric Kruskal-Wallis test with α = 0.05, using the XLSTAT software.

RESULTS

Development of Quantitative Real-Time PCR Assays

Novel qPCR assays targeting the four species relevant in our bioleaching experiments (A. caldus, L. ferriphilum, S. thermosulfidooxidans, S. benefaciens) were especially designed for this study. Melt curve analysis proved the specificity of the designed assays for the target species. Only the L. ferriphilium assay yielded an amplification product with L. ferrooxidans DNA but all of the other assays were clearly species-specific, as also confirmed by gel electrophoresis. Different concentrations of DNA were tested to establish the detection limit with linear calibration curves being obtained over seven orders of magnitudes ranging from 10 3 -10 9 16S rRNA gene copies. All four designed qPCR assays reached appropriate amplification efficiencies ( 94%) and r 2 values of 0.99. The precision of the assays was measured by calculating the variation in C t values across three replicate samples, and an average standard deviation of less than 0.16 units proved the reproducibility of the realtime PCR assays. Successful application of the novel qPCR assays was confirmed at BGR and BRGM by using different qPCR machines, master mixes, and application to various DNA extracts of bioleaching samples.

Comparison of Different Molecular Monitoring Methods

T-RFLP and CE-SSCP fingerprinting analyses were performed on the same DNA extracts as used for qPCR and the quantitative data gained, expressed as relative abundance of a defined species in the total bacterial community, were compared to evaluate the most accurate and efficient method. All three methods gave the same trend for the relative abundance of the different species.

Relative abundances measured by qPCR and T-RFLP yielded to similar results for each of the species L. ferriphilum, A. caldus, and S. thermosulfidooxidans, with no significant differences detected by a Kruskal-Wallis test (p < 0.005; Figure 1). The abundance of S. benefaciens was usually below the detection limit for T-RFLP analysis (Figure 1), while qPCR succeeded to detect this species even if only present in minor amounts (<8%); again, differences were not significant (Kruskal-Wallis test p < 0.005; Figure 1).

CE-SSCP allowed the detection of all the four target species, and a reasonable correlation between CE-SSCP and qPCR was found (Figure 2). But species-specific qPCR showed usually slightly lower relative abundances than the ones deduced from CE-SSCP profiles. However, as obtained for T-RFLP and qPCR results, differences were not found to be significant (Kruskal-Wallis test p < 0.005).

Total Bacterial Counts Using SYBR Green Staining

When using the standard SYBR Green staining protocol as described by [START_REF] Lunau | An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy[END_REF] for monitoring total cell numbers during bioleaching, we found an inhomogeneous distribution of the cells which was mostly due to cell attachment to particles and formation of clusters on the filter. The cells were visible in two levels of focusing as some were overlapping. Also a very fast fading of the fluorescent signal made it almost impossible to count the cells. Therefore, we tested three different pre-treatment methods with the aim to improve the fluorescent signal as well as the distribution of the cells on the filter.

While washing the samples with acidified basal salt medium did not improve the SYBR Green staining method, pre-treatment with detergents made a change. Treatment with 0.05% Triton X led to a much better microscopic image, the sample distribution was more homogeneous, and cells did not overlap and were better visible. One disadvantage was that the fluorescence signal was very strong and the background too, however, the contrast improved after a few minutes under the microscope. Also the cells and particles seemed somehow swollen, lower concentrations of Triton X did not overcome this side effect.

The pre-treatment with Tween 20 resulted in a better microscopic observation with a homogeneous distribution of the cells on the filter which allowed more accurate cell counting. Cell numbers were equal to the pre-treatment with Triton X but no side effects were visible when using Tween 20. Thus, for further SYBR Green staining and total cell counts of such bioleaching samples the Tween 20 pre-treatment was selected.

Monitoring of Bioleaching Reactors

Batch bioreactors housing the KCC consortium of moderately thermophilic and acidophilic bacteria utilized for bioleaching of a copper concentrate served as model system for microbial monitoring studies with the aim of evaluating the selected monitoring methods. Several experiments with the same set up were conducted in order to generate various samples for comparative microbial community studies. Besides detailed molecular monitoring only the chemical results of one standard experiment are reported.

Physico-Chemical Parameters

Each bioleaching experiment was started by adding sulfuric acid to the medium after adding the ore and before inoculation. The acid neutralized most of the carbonates and kept the pH below 2.0 instead of the initial pH 4.1 to avoid inhibition of the acidophilic bacteria. This already caused some copper and iron dissolution (Figure 3A). Solution pH dropped from 2.0 to 1.3 under biological leaching conditions. The redox potential increased from initial 545 mV to about 903 mV during the course of the bioleaching experiment (Figure 3B) whereas it remains rather constant in the chemical control experiment.

The kinetics of metal release improved constantly from bioleaching adaptation step 2 to 5 as shown in Table 2. The increase in bioleaching activity due to the adaptation procedure could also be noted by a decrease in solution pH and increase in redox potential (data not shown).

The dissolved copper concentration in the medium of bioleaching step 5 strongly increased until day 4 and only increased slightly in the following days to about 14440 mg/L Cu after 10 days (Figure 3A). Similar kinetics were observed for iron dissolution with a maximum of 4180 mg/L Fe on day 4 of the bioleaching.

Final copper recovery from the copper concentrate during bioleaching under standard conditions in step 5 was about 94% compared to 53% in chemical control experiments (data not shown). The overall Fe and total S extraction (54 and 62%, respectively) was lower than sulfide (88%) and Cu recovery (94%). In this kind of system Fe and S leaching yields are usually underestimated because of the formation of precipitates (e.g., jarosite, gypsum).

Microbial Community Monitoring Using qPCR

The microbial community in the bioleaching reactors was represented by L. ferriphilum, A. caldus, S. thermosulfidooxidans, and S. benefaciens. The species A. caldus and S. thermosulfidooxidans were dominant at the end of bioleaching steps 2 and 3 and still comprised a major part of the community at steps 4 and 5 (Figure 4). The relative abundance of L. ferriphilum in the microbial community increased in step 4 and 5 of the experiment making it the key player in these two steps besides A. caldus and S. thermosulfidooxidans. S. benefaciens only represented a minor proportion of the leaching community throughout all bioleaching steps. In some experiments, S. benefaciens was even under the detection limit for qPCR

Cu leaching rate (mgCu/L/h)

Step 2

Step 3

Step 4

Step 5

81.2 107.9 116.6 120.2

but could be detected again in later bioleaching steps from the same inoculum (data not shown). There was no pronounced change in community composition between adaptation steps 4 and 5.

Figure 5 shows the monitoring of the microbial community through the course of the experiment in step 5. The iron-oxidizer L. ferriphilum dominated the experiment at the beginning and end and was strongly accompanied by the sulfur-oxidizers A. caldus and S. thermosulfidooxidans. S. benefaciens was only present in very low numbers during the whole experiment.

Figure 6 shows the total cell numbers for step 5 using SYBR Green staining in comparison with total cell numbers deduced from bacterial 16S rRNA gene copy numbers determined by qPCR. The later were transferred into cell numbers by taking into account the specific 16S rRNA gene copies of the strains in the KCC consortium. It confirmed an increase in cell numbers over time of the bioleaching experiment from about 10 6 to 10 9 cells/mL. There was no difference between the analyses of samples fixed in formaldehyde and stored at -20 • C or samples which were directly processed without formaldehyde fixation. Total cell numbers determined by SYBR Green staining and total bacteria via qPCR in Figure 6 show a strong correlation between the two monitoring methods.

Microcalorimetric Activity Measurements

The biological activity at the beginning of bioleaching steps 2-4 was very similar at about 190 µW/g and increased toward the end of the experiment (Figure 7). While the final heat output in step 2 was higher compared to steps 3 and 4, the biological activity reached a maximum in bioleaching step 5 off around 345 µW/g. Overall, the bioleaching activity progressively increased between the bioleaching steps. Heat output measured in the chemical control (Figure 7) also increased during the experiment but was always below that of the biological setups.

DISCUSSION

The aim of this study was to develop and evaluate various methods for monitoring bioleaching microbial communities and investigate changes in the microbial community composition and its performance during bioleaching applications. Bioleaching experiments with copper concentrate and the acidophilic, moderately thermophilic KCC consortium in 2 L stirred tank bioreactors following a 5-step adaptation protocol served as a model system for biomining operations in this study.

According to the main aim of this study, we focused on the quantitative monitoring of the microbial community during the bioleaching process. The bioreactor system was inoculated with the mixed KCC culture of the iron-oxidizer L. ferriphilum, the sulfur oxidizer A. caldus and the iron-sulfur-oxidizing S. thermosulfidooxidans and S. benefaciens. These organisms are commonly found in stirred tank operations [START_REF] Olson | Bioleaching review part B: progress in bioleaching: applications of microbial processes by the minerals industries[END_REF][START_REF] Schippers | Microorganisms involved in bioleaching and nucleic acid-based molecular methods for their identification and quantification[END_REF]d'Hugues et al., 2008;[START_REF] Spolaore | Relationship between bioleaching performance, bacterial community structure and mineralogy in the bioleaching of a copper concentrate in stirred-tank reactors[END_REF] where they combine autotrophic and mixotrophic growth as well as iron-and sulfur-oxidizing abilities to efficiently carry out bioleaching of sulfide ores. In order to better understand the behavior and role of these organisms in the bioleaching process, a quantitative and species-specific monitoring of each member of the community during the bioleaching process is essential.

We chose to focus on qPCR as it is currently the most common method for quantitative microbial community monitoring. We tested qPCR assays reported in the literature to target Acidithiobacillus, Leptospirillum and Sulfobacillus species (e.g., [START_REF] Liu | Rapid specific detection and quantification of bacteria and archaea involved in mineral sulfide bioleaching using real-time PCR[END_REF][START_REF] Remonsellez | Dynamic of active microorganisms inhabiting a bioleaching industrial heap of low-grade copper sulfide ore monitored by realtime PCR and oligonucleotide prokaryotic acidophile microarray[END_REF][START_REF] Zhang | Application of real-time PCR to monitor population dynamics of defined mixed cultures of moderate thermophiles involved in bioleaching of chalcopyrite[END_REF] and found that most of them were only specific on genus-level or not specific for the desired species (results not shown). Thus we designed new qPCR assays targeting the species of the KCC consortium. The assays were confirmed to be specific for A. caldus, S. thermosulfidooxidans and S. benefaciens and L. ferrooxidans/ferriphilum by carrying out various cross-checks with closely related species and strains. The novel assays were successfully applied to monitor the microbial community during bioleaching in stirred-tank reactors.

The qPCR results were also compared with data from the commonly used molecular fingerprinting methods T-RFLP and CE-SSCP. Both methods are classified as semi-quantitative since they are based on end-point PCR, while qPCR gives accurate quantification and absolute gene copy numbers. Indeed, the very beginning of the amplification is monitored online when the fluorescence intensity is proportional to the copy number of the target gene initially present in the DNA extract [START_REF] Heid | Real time quantitative PCR[END_REF][START_REF] Wittwer | Continuous fluorescence monitoring of rapid cycle DNA amplification[END_REF]. T-RFLP and CE-SSCP allow relative quantification of all species present in one sample at the same time, since they are based on the amplification of the 16S rRNA gene of the total community, and therefore also allow the detection of previously unexpected species in the community. qPCR requires separate amplification of each taxon using species-specific primers and allows only the quantification of the abundance of the target species or comparison of their relative abundance with the number of total bacteria determined by another assay, but will not detect other unexpected species in a sample. Fingerprinting and qPCR are thus complementary monitoring methods. Even though qPCR is more expensive and time consuming, species-specific quantification is often more accurate and, if the number of gene copies on the genome of the target strain is known, cell numbers can be deduced from a standard reporting known gene copy numbers.

Statistical analysis confirmed that there are no significant differences between the relative abundances of the various species determined by species-specific qPCR and TRFLP or CE-SSCP molecular fingerprints. The data lead to comparable results and trends appeared suitable to monitor bioleaching species abundances in the studied bioleaching reactors. Contrary to molecular fingerprint, qPCR gives also absolute cell numbers, expressed, e.g., as cells per mL of culture, thus data on the multiplication or decline of the number of each species in a reactor. Molecular fingerprinting, however, as it uses universal bacterial primers, may allow the detection of other taxa developing in a reactor, initially not inoculated but introduced by the ore. The specific characteristics of each technique make them complementary and suitable for monitoring bacterial communities in bioleaching processes.

All PCR-based molecular monitoring methods require the extraction of nucleic acids and, as in the presented study when DNA is used, active as well as inactive cells are detected, and therefore no information about the activity status of the microorganisms is determined. However, quantitative monitoring on active cells using the described methods could be achieved by targeting RNA rather than DNA. When running reactors in continuous mode for large-scale bioleaching operation, DNA-based monitoring should also be informative on active cells because of the wash-out of inactive ones.

In complement, we also applied two methods, SYBR Green staining for total cell counting and microcalorimetry, which target whole cells and do not require any extraction of nucleic acids, proteins or lipids. Both methods can be directly applied to the pulp samples and allow a quick and easy monitoring of cell abundance.

For SYBR Green staining of bioleaching pulp samples, a special pre-treatment protocol was established, which greatly improved the quality of the microscopic observation in terms of both the fluorescence intensity and the distribution of the cells on the filter. A constant increase in cell numbers over the experimental time was shown confirming that the cells grew during bioleaching by the oxidation of reduced sulfur compounds and ferrous iron. SYBR Green staining, according to the newly adapted protocol, is therefore a quick and reliable method to monitor and detect changes in the total cell number and therefore indirectly overall bioleaching activity as it has already been routinely used in several other studies (e.g., [START_REF] Schippers | The use of FISH and real-time PCR to monitor the biooxidation and cyanidation for gold and silver recovery from a mine tailings concentrate (Ticapampa, Peru)[END_REF]. The strong correlation of cell counts determined by SYBR Green staining and those deduced from the total bacteria qPCR assay clearly shows the power and reliability of these two complementary methods for the determination of total cells in bioleaching experiments.

Isothermal microcalorimetry determines the heat output from exothermic chemical reactions and is therefore a tool for microbial activity measurements if the chemical reactions are catalyzed by microorganisms [START_REF] Braissant | Use of isothermal microcalorimetry to monitor microbial activities[END_REF]. This has shown to be the case for bioleaching, e.g., metal sulfide oxidation by acidophhilic bacteria, and a correlation between heat output and the metal sulfide dissolution rate as well as cell numbers of acidophiles was found for laboratory and environmental samples [START_REF] Schippers | Microbial diversity in uranium mine waste heaps[END_REF][START_REF] Rohwerder | Determination of reaction energy values for biological pyrite oxidation by calorimetry[END_REF]. In a few cases, microcalorimetry was used for the monitoring of bioleaching operations [START_REF] Sand | Controlled microbiological in-situ stope leaching of a sulphidic ore[END_REF] or field experiments for the inhibition of biological metal sulfide oxidation for acid mine drainage prevention [START_REF] Sand | Longterm evaluation of acid rock drainage mitigation measures in large lysimeters[END_REF]. When determining the biological activity at the beginning (low cell abundance) and end (high cell abundance) of the last bioleaching step in our experiments, the heat output data confirmed that the activity increased during the experiment. Furthermore, an increase of the activity of the cells between the five bioleaching steps was confirmed, which correlates with the increase in SYBR Green counts as well as in copper dissolution and iron oxidation rate. The heat output measured in the chemical control was most likely due to the acid dissolution of some mineral phases as also confirmed by the copper and iron dissolution data of the chemical control.

Following copper and iron dissolution from the copper concentrate over time confirmed that bacteria strongly catalyzed the bioleaching of the concentrate with a maximum copper dissolution of 94%. Metal bioleaching was enhanced by applying the 5-step adaptation protocol since the bacteria were adapted to the ore and the physico-chemical conditions and were therefore able to achieve maximum performance. Final copper recovery was achieved after about 6-7 days, afterward there was no significant change in soluble metal concentration and pH. Solution pH dropped from about 2.0 at the beginning to 1.3 under biological leaching conditions due to the oxidation of sulfides and formation of sulfuric acid by bacteria. The redox potential increased from initial 545 mV to about 903 mV, mainly caused by the dissolution of ferrous iron and oxidation to ferric iron. The overall Fe (54%) and total S (62%) extraction was lower compared to sulfide (88.3%) and Cu recovery (94%), which is probably due to Fe and sulfate precipitation as jarosite.

The achieved results correlate with earlier reports on this system [START_REF] Spolaore | Relationship between bioleaching performance, bacterial community structure and mineralogy in the bioleaching of a copper concentrate in stirred-tank reactors[END_REF] where a maximum copper recovery of 95% was achieved after 6.25 days with a similar concentrate and the same laboratory setup. In this work the authors showed that incomplete copper dissolution was mainly due to remaining chalcopyrite in the concentrate which is more recalcitrant to bioleaching than the other copper sulfides.

S. benefaciens was only detected in lower numbers throughout the course of the experiment, but its presence clearly showed that there must be a direct role in the bioleaching process. This example perfectly shows how the bioleaching consortium works together by the sulfur-oxidizer initiating the pH decrease and thereby the bioleaching of metals including copper and ferrous iron and the iron oxidizers later contributing to the bioleaching process by converting ferrous to ferric iron.

Microbial community analysis at the end of each bioleaching step confirmed that A. caldus and S. thermosulfidooxidans dominated in step 2 and 3 but were overtaken by L. ferriphilum in step 4 and 5 of the adaptation procedure. A. caldus and S. thermosulfidooxidans both oxidize reduced sulfur compounds and thereby generate acidity which was clearly detected as the initial dominant phase in the bioleaching reactors whereas enhanced iron release and oxidation took place after 2-3 days and was more enhanced in later adaptation steps. Therefore, this could be an explanation for the dominance of L. ferriphilum in step 4 and 5 of the bioleaching compared to earlier adaptation steps together with the ability of L. ferriphilum to thrive at very low pH values and high redox potentials. This again clearly shows that it is necessary to adapt the microorganisms to the ore and increasing solid load to achieve most efficient bioleaching of relevant metals.

This study has enhanced our knowledge and the "toolbox" for the quantitative monitoring of bioleaching operations, by successfully applying four novel qPCR assays for measuring the abundance of common bioleaching species. It confirmed that standard fingerprinting methods and qPCR give similar results, but only when expressed as a relative measure of the abundance as fingerprinting methods cannot determine cell numbers. The additional application of two quick and convenient whole cell methods, SYBR Green staining and microcalorimetry helped to follow changes in cell number and activity during the bioleaching experiment. Altogether, the applied methods are well suited for microbial community monitoring and will help to understand the bioleaching process and react to optimize bioleaching performance.

FIGURE 1 |

 1 FIGURE 1 | Relative abundances of the dominant bioleaching species at step 5 as determined by T-RFLP (plain) and qPCR (hatched). Data are mean values of triplicates ±SD. Asterisk above the plots indicate no significant difference between T-RFLP and qPCR results (Kruskal-Wallis-Test, p < 0.005).

FIGURE 2 |

 2 FIGURE 2 | Relative abundances of the dominant bioleaching species at step 5 as determined by CE-SSCP (plain) and qPCR (hatched). Data are mean values of triplicates ±SD. Asterisk above the plots indicate no significant difference between CE-SSCP and qPCR results (Kruskal-Wallis-Test, p < 0.005).

FIGURE 3 |

 3 FIGURE 3 | Kinetics of batch bioleaching step 5 in stirred bioreactors with copper concentrate and the acidophilic, moderately thermophilic bioleaching KCC consortium. (A) Copper (triangles) and iron (squares) dissolution in bioleaching (solid line) and chemical (dotted line) experiments. (B) pH (squares) and E h (triangles) in bioleaching (solid line) and chemical (dotted line) experiments. Data are mean values of triplicates ±SD.

FIGURE 4 |

 4 FIGURE 4 | Relative abundance of the four different species (Leptospirillum ferriphilum-hatched bars, Acidithiobacillus caldus-dotted bars, Sulfobacillus benefaciens-gray bars, Sulfobacillus thermosulfidooxidans-black bars) in the bioleaching reactors at the end of each bioleaching experiment determined by qPCR. Data are mean values of triplicates ±SD.

FIGURE 5 |

 5 FIGURE 5 | Monitoring of the microbial community in step 5 of triplicate bioleaching experiments using qPCR. L. ferriphilum-hatched bars, A. caldus-dotted bars, S. benefaciens-gray bars, S. thermosulfidooxidans-black bars. Data are mean values of triplicates ±SD.

FIGURE 6 |

 6 FIGURE 6 | Total cell numbers determined by SYBR Green staining (gray bars) and qPCR (hatched bars) during step 5 of the copper concentrate bioleaching. Error bars represent data from three identical bioreactor runs and triplicate measurements of each.

FIGURE 7 |

 7 FIGURE 7 | Biological activity as determined by microcalorimetry at the start and end of each bioleaching step at 42 • C. The chemical control measurements were conducted with the same set up. Error bars represent 2-3 identical bioleaching tests with triplicate measurements in the microcalorimeter.

TABLE 1 |

 1 Designed primers for the quantification by qPCR of bacterial species of the KCC consortium. in at least two 10-fold dilutions to check for PCR inhibition. Purified 16S rRNA gene PCR products of pure strains or linearized plasmids carrying a target 16S rRNA gene were used as standards for qPCR. A seven-point serial decimal dilution of the respective standard was run in duplicate or triplicate with each set of reactions to generate the standard curve of C t (threshold cycle) versus the number of gene copies.

	Target	Name	Sequence (5 →3 )	Product size (bp)
	Acidithiobacillus caldus	Acaldus_F	CGGATCCGAATACGGTCTG	396
		Acaldus_PC1_RC *	TCAGCACCTAAGGCGCCAA
	Leptospirillum ferriphilum	Lfp392F	GAAGGCTTTCGGGTTGTAAACCAC	210
		Lfp601R	TTAAGCCACGGCCTTTCACCAA
	Sulfobacillus thermosulfidooxidans	St_F	TGTCGTGAGATGTTGGGTTAG	225
		St_R	AGATCCCGTTTGAAGGATTGG
	Sulfobacillus benefaciens	Sb1030F	CAGCTCGTGTCGTGAGATGT
		Sb1265R	ACTGAGGATCCGTTTGCGG

triplicate

Frontiers in Microbiology | www.frontiersin.org 1 December 2016 | Volume 7 | Article 2044

Frontiers in Microbiology | www.frontiersin.org

December 2016 | Volume 7 | Article 2044

* Modified from[START_REF] Liu | Rapid specific detection and quantification of bacteria and archaea involved in mineral sulfide bioleaching using real-time PCR[END_REF]. Frontiers in Microbiology | www.frontiersin.org 3 December 2016 | Volume 7 | Article 2044

ACKNOWLEDGMENTS

We thank I. Kruckemeyer and J. Jacob for help in conducting bioleaching experiments, I. Kruckemeyer and D. Frohloff for DNA extraction, qPCR analysis and help with SYBR Green staining. J. Hellal is kindly acknowledged for her expertise on statistical analyses. We also thank F. Bodenan for project coordination at BRGM and KGHM for providing the copper concentrate samples.

FUNDING

This work is part of the collaborative bilateral research project Ecometals co-funded by the German Federal Ministry of Education and Research (BMBF under the grant ID 033RF001) and the French National Research Agency (ANR-13-RMNP-0006).

AUTHOR CONTRIBUTIONS

SH designed the study, conducted, and supervised the experiments at BGR, analyzed the data and wrote the manuscript. A-GG designed and supervised bioleaching experiments and analyzed the data at BRGM and contributed to the manuscript. MC conducted molecular experiments and analysis at BRGM. AS discussed data at BGR and contributed to th manuscript. CJ conducted and analyzed molecular experiments at BRGM and wrote the manuscript together with SH.

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Hedrich, Guézennec, Charron, Schippers and Joulian. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


Anne-Gwénaëlle Guezennec
email: guezennec@brgm.fr

C Joulian

J Jacob

A Archane

D Ibarra

R Gregory

F Bodénan

P D'hugues

Influence of dissolved oxygen on the bioleaching efficiency under oxygen enriched atmosphere

Keywords: bioleaching, oxygen, dissolved oxygen concentration, sulfide, bacteria monitoring, stirred tank reactor

The use of oxygen enriched air is a common practice in high-temperature bioleaching tests (> 70°C) to overcome oxygen solubility limitation and reduced the energy costs of the process. Air is usually preferred in medium and low-temperature operations mainly for technical and economic constraints. Nevertheless, under high-sulfide loading conditions -high-grade metal sulfide concentrates and high solids concentration -the microbial and chemical demand for oxygen is significantly increased during the bioleaching process. If not satisfied, this high oxygen demand might limit the oxidation efficiency. Therefore it requires the injection of large amounts of air. Sparging with oxygen enriched gas instead of air may offer an interesting alternative process option to improve gas transfer in the bioleaching reactor and to provide an adequate oxygen supply in order to satisfy the oxygen demand. It might be useful to develop innovative alternative to the classical stirred tank reactor (STR) technology. However, the use of such conditions can lead to much higher dissolved oxygen (DO) concentrations than those encountered with air. Very few papers have been devoted to the study of the optimal range of DO concentrations for bioleaching processes. Most of them reported an inhibitory effect of DO concentrations above 5 ppm. The purpose of this study was to investigate the influence of DO on the bioleaching efficiency under oxygen-enriched atmosphere in 21 L stirred tank reactor at 42°C. Bioleaching experiments were performed in continuous mode with sulfide-rich tailings wastes composed mainly of pyrite (51%) and quartz using the "BRGM-KCC" bacterial consortia. The solid load was close to 20% (w/w). Using various oxygen supply conditions (partial pressure, gas rate), the DO concentration in the reactor varied between 4 and 17 ppm. For a DO ranging from 4 to 13 ppm, a good bacterial oxidizing activity was observed and the sulfide dissolution efficiency increased with the DO concentration. It is assumed that this improvement of the bioleaching efficiency was linked to an increase of the oxygen transfer rate from the gas phase to the liquid phase rather than a direct effect of the DO level. When the DO concentration reached 17 ppm a significant decrease of the microbial activity and consequently of the oxygen consumption was noticed. These results show that there is a critical value above which the DO concentration is detrimental to the activity of the bioleach microorganisms present in the "BRGM-KCC" consortia but this value is much higher than the one usually mentioned in the literature.

1-INTRODUCTION

Biohydrometallurgy is well established for the treatment of certain sulfide minerals, where iron and sulfur-oxidising bacteria are used for the leaching of low grade copper ores and the pretreatment of pyritic gold ores and concentrates. However part of the mining industry remains skeptical and reluctant to adopt biohydrometallurgical techniques as a reliable alternative option. Heap leaching is sometimes considered unsuitable due to space constraints, slow leaching kinetics and low recovery rate. The interest of using stirred tank reactor (STR) for the treatment of other minerals than refractory gold ores, such as the base metal sulfides, has already been demonstrated but some improvements are still needed to meet economic viability (d'Hugues et al., 2008;[START_REF] Spolaore | Bioleaching of an organic-rich polymetallic concentrate using stirred-tank technology[END_REF][START_REF] Kutschke | Bioleaching of Kupferschiefer blackshale -A review including perspectives of the Ecometals project[END_REF]. The main costs of bioleaching operations in STR are the costs associated with the leaching tanks and the gas mass transfer to the pulp. The main capital costs are the ones for the agitators and for the gas injection devices. The main operating costs are associated with the energy consumption required for slurry agitation and air compression, since air is usually used to provide oxygen in bioleaching operations (Rossi, 2001;[START_REF] Morin | Bioleaching of a cobalt-containing pyrite in stirred reactors: a case study from laboratory scale to industrial application[END_REF][START_REF] Van Aswegen | The BIOX process for the treatment of refractory gold concentrates[END_REF]. The microorganisms involved in bioleaching processes get their energy through the oxidation of reduced sulfur compounds and iron (II). In these reactions oxygen is used as electron acceptor. In the case of pyrite bioleaching mechanisms involve the following oxidation reactions:

FeS2 + 2 Fe 3+ → 3 Fe 2+ + 2 S 0
Eq. 1 2 Fe 2+ + 2 H + + 1/2 O2 → 2 Fe 3+ + H2O Eq. 2 S 0 + 3/2 O2+ H2O → H2SO4 Eq. 3

The reactions (2) and ( 3) are biologically catalyzed by acidophilic Fe-and S-oxidizing bacteria, whereas the reaction (1) occurs through chemical oxidation. The combination of the three reactions leads to the overall bioleaching reaction (Eq. 4) which shows that 3.5 mols of oxygen is required to oxidize 1 mol of pyrite.

2 FeS2 + 4 H + + 7 O2 + 2 H2O → 2 Fe 2+ + 4 H2SO4 Eq. 4

Oxygen supply is thus a key issue, particularly in bioleaching processes with high-sulfide loading conditions where the microbial and chemical demand for oxygen is significantly increased. As a consequence, there is a need to inject large amounts of air which can be technically difficult and increases the costs of the process. Sparging with oxygen enriched gas instead of air may offer an interesting alternative process option to improve gas transfer and bioleaching efficiency by providing an adequate oxygen supply in order to satisfy high oxygen demand.

Gas mass transfer in bacterial leaching systems has been widely studied and is well documented (Bailey and Hansford, 1993a;[START_REF] Bailey | Oxygen mass transfer limitation of batch bio-oxidation at high solids concentration[END_REF][START_REF] Boon | Gas-liquid mass transfer phenomena in bio-oxidation experiments of sulphide minerals. A critical review of literature data[END_REF][START_REF] Savic | Effect of the oxygen transfer rate on ferrous iron oxidation by ThiobaciUus ferrooxidans[END_REF][START_REF] Veglio | Empirical models for oxygen mass transfer: a comparison between shake flask and lab-scale fermentor and application to manganiferous ore bioleaching[END_REF]. Transfer to liquid theory (illustrated schematically in Fig. 4) indicates that the O2 mass transfer rate, called RO2 (quantity of oxygen transferred in liquid phase per unit of time) is given by:

RO2 = KLa × (C* -CL)

Eq. 5

where: RO2 is O2 transfer rate (mol m -3 s -1 ) KLa is the volumetric oxygen mass transfer coefficient (s -1 ) C* is the oxygen solubility in reactor conditions (mol m -3 ) CL is the oxygen concentration in the liquid phase (mol m -3 ) Several authors [START_REF] Lui | Oxygen transfer to Thiobacillus cultures[END_REF][START_REF] Bailey | Oxygen mass transfer limitation of batch bio-oxidation at high solids concentration[END_REF][START_REF] Jordan | Acidophilic bacteria, their potential mining and environmental applications[END_REF][START_REF] Myerson | Oxygen mass transfer requirements during the growth of Thiobacillus ferrooxidans on iron pyrite[END_REF]Bailey and Hansford, 1993b) have pointed out that the lack of adequate gas mass transfer is a rate limiting step in many bacterial leaching processes. The gas-liquid mass transfer rate depends on a number factors [START_REF] Van Weert | Transfer of oxygen from air to mineral slurries in a Rushton turbine agitated tank[END_REF], d'Hugues et al., 1997[START_REF] Boon | Gas-liquid mass transfer phenomena in bio-oxidation experiments of sulphide minerals. A critical review of literature data[END_REF] such as the reactor type, geometry and size, the gas-flow rate, sparger design and depth, the stirring speed, particle shape and size, pulp density and viscosity. However oxygen can become a limiting factor in bacterial leaching because of its low solubility: only 0.26 mM O2 (8.32 ppm) can dissolve per liter of water at 25°C in an air/water mixture. As mentioned by [START_REF] Witne | Bioleaching of Ok Tedi copper concentrate in oxygen-and carbon dioxide enriched air[END_REF], one way of increasing the solubility of oxygen in water or media solution is by increasing the driving force, i.e. raising the oxygen partial pressure in the gas stream supplied to the leach pulp. This mechanism is described by Henry's gas law which gives the solubility of oxygen in solution in relation to the oxygen partial pressure in the gas phase:

C* = (P°/H)
Eq. 6

where: C* is the oxygen concentration of the nutrient solution; P° is the partial pressure of the gas in the gas phase; H is Henry's constant, which is specific for the gas and the liquid phase.

Henry's gas law shows that the solubility of O2 in the leach pulp increases with increasing oxygen partial pressure in the gas stream. Higher O2 partial pressures could be attained when the bioreactor air or gas stream is enriched with added pure oxygen. This approach was used for the development of processes using high thermophiles culture by teams working on the development of STR processes for the bioleaching of chalcopyrite concentrates [START_REF] Dew | Bioleaching of base metal sulphide concentrates: a comparison of mesophile and thermophile bacterial cultures[END_REF]d'Hugues et al, 2002). In these studies, oxygen was used instead of air because of the low solubility of O2 at high temperature. When working with mesophilic or moderately thermophilic microorganisms, they might be various interests of using O2 enriched air such as (i) to decrease the flow of gas stream injected in the pulp, reducing energy consumption linked to agitation and compression of air (ii) to improve gas transfer to satisfy higher oxygen demand associated with higher sulfide concentration. Using O2 enriched air is a promising alternative that might be useful to develop innovation to improve the classical STR technology. Air Liquide, Milton Roy Mixing and BRGM are currently testing an innovative bioleaching process using floating agitators to mix and to suspend solids in the solution as well as to inject gases in the pulp. This new concept enables to decrease the costs of bioleaching processes by operating in lagoons or ponds instead of using costly tanks, and at higher solid loading (>20%) than in conventional stirred tank bioreactors. In these conditions of high solid load, the microbial and chemical demand for oxygen is significantly increased and air could be replaced by oxygen enriched gas to provide an adequate oxygen supply in order to satisfy the oxygen demand.

However the use of such conditions can lead to much higher dissolved oxygen (DO) concentrations than those encountered with air sparging. Very few papers have been devoted to the study of the optimal range of DO concentrations for bioleaching processes. However most of them reported an inhibitory effect of DO concentrations above 5 ppm [START_REF] De Kock | Oxygen and carbon dioxide kinetic challenges for thermophilic mineral bioleaching processes[END_REF][START_REF] Wang | Effects of dissolved oxygen and carbon dioxide under oxygen-rich conditions on the biooxidation process of refractory gold concentrate and the microbial community[END_REF].

The purpose of this study was to investigate the influence of DO on the bioleaching efficiency of a mesophile to moderate thermophile consortium. The bioleaching tests were carried out under oxygenenriched atmosphere in a 21L stirred tank reactor at 42°C in continuous mode with sulfide-rich tailings wastes composed mainly of pyrite (51%) and quartz. The solid load was closed to 20% (w/w).

The DO concentration in the reactor was varied between 4 and 18 ppm by increasing the gas flow rate. The influence of the DO level on the bioleaching efficiency was investigated through the monitoring of the sulfide leaching kinetics, the sulfide dissolution yield and the structure and the abundance of the microbial community.

2-MATERIALS AND METHODS Characterization of the sulfidic materials

The experiments were performed using flotation tailings coming from a European copper mine. The mineral of economic interest in the ore body is chalcopyrite (CuFeS2). At site, the ore is ground and valuable chalcopyrite is then separated from pyrite by flotation. Copper contained in the chalcopyrite is recovered by smelting whereas pyrite is discharged in tailings, from which the material used in this study was sampled. The tailings are mainly composed of pyrite (51%) containing cobalt (0.06%), copper (0.19%) and gold (1 g/t). This waste has been chosen as test materials for its high content of pyrite, which makes it particularly suitable for bioleaching.

Bacterial culture and nutrients

The experiments were carried out using the BRGM-KCC microbial consortium, which has already been fully described (d'Hugues et al., 2003). The predominant organisms in the culture are affiliated to Leptospirillum (L.) ferriphilum, Acidithiobacillus (At.) caldus and Sulfobacillus (S.) benefaciens. The culture used as an inoculum originated from BRGM stock culture, stored at -80 °C. The culture was subcultured several times in batch mode from 2 mL up to 21 L prior to the beginning of the test. The culture was grown in a nutrient medium called "0Km". This is a modified "9K" medium (9K without iron, "m" indicating modification of the basal salts) and it was optimised for bacterial growth on cobaltiferous pyrites. Its standard composition is the following: (NH4)2SO4, 3.70 g L -1 ; H3PO4, 0.80 g L -1 ; MgSO4•7H2O, 0.52 g L -1 ; KOH, 0.48 g L -1 .

Laboratory apparatus and experimental conditions

Bioleaching tests were carried out in continuous mode using a 21 L (working capacity) laboratoryscale stirred tank. This tank is made from 316L stainless-steel and has a height/diameter ratio equal to 1. The top of the reactors was connected to a gas cooling system to prevent excessive evaporation. The bioleach slurry was mechanically stirred by a mixed (axial/radial) system (so-called BROGIM® -BRGM/MRM) mounted on a rotating shaft. The gas stream made of N2, CO2 and O2 supplied by Air Liquide gas cylinder was injected beneath the turbine at the bottom of the reactor via a stainless steel pipe. Gas mixture composition was controlled by valves and mass flow regulators for each gas. Gases went through a gas mixer before injection in the reactor. The feed was made up of a high density pulp flow (40% wt/wt of sulfidic materials in water) and a concentrated nutrient medium flow. The two feed flows were pumped separately and injected into the tank in a ratio corresponding to the solids ratio (20% w/w) required for the feeding pulp. The combination of the two flows resulted in nutrient concentrations corresponding to 0Km medium. The residence time of the pulp in the tank was closed to 2.2 days. The pH was regulated between 1.2 and 1.5 by adding limestone slurry of 500 g L -1 CaCO3. The standard temperature was 42°C and was maintained constant by circulating cold water through an internal stainless-steel coil.

Five conditions of gas flow rates and compositions were tested (Table 1). CO2 was maintained close to 1% during the whole assay. O2 partial pressure was first set to 56% and the gas flow rate was progressively increased from 50 to 500 NL h -1 (conditions 1 to 4, see Table 1) in order to increase the concentration of dissolved oxygen in the liquid phase. In the last condition the gas flow rate was decreased to 250 NL h -1 and the oxygen partial pressure was set to 70%. For each condition, the experiment was carried out for an average time of 20 days, which enabled to monitor the bioleaching test during several residence times and reach a steady state.

Daily monitoring

Electrochemical potential, pH and dissolved oxygen (DO) were measured directly in the pulp. Samples of pulp were collected daily in the bioreactor and filtered (syringe filters in cellulose acetate, diameter 30 mm, porosity 0.45 µm). Cobalt and total iron concentrations were measured by atomic absorption spectroscopy (Varian SpectrAA-300) in the filtered fraction. The O2 and CO2 concentrations in the inlet and outlet gas of the reactor were measured using a paramagnetic analyser and an infrared analyser (ADC 7000 -Analytical Development Company Ltd.) respectively. The gas balancing method was used to calculate the oxygen uptake rate (OUR). The oxygen transfer rate (OTR) and the OUR are linked by the following equation:

𝑶𝑻𝑹 = 𝑶𝑼𝑹 + 𝒅𝑪 𝑶𝟐 𝒅𝒕 = 𝑸 𝑶𝟐 𝒊𝒏 -𝑸 𝑶𝟐 𝒐𝒖𝒕 𝑽

Eq. 7

where: dCO2/dt is the accumulation oxygen rate in the liquid phase, QO2 in and QO2 out are the oxygen gas flow at the bioreactor inlet and outlet, V the volume of the liquid phase in the bioreactor.

At steady state, when the DO concentration is constant, there is no accumulation of oxygen in the reactor and the OUR is equal to the OTR. Assuming that the nitrogen gas flow is the same at the inlet and outlet of the reactor (i.e. is not consumed neither transferred to the liquid phase), Eq. 7 becomes:

𝑶𝑻𝑹 = 𝑶𝑼𝑹 = 𝑸 𝒊𝒏 * (𝑷 𝑶𝟐 𝒊𝒏 - 𝑷 𝑵𝟐 𝒊𝒏 𝑷 𝑵𝟐 𝒐𝒖𝒕 ⁄ .𝑷 𝑶𝟐 𝒐𝒖𝒕 )

𝑽

Eq. 8

where: Q in is the gas flow rate at the inlet of the bioreactor, PN2 in and PN2 out are the partial pressure of nitrogen in the gas at the inlet and outlet of the bioreactor.

Analysis of the solid fractions and mass balances

When the bioleaching reactor was operating at steady state, samples were collected from the overflowing pulp at the exit of the reactor and from the initial pulp feed. The samples of pulp were first filtered with a Büchner funnel to separate liquid and solid phases. The filtered solid material was then rinsed with a sulfuric acid solution at pH 1.8 and dried. Representative aliquots of the dried materials (<80 µm) were analysed for metal contents (namely Fe, Co, Cu), total carbon and sulfur speciation. Major and trace metal contents were determined by ICP-AES (Inductively Coupled Plasma-Atomic Emission Spectrometry) after oxidizing digestion according to protocols dedicated to ore analysis (degradation of all sulfides). Total carbon and total sulfur were determined using a Leco analyzer: i.e. non-dispersive infrared analysis of CO2 and SO2 gas respectively, after heating and passing of an oxygen stream. The same technique was used for sulfide (S 2-) determination, once the sample has been leached in warm sodium carbonate solution to convert the metal sulfate into insoluble carbonates and soluble sulfate. Sulfate was determined by gravimetry after the sample being leached at boiling temperature with a sodium carbonate solution, ferric ion reduced to ferrous iron by the addition of hydroxylamine hydrochloride, and the filtrate being precipitated with barium chloride. The analytical data were then used to calculate the mass balance of the operation and to assess the bioleaching performances (sulfide oxidation rates and yields).

Bacterial community monitoring

Homogeneous 2 mL pulp samples were taken at the end of the steady state of each of the five operating conditions. After centrifugation, the pellet was washed in Tris buffer (100 mM, pH 8) until acidity was neutralized, and genomic DNAs were extracted with the FastDNA Spin Kit for Soil and manufacturer's protocol (MP Biomedicals), except for a speed of 5 for 30 s for mechanical lyses. DNA was also extracted with the same procedure from filtrate (2 mL) and solids (about 0.7 g) obtained after filtration of homogeneous pulp, in order to characterize bacteria located in the liquid phase or associated with the solids. The abundance of each inoculated strains, i.e. L. ferriphilum, At. caldus, and S. benefaciens, was determined by measuring abundance of their 16S rRNA gene using species-specific real-time quantitative PCR (qPCR) performed with the newly developed assays described by [START_REF] Hedrich | Development of molecular assays to efficiently monitor and quantify microbial species during bioleaching of copper-rich ores[END_REF]. Results were expressed as numbers of gene copies per mL of bioleaching pulp.

3-RESULTS

3.1-Influence of the dissolved oxygen on the dissolution rate and the OUR

The oxygen partial pressures analyzed in the gas inlet and outlet for the five tested conditions and the corresponding oxygen solubility calculated using Henry's law (Eq. 6) are gathered in Table 2. This table presents also the DO concentration measured in the reactor. The gas injection conditions selected enabled to test a wide DO concentration range from 4.7 ppm to 17.7 ppm. The variation in OUR and pyrite dissolution rate for the five tested conditions is shown in Fig. 1. Both increased when the DO concentration is increased from 4.7 ppm to 12.9 ppm, which corresponds to the conditions 1 to 4. OUR values are close to the values of pyrite dissolution rate as expected from Eq. 4 which shows that the bioleaching of 1 kg of pyrite consumes 1 kg of oxygen.

For the condition 5 where the DO concentration reached 17.7 ppm a sharp decrease of the pyrite dissolution rate was observed. In that case it was not possible to calculate the corresponding OUR because of the lack of precision in the analysis of the gas composition.

Figure1: Pyrite dissolution rate and OUR vs. DO concentration in continuous pyrite bioleaching experiments

3.2-Influence of the dissolved oxygen on the dissolution yields

In table 3 are reported the sulfide dissolution yields obtained for each tested conditions and the corresponding residence time. The slight variation in residence time can be explained by variation in the pumps flows for the calcite as it varies with the leaching efficiency and the corresponding production of sulfuric acid (see Eq. 4). It explains why the residence time is not constant especially for the conditions 1 and 5 for which the oxidizing activity was lower (as shown by the lower values of OUR). In both cases the amount of calcite introduced in the reactor was much lower and the residence time increased. When looking at the conditions 2, 3 and 4 for which the residence time is the same, the sulfide dissolution yield increased with the DO concentration. For the condition 4 the sulfide dissolution yield reached 77% for a DO concentration of 12.9 ppm. These results as well as the corresponding values of OUR and pyrite dissolution rates show (i) that the oxidizing activity in the bioleaching reactor is high from 5 to 12.9 ppm DO concentration and (ii) this activity is enhanced when the DO concentration increases.

For the condition 1 the sulfide dissolution yield is higher than for the conditions 2 and 3. But it is likely that this result is linked to the higher residence time in the reactor rather than to an effect of the DO considering the OUR and the pyrite dissolution rate which are both lower for the condition 1 than for the conditions 2 and 3. For the condition 5 the sulfide dissolution yield drops to 29% despite the higher residence time. This result is in accordance with the decrease of pyrite dissolution rate observed for this condition and seems to confirm the appearance of an inhibitory effect of the DO concentration on the biological activity when the DO becomes too high.

3.3-Influence of the dissolved oxygen on the microbial community (abundance and structure)

The bacterial community composition shows that the strains present in the bioleach pulp were the same as the three main strains which make up the BRGM-KCC consortium used to inoculate the bioreactors. They are all found in the five tested condition, L. ferriphilum being dominant and accounting for up to 80% of the total community (Fig 2)

.

The influence of DO on the biomass was studied, using 16S rRNA gene abundances as a measure of total biomass as well as strain specific abundances. The total biomass was negatively affected by an increase of DO, as shown by the decrease especially marked at a DO of 17.7 ppm where about one log of gene copy numbers was lost (Fig. 2). The strains composing the community followed the same trend. Especially, At. caldus abundance, although high at low DO (4.7 and 5 ppm), showed a regular diminution all along the experiment as well as the sharper decrease (1 log) as soon as DO increased from 5 to 9.5 ppm. Interestingly, At. caldus represented the large majority of planktonic bacteria recovered from the liquid compartment (see Fig. 3) and accounted for more than 96% of the total planktonic community at DO of 4.7 and 5 ppm, respectively. At 17.7 ppm however, the biomass of all strains had decreased, and L. ferriphilum and S. benefaciens appeared to be only slightly less affected (1.9 log between conditions 1 and 5) than At. caldus (2.3 log).

DISCUSSION

It is likely that a dissolved oxygen concentration between 13 and 17 ppm is critical for the bioleaching efficiency. Below this critical value bioleaching efficiency increases with the DO concentration. It is assumed that this improvement of the bioleaching efficiency is linked to an increase of the oxygen transfer rate from the gas phase to the liquid phase rather than a direct effect of the DO level. This hypothesis is partially confirmed by the increase of the oxygen uptake rate which is equal to the oxygen transfer rate in steady state mode. Above this critical value the DO concentration starts to be detrimental to the microbial activity and a sharp decrease of bioleaching efficiency (associated with a significant decrease of the bacterial population) is observed. This critical value is difficult to determine since the DO concentration depends on the rate of oxygen transferred from the gas phase to the liquid phase and from the oxygen consumed by the bio-oxidation. As soon as the DO concentration is above this critical value, the microbial activity decreases as well as the oxygen consumption, which entails an increase of the DO concentration.

The DO level which affects negatively the efficiency of bioleaching with the KCC consortium is quite different from those previously mentioned in the literature which reports an inhibitory effect for much lower DO levels. Especially this study can be compared to the results published recently by [START_REF] Wang | Effects of dissolved oxygen and carbon dioxide under oxygen-rich conditions on the biooxidation process of refractory gold concentrate and the microbial community[END_REF] which studied the effect of dissolved oxygen on bioleaching using a sulfidic material and a microbial community similar to the ones used in this study. The optimum activity of the microbial population was obtained at 3.75 ppm concentration of DO. Above and below this value the OUR, the oxidation rate and the oxidation ratio decreased. It must be noted that Wang et al. carried out their experiments at 10% solids and in batch mode. The maximum OUR measured was closed to 160 mg L - 1 h -1 , which is more than ten times lower than the maximum OUR measured in this study. The microbial oxidizing activity was thus much lower. In the absence of data regarding the gas flow rate and its composition (especially the partial pressure of oxygen) it is difficult to explain the discrepancies observed between both studies, but it can be assumed that the influence of DO concentration on the bacterial activity is very dependent on the other operating conditions such as the solid concentration and the culture mode. It might also be bacterial strain dependent.

The increase of DO concentration has also a noticeable influence on the biomass which progressively decreases when the DO concentration increases. Below 13 ppm At. caldus seems to be the most affected by the increase of DO concentration. This might suggest that At. caldus is less tolerant to high DO concentrations than the other strains of the KCC consortium. Nevertheless other factors must also be considered and especially the fact that At. caldus is the most abundant strain in the liquid phase compared to L. ferriphilum and S. benefaciens which are mainly located on the particles. Because of the presence of these bacteria on the solid phase it can be assumed that the oxidation of FeII and sulfur occur also at the surface of the pyrite particles. Oxygen is thus consumed at the liquid/solid interface and its transfer in the liquid/solid boundary layer is governed by the same theory as the one presented in the introduction:

RO2 = KS . as . (CL -CS)

Eq. 7

where: RO2 is O2 mass transfer rate (mol m -3 s -1 ) Ks is the mass transfer coefficient (m s -1 ) as is the solid specific area (m -1 ) CL is the oxygen concentration in the liquid phase (mol m -3 ) CS is the oxygen concentration at the solid surface (mol m -3 )

This phenomenon is illustrated in Fig. 4 and shows that the oxygen concentration in the liquid phase is higher than the one at the surface of the particles. The transfer rate being closely linked to the oxygen consumption in the system the difference (also called "driving force") is higher when the oxygen consumption rate increases. Planktonic cells are thus submitted to higher DO concentrations than the cells attached to the particles, which might explain that At. caldus abundance decreases more quickly than L. ferriphilum and S. benefaciens. The level of detrimental effect may also be linked to the capacity of a cell to handle high DO concentration. The toxicity of the oxygen towards the bacteria involved in bioleaching might be linked to the formation of reactive oxygen species (ROS) by a mechanism involving Fenton and Haber-Weiss reactions. By switching oxidation states metal ions further activate species like hydrogen peroxide (H2O2) and superoxide (O2˙) to the highly reactive hydroxyl radical (˙OH) [START_REF] Imlay | Cellular defenses against superoxide and hydrogen peroxide[END_REF]. ROS are known to have deleterious effects on cells through a mechanism called oxidative stress [START_REF] Halliwell | Biochemistry of oxidative stress[END_REF]. Several authors [START_REF] Borda | A mechanism for the production of hydroxyl radical at surface defect sites on pyrite[END_REF][START_REF] Cohn | Pyrite-induced hydroxyl radical formation and its effect on nucleic acids[END_REF][START_REF] Schoonen | Mineral-Induced Formation of Reactive Oxygen Species[END_REF][START_REF] Schoonen | Role of hydrogen peroxide and hydroxyl radical in pyrite oxidation by molecular oxygen[END_REF] have shown that the oxidation of pyrite in solutions containing dissolved oxygen leads to the formation of ROS through several mechanisms that are synthetized in Fig. 5. As can be seen the ROS concentration in the leaching solution would increase with the DO concentration. Recently, evaluations in tank reactors have also shown the negative influence of ROS generation (due to fine grinding of pyrite) on bioleaching [START_REF] Jones | The generation of toxic reactive oxygen species (ROS) from mechanically activated sulphide concentrates and its effect on thermophilic bioleaching[END_REF], suggesting the direct effect of external ROS generation on bioleaching organisms. Bioleaching bacteria harbor genetic systems for the defense against oxidative stress by the production of various proteins involved in anti-oxidative protection and biomolecules repair mechanisms (Cardenas et al., 2012). If there is evidence that Leptospirillum, Sulfobacillus and Acidithiobacillus strains possess such systems, the mechanisms are not fully understood yet and their efficiencies depending on genus or strain during bioleaching are not known.

CONCLUSION

The BRGM-KCC microbial consortium can tolerate DO levels up to 13 ppm, and probably higher level with a critical limit between 13 and 17.7 ppm, during continuous bioleaching and still maintain a high bioleaching activity. This value is much higher than the maximal DO levels reported in previous papers and is also higher than the solubility of oxygen in an air/water mixture (8.3 ppm at 25°C, 6 ppm at 40°C). The bioleaching efficiency (oxidation rate and dissolution yield) increases with the increase of the oxygen supply (and oxygen transfer) up to a DO concentration of 13 ppm. However the microbial activity and consequently the oxygen consumption significantly decreased at DO concentration of 17 ppm. These results confirm the applicability of using oxygen enriched gas instead of air to efficiently supply oxygen in bioleaching reactors without inhibitory effect on the microbial oxidizing activity. However the oxygen partial pressure and the gas flow must be carefully adapted to the oxygen requirements of the system to avoid too high DO concentrations in the bioreactors. Acknowledgments: This work is part of the collaborative bilateral research project Ecometals cofunded by the German Federal Ministry of Education and Research (BMBF under the grant ID 033RF001) and the French National Research Agency (ANR-13-RMNP-0006). The authors thank M. Djemil, H. Tris for their technical involvement during the pilot operation.

Figure 2 :Figure 3 :

 23 Figure 2: Influence of DO concentration on the microbial community structure and the quantity of biomass present in the reactor (assessed using the number of 16S rRNA gene copy)

Figure 4 :

 4 Figure 4: Schematic description of oxygen transfer and oxygen concentration profile in a triphasic media with reactions consuming oxygen at the solid surface

Figure 5 :

 5 Figure 5: ROS formation mechanisms at pyrite surface (Schoonen et al. 2010)

Table 1 : Main operating conditions for the gas injection (the partial pressures are measured in the gas inlet after mixing of the N2, O2 and CO2 streams)

 1

		Gas flow rate	O2 partial pressure	CO2 partial pressure
		NL h -1	%	%
	Condition 1	50	57.4	1.10
	Condition 2	100	56.9	1.10
	Condition 3	250	55.4	0.99
	Condition 4	500	57.1	1.12

Table 2 : Oxygen concentration in the gas phase and in the liquid phase in continuous pyrite bioleaching experiments (O2 partial pressure measured in the gas inlet (PO2 in) and outlet (PO2 out) and corresponding O2 solubility calculated using Henry's law; DO concentration measured in the reactor)

 2

		PO2 in	O2 solubility* corresponding to PO2 in	PO2 out	O2 solubility* corresponding to PO2 out	measured DO concentration
		%	ppm	%	ppm	ppm
	Condition 1	57.4	17.6	41.1	12.6	4.7
	Condition 2	56.9	17.5	48.3	14.8	5.0
	Condition 3	55.4	17.0	51.7	15.9	9.5
	Condition 4	57.1	17.5	55.2	16.9	12.9
	Condition 5	70	21.8	69	21.3	17.7

Table 3 : Residence time and sulfide dissolution yield

 3

		measured DO concentration	Residence time	Sulfide dissolution yield
		ppm	d	%
	Condition 1	4.7	2.5	63%
	Condition 2	5.0	2.2	56%
	Condition 3	9.5	2.2	61%
	Condition 4	12.9	2.2	77%
	Condition 5	17.7	2.4	29%