Effect of elevated pressure on ferric iron reduction coupled to sulfur oxidation by biomining microorganisms

Accepted Manuscript Effect of elevated pressure on ferric iron reduction coupled to sulfur oxidation by biomining microorganisms

Ruiyong Zhang, Sabrina Hedrich, Christian Ostertag-Henning, Axel Schippers PII: DOI: Reference:

S0304-386X(18)30188-9 doi:10.1016/j.hydromet.2018.05.003 HYDROM 4809

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Hydrometallurgy

Received date: Revised date: Accepted date:

1 March 2018 27 April 2018 3 May 2018

Please cite this article as: Ruiyong Zhang, Sabrina Hedrich, Christian Ostertag-Henning, Axel Schippers , Effect of elevated pressure on ferric iron reduction coupled to sulfur oxidation by biomining microorganisms. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Hydrom(2017), doi:10.1016/j.hydromet.2018.05.003

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ACCEPTED MANUSCRIPT Effect of elevated pressure on ferric iron reduction coupled to sulfur oxidation by biomining microorganisms Ruiyong Zhang*, Sabrina Hedrich, Christian Ostertag-Henning and Axel Schippers* Federal Institute for Geosciences and Natural Resources (BGR), Stilleweg 2, 30655 Hannover, Germany

Mailing Address:

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Bundesanstalt für Geowissenschaften und Rohstoffe (BGR) Geozentrum Hannover

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Stilleweg 2 30655 Hannover, Germany.

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Tel: +49 (0)511-643-3103 Fax: +49 (0)511-643-2304

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email: [email protected]; [email protected]

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*Corresponding author.

ACCEPTED MANUSCRIPT Abstract Acidophiles, including a mixed mesophilic, iron-oxidizing culture (FIGB) and a thermophilic enrichment culture (TK65) from a black shale and sandstone copper ore (Kupferschiefer), were used to evaluate microbial ferric iron reduction coupled to oxidation of reduced inorganic sulfur compounds (RISCs) at elevated pressure to simulate potential biogeochemical processes

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in deep in situ leaching operations. Experiments were performed from atmospheric pressure (0.1 MPa) up to 36 MPa in different incubation chambers and high-pressure reactors. Microbial

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abundance and activity were determined by quantifying iron and sulfur species concentrations, by direct cell counting, terminal restriction fragment length polymorphism (T-RFLP) analysis

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and quantitative PCR (qPCR). The data indicate that the tested acidophilic cultures were able to reduce soluble ferric iron coupled to oxidation of sulfur compounds under anaerobic

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conditions. Even at high pressure (up to 36 MPa) these acidophiles were capable of growing, and microbial ferric iron reduction was only partially inhibited. These results show that the

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tested acidophiles are barotolerant and their activities may contribute to sulfur and iron cycling in anaerobic environments including deep ore deposits, which is relevant for in situ leaching

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operations.

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Keywords: elevated pressure; biomining microorganisms; ferric reduction; sulfur oxidation;

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in situ leaching

ACCEPTED MANUSCRIPT 1 Introduction Microbial leaching (indirect mechanism) of sulfide minerals relies on the main oxidant ferric iron which causes metal sulfide (MS) dissolution. Biomining microorganisms contribute to MS dissolution by providing ferric iron and/or protons because of their iron/sulfur oxidation activities in the presence of oxygen (Johnson, 2014; Vera et al., 2013). In some leaching

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environments such as heap leaching or large-scale in situ leaching (ISL) operations the concentration of dissolved ferric iron may be several orders of magnitude greater than that of

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molecular oxygen. The relevance of microbial ferric iron reduction for the leaching of MS and the generation of acid mine/rock drainage (AMD/ARD) (Hedrich and Schippers, 2016) is of

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considerable interest.

As various metal resources at Earth’s surfaces continue to diminish, the exploration of

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mineral deposits is shifting to the deeper subsurface (Johnson, 2015). In situ leaching (ISL) is receiving renewed attention as a technology that could be used to extract metals from deep

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underground ores cheaper, with less environmental impact and using far less energy than conventional mining (Huff and Huska, 1975; Sand et al., 1993; Tuovinen and Bhatti, 1999;

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Wadden and Gallant, 1985). The sulfidic Kupferschiefer deposits of northern central Europe

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represent the largest copper reserve in Europe (Kutschke et al., 2015). Mining of these ore deposits is of economic interest (Kamradt et al., 2018; Kutschke et al., 2015). The EU Horizon 2020 project BIOMOre aims to explore in situ bioleaching for extracting valuable

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metals from deep mineralized zones (>1 km) in Europe by coupling solution mining and bioleaching technology (http://www.biomore.info/).

The

main

role

envisaged

for

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microorganisms in the BIOMOre concept is to generate acidic ferric sulfate in bioreactors located at the mine surface (Fig. 1). Microorganisms could e.g. catalyze anaerobic oxidation of elemental sulfur, which is predicted to form during the indirect oxidation of metal sulfides by ferric iron (Schippers et al., 1996; Schippers and Sand, 1999) in the fractured ore body. Solid sulfur could inhibit lixiviant flow by clogging pores. Various kinds of (bio-)chemical reactions may occur once an acidic ferric solution comes in contact with the ore (Fig. 1). Hydrostatic pressure and temperature increase with depth from the earth’s surface, which, together with the exothermic leaching reactions, may influence the bacterial activity (Dixon, 2000; Franzmann et al., 2007). Biochemical processes are influenced by physical effects of

ACCEPTED MANUSCRIPT high hydrostatic pressure, because the thermal expansion coefficient as well as viscosity and fluidity of water and solubility of gases affect chemical reactions and cellular processes. Basic parameters affecting bacterial activities in biomining processes are temperature, pH (Baker-Austin and Dopson, 2007), substrate availability (Schippers et al., 2014; Shiers et al., 2013), ionic strength (Bellenberg et al., 2015; Ojumu et al., 2008), redox potential, toxic

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metal concentration (Dopson et al., 2014). The effect of temperature on growth and activity of mineral degrading acidophiles has been relatively widely studied (Barahona et al., 2014;

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Brierley and Brierley, 2013; Christel et al., 2016), but little is known how their metabolic

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activity is affected by elevated pressure - as this is not an issue in current biomining operations.

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The effects of pressure on microorganisms involve several aspects. Growth kinetics and cell metabolism such as DNA synthesis and cellular anaerobic respiration have been shown to be

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pressure-affected and -regulated (Abe et al., 1999; Fang et al., 2010; Wang et al., 2008). Some data demonstrate that cell morphology is controlled by elevated pressure (Mañas and

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Mackey, 2004). E. coli and other bacteria form filaments at elevated hydraulic pressure

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(Ghosh and Aertsen, 2013; Tamura et al., 1992; Terry et al., 1966; Zobell and Cobet, 1964). This study focused on the evaluation of anaerobic ferric iron reduction coupled to sulfur oxidation by acidophilic bacteria and archaea at elevated temperatures and high hydrostatic

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pressure. The mixed mesophilic culture FIGB and a thermophilic enrichment culture from Kupferschiefer sandstone/blackshale ore material were chosen as test organisms.

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2 Materials and Methods

2.1 Medium and cultivation Acidophilic microorganisms commonly found in bioleaching environments (Table 1) were cultivated in basal salts medium (Wakeman et al., 2008). The thermo-acidophilic TK65 culture was enriched from Kupferschiefer black shale/sandstone ore (KGHM Poland). The mixed meso-acidophilic, iron-oxidizing culture FIGB was provided by Bangor University, Wales, UK (Pakostova et al., 2017). All cultures were cultivated and maintained in the BGR strain collection. The FIGB culture was maintained aerobically in liquid medium containing 50 mM ferrous sulfate at 30 °C. The TK65 culture was anaerobically cultivated with 25 mM

ACCEPTED MANUSCRIPT ferric sulfate, 10 g/L S0, and 10 g/L Kupferschiefer-associated sandstone (SS; Cu 2.48%, Fe 0.44%, S 0.14%) and 0.04% yeast extract (YE) at 55-60 °C. For cultures grown anaerobically at atmospheric pressure, standard anaerobic cultivation techniques were applied. Briefly, sterilized medium was poured into individual serum bottles (25 mL-100 mL). The medium was supplemented with 50 mM ferric iron and 5 mM

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potassium tetrathionate or 10 g/L S0. The serum bottles were capped with butyl rubber stoppers and sealed with aluminium crimps. The solution was vigorously bubbled with N2 for

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25 min to strip dissolved oxygen from the aqueous solution. Afterwards, CO2 was injected into the serum bottles to a final concentration of 10%. The static cultures were incubated at

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30 °C for mesophiles and 55 °C for the thermophiles in the dark. 2.2 Pressure incubation systems

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To achieve elevated pressure, three types of experimental setups were used, namely a high-pressure incubation chamber, a Dickson-type (Seyfried Jr et al., 1987) flexible

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gold-titanium cell in a rocking high pressure reactor, and a static high-pressure reactor with a glass liner (Fig. 2 and Supplementary Fig. S1).

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For the high-pressure incubation chamber, Hungate glass tubes (20 mL; Fig. 2b) were loaded

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with 10 mL of culture with substrates inside. The tubes were sealed with rubber stoppers and secured with aluminum caps to assure anoxic conditions. Afterwards, 1 mL of CO2 (final concentration ~10%) was injected into the tubes after flushing with nitrogen for 25 min.

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These tubes, connected with hypodermic needles with sterile syringes (10 mL) which were fully filled with culture with substrates (in case of soluble substrates) inside, were placed in a

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static high-pressure vessel (DUSTEC High Pressure Technology, 6.8 L/2.0 L). The limit of working temperature and pressure of the two kinds of vessels were 90 °C/45 MPa and 55 °C/25 MPa, respectively. The pressure inside the pressure vessel was attained by addition of water with a pump (DUSTEC High Pressure Technology, up to 36 MPa). This pressure was transmitted by the moving syringe plungers to the individual culture tubes as described elsewhere (Nauhaus et al., 2002). The high-pressure vessel was placed in an incubator at 30 °C or 55 °C. In the Dickson-type flexible gold-titanium cells (Fig. 2 d-f), pressure was transmitted to the reaction cell via the high deformability of the 0.5 mm thin gold foil forming the cell, which

ACCEPTED MANUSCRIPT ensures less than 0.1 MPa pressure difference between the inside of the gold-titanium cell and the outside in the water-filled high-pressure reactor (PARR Instruments, model 4871). Due to this deformability and the comparably large volume of the cell (up to 130 mL), up to 40% of the original solution could be sampled throughout the course of the experiment using high-pressure titanium tubing and valves fitted to the titanium head of the reaction cell (Fig.

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2d). During sampling no pressure changes occurred as the withdrawn sample volume was added as water volume to the outside of the cell in the high-pressure reactor. The rotation of

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the high-pressure reactors by 120 ° (“rocking”) maintained a thoroughly mixed suspension inside the gold-titanium cell (Fig. 2f). The high-pressure reactor – and hence the

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gold-titanium reaction cell was pressurized by a high-pressure syringe pump (DM100s, Teledyne Isco). The temperature was controlled by a band heater outside the high-pressure

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reactor. Pressure and temperature were constantly logged.

For experiments in the static high-pressure reactor (Supplementary Fig. S1), 100 mL of

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bacterial cultures were inoculated in a glass liner (total volume ~130 mL). The inoculated liner was placed in a cylindrical pressure-proven steel vessel. A pressure of 10 MPa in the

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pressure reactor was reached by adding 0.1-1 MPa of CO2 and then the rest by adding

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pressurized N2 and the pressure was maintained by an air-driven pump (Maximator GmbH, model M111, Germany).

2.3 Physico-chemical methods

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The pH values and oxidation/reduction potentials (ORP) were analyzed with digital pH- / redox meters (Mettler Toledo and WTW) with semi-micro pH electrodes (Blue Line pH 16,

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SI Analytics), and an ORP electrode (Ag/AgCl reference, PCE Instruments), respectively. Planktonic cells were counted directly by using a light microscope (Leica DM3000) in phase contrast mode (400× magnification) with a Thoma Chamber

(Hecht-Assistent).

Concentrations of inorganic sulfur species (S0, sulfate and tetrathionate) were quantified according to the methods described in previous studies (Schippers et al., 1996). S0 was extracted from solid samples in three steps with ethanol. For this purpose, 1 ml of ethanol was added to the pellet after centrifugation (9839 × g, 15 min) of a sample and the tube was whirled until the pellet was totally suspended. Thereafter the suspension was sonicated in ultrasonic bath (Sonorex Digiplus, BANDELIN electronic GmbH) for 15 min at 35 KHz and

ACCEPTED MANUSCRIPT 640 W and centrifuged at 9839 × g for 20 min (Eppendorf 5430). The supernatant was quantitatively decanted, and the procedure was repeated two times by addition of fresh ethanol. The collected supernatants were used for the quantification of the sulfur content. The measurement was done by reversed-phase chromatography with a Zorbax ODS column (4.6 by 12.5mm; 5µm) with pure methanol (Merck) as eluent at a flow rate of 1 mL/min. For the

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determination of tetrathionate, liquid samples were centrifuged in 2 mL-tubes at 9839 × g for 10 min (Eppendorf 5430). Then the supernatant was quantitatively decanted, and 250 µL of

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the supernatant was mixed with 750 µL of a 0.1 M, pH 7.0 phosphate buffer (0.0615 M K2HPO4 and 0.0385 M KH2PO4) to precipitate ferric iron. After the removal of the

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precipitates by centrifugation, the concentration of tetrathionate was measured by high-pressure liquid chromatography (HPLC) with an Eclipse Plus C18 column (4.6 by

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12.5mm; 5µm) with TBAH-buffer and acetonitrile as eluent at a flow rate of 1 mL/min. For analysis of both, S0 and tetrathionate, a high-pressure liquid chromatography system (Agilent

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1200 HPLC) from Agilent Technologies was used. It consisted of a binary pump G1312A, a thermostated autosampler G1329A and a diode array detector G1315D. S0 and tetrathionate

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were measured by UV detection (S0 at 254 nm and tetrathionate at 215 nm). The assignment

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of the peaks was made by using pure substances. S0 and potassium tetrathionate were obtained from Merck. For measuring sulfate, 100 µL of the supernatant was diluted with 900 µL of a 20 mM NaOH solution instead of phosphate buffer (phosphate ions interfere with the

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detection of sulfate). After removal of the precipitates by centrifugation, the concentration of sulfate was measured by ion chromatography (IC, Dionex Type ICS-3000) with an AS 19

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column and KOH as eluent.

Determination of dissolved iron(II) and total iron followed the ferrozine assay (Lovley and Phillips, 1987).

2.4 T-RFLP, quantitative real-time PCR and sequencing For DNA extraction from the cells associated with solid substrates (S0 or ore), solid samples were collected and washed twice with sulfuric acid (pH 1.8) to remove the planktonic cells. DNA extraction was achieved by using the FastDNA Spin Kit for Soil (MP Biomedicals) according to a modified protocol (Webster et al., 2003). Copy numbers of bacterial and archaeal 16S rRNA genes were determined by quantitative real-time PCR (qPCR) using

ACCEPTED MANUSCRIPT universal bacterial- and archaeal-specific primers (Supplementary Table S1) as described previously (Nadkarni et al., 2002; Takai and Horikoshi, 2000). Amplification of an approximate 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 (Frank et al., 2007) and 907R (Amann et al.,

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1992) for bacteria and a Cy5-labeled 20F forward primer (Massana et al., 1997) and 915R (Stahl and Amann, 1991) for archaea, respectively (Supplementary Table S1). Up to 3 µL of

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PCR product were digested in a 10 µL reaction with 1 U of the restriction endonucleases HaeIII, CfoI, RsaI and AluI (Thermo Scientific) and 1 µL appropriate buffer. The reactions

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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

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600 bp size standard and identified by reference to the databank of acidophilic microorganisms held at BGR (Hedrich et al., 2016).

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For 16S rRNA sequencing, bacterial primers (341F-785R) and archaeal primers (A340F-A915R) (Supplementary Table S1) were used for amplification, respectively.

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Purification, cloning and sequencing of the 16S rRNA PCR products was conducted at LGC

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Genomics GmbH, Berlin. Amplicon sequencing was performed using Illumina MiSeq V3. Demultiplexing of all libraries for each sequencing lane was done using the Illumina bcl2fastq 1.8.4 software.

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Forward and reverse reads were merged using BBMerge 34.48. 16S pre-processing and OTU

3 Results

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picking from amplicons was done with Mothur 1.35.1.

3.1 Batch culture in the static high-pressure incubation chamber 3.1.1 Mesophilic acidophiles Fig. 3 shows the planktonic cell numbers, community composition and ferric iron reduction by the FIGB culture after one week incubation at 30 ˚C at 0.1 MPa (atmospheric pressure) or 10 MPa. It is obvious that cells readily grew on different substrates at 0.1 MPa. As indicated by both 16S rRNA gene copy numbers and direct cell counts, the cell density was highest when grown on 5 mM tetrathionate (Fig. 3a and b). In presence of SS the planktonic cell numbers were considerably lower, likely due to cell attachment. The cell numbers in the

ACCEPTED MANUSCRIPT assays with S0 or tetrathionate plus sandstone were similar. Iron reduction was comparable for cells grown on all three substrates (elemental sulfur, tetrathionate and tetrathionate + sandstone), with 33-37 mM ferrous iron generated after one week of incubation. For assays at 10 MPa, significant cell growth was observed too. However, all cell and gene copy numbers were lower than for the respective 0.1 MPa experiments reflecting a partial inhibition of

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growth for all three substrates. This inhibition was up to 38% for cells grown on tetrathionate + sandstone at 10 MPa compared to those at 0.1 MPa, considering both, gene copy numbers

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and direct cell counts. Consistent with the reduced cell numbers at 10 MPa, ferric iron reduction decreased by 50% if compared to the 0.1 MPa assays. A significant pH drop from

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(0.4-0.6 pH units) indicated the oxidation of sulfur compounds to sulfuric acid in all the biotic assays (not shown). In both control experiments, chemical ferric iron reduction at 0.1

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MPa or 10 MPa was not evident under tested conditions.

When grown aerobically on ferrous iron, the FIGB culture mainly comprised representatives

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of the genera Leptospirillum, Sulfobacillus (Sb.), Acidithiobacillus (At.) with a small proportion of Ferroplasma (F.) acidiphilum (Table 1). This result is consistent with the

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original FIGB composition (Pakostova et al., 2017). Under anaerobic conditions, the FIGB

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culture mainly consisted of At. ferrooxidans, Sb. thermosulfidooxidans and the archaeon F. acidiphilum, with the first one dominating (67-90%) in all assays (Fig. 3c). Cells of F. acidiphilum accounted for 8.3% of the community when grown on S0 under anaerobic

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conditions. However, the cell proportion decreased to 2.4% when the pressure increased to 10 MPa (Fig. 3c). The abundance of Sb. thermosulfidooxidans increased from 3.8% at 0.1 MPa

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to 7.0% at 10 MPa. However, cells were hardly detected in the culture grown on tetrathionate. Cells might form spores under high pressure conditions. The Gram-positive bacterium Sb. thermosulfidooxidans is well known to form spores under harsh conditions (Kovalenko and Malakhova, 1983). The pure culture of several meso-acidophiles showed considerable activity at 10 MPa (Table 2). Ferric iron reduction by At. ferrooxidansT and the FIGB culture was retarded at 10 MPa compared to the one at 0.1 MPa by 16% and 10%, respectively. Ferric reduction by both At. thiooxidansT and At. thiooxidans Ram8T at 10 MPa was much slower (by 54% and 80%, respectively) compared to assays at 0.1 MPa.

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3.1.2 Thermophilic acidophiles The pressure effect on the thermophilic enrichment culture TK65 is shown in Table 3. When the pressure increased from 0.1 MPa to 10 MPa, cell growth was inhibited by 27 up to 39% as indicated by direct cell counts and qPCR data. Accordingly, cells showed 24% decreased

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ferric iron reduction activity at 10 MPa. The TK65 culture was mainly composed of Thermoplasma (T.) acidophilum (~85%) and

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Acidianus (A.) brierleyi (3.5%) and other unidentified archaea when grown anaerobically on S0 at 0.1 MPa (Table 3). T. acidophilum was the only detected archaeal species when the

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TK65 culture grew under 10 MPa. The detection of hydrogen sulfide odour and pH drop (~0.3 pH units) at the end of the biotic experiments indicated that sulfur reduction and

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disproportionation were carried out by the culture TK65. This sulfur disproportion has been reported in several thermophilic archaea (Kletzin et al., 2004). The generated hydrogen

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sulfide might react with metal ions such as ferrous iron and form black FeS precipitates. However, FeS precipitates were not observed at the tested conditions. Approximately 1.9 and

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0.9 mM ferrous iron were detected in abiotic controls at 0.1 MPa and 10 MPa, respectively.

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The chemical reduction of ferric iron occurred most likely due to the presence of certain organic compounds in yeast extract.

The pressure response of several representatives of thermo-acidophiles grown anaerobically

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on S0 and ferric iron was tested and compared. As shown in Table 4, the relative order of the amount of ferrous iron produced due to microbial activity at 23-25 MPa was TK65 culture >

4).

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T. acidophilumT > S. metallicusT ≥ A. brierleyiT under the tested conditions (Fig. 3 and Table

3.2 The effect of elevated pressure on ferric iron reduction The growth of the FIGB culture on tetrathionate in a static high-pressure reactor with a glass liner (Supplementary Fig. S1) with the possibility of sampling at different time intervals was tested. The data are shown in Fig. 4. An increase of the ferrous iron concentration from 3 mM to 20 mM together with the decrease of tetrathionate indicated the active growth of the FIGB cultures at 0.1 MPa. The maximum cell number reached 6.5×107 cells/mL. The cells oxidized tetrathionate in the first eight days and the tetrathionate concentration remained almost

ACCEPTED MANUSCRIPT constant at the end of the test. A net increase of the sulfate concentration of 7.1 mM was measured till the end of experiments (not shown). The overall mole ratio of net increase of ferrous iron to consumed tetrathionate was approximately 5.5, which is below 14 for a complete oxidation of tetrathionate to sulfate by ferrous iron reduction (Fig. 1). Small concentrations of S0 (below 0.2 mM) were occasionally detected at day 9 and day 13 at 0.1 MPa and 10 MPa, respectively (not shown). In the assays performed in the Hungate tubes,

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maximum concentrations of 3.6 mM and 4.5 mM of S0 at 0.1 MPa and 10 MPa were detected,

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respectively (Supplementary Table S2). Thus, it seems that tetrathionate oxidation by acidophiles under anaerobic conditions may involve similar pathway as under aerobic

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conditions (Schippers et al., 1996).

Cells grown at 10 MPa showed much slower growth and had a long lag phase (approximately

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10 days). Cell numbers reached the maximum of 3.7×107 cells/mL after 15 days. The tetrathionate concentration slightly decreased from 5 mM to 4.3 mM. Both sterile control

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experiments at 0.1 MPa and 10 MPa did not show a chemical oxidation of tetrathionate (Supplementary Table S2).

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For the experiments in the flexible gold-titanium cells in the rocking high-pressure reactor the

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data of the FIGB growth on S0 with ferric iron at 0.1 MPa and 10 MPa are presented in Fig. 5. Under both conditions the cultures had a lag phase of ca. 10 days when grown in the gold-titanium reaction cells. After that period, a rapid increase of the ferrous iron

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concentration from ~9 mM to 32 mM occurred in the culture grown at 0.1 MPa. Meanwhile, the cell numbers exponentially increased to 1.3×108 cells/mL. In contrast, cell growth at 10

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MPa was significantly inhibited during experiments. A slight but consistent increase of cell numbers up to 4.5×107 cells/ml was observed. The increased amount of the ferrous iron was 9.7 mM, which was approximately one-third of the generated ferrous iron in the 0.1 MPa assays. The total iron in both 0.1 MPa and 10 MPa assays was stable at approximately 50 mM before ca. 10 days. Afterwards, total iron showed slightly decreased and this might be due to the formation of ferric precipitates in the system.

ACCEPTED MANUSCRIPT 3.3 Planktonic populations vs. attached populations at elevated pressure The proportion of cells in planktonic (in the bulk solution) and biofilm (associated with S0 particles) phases at both 0.1 MPa and 10 MPa in the flexible gold-titanium cells was evaluated. It was found that the biofilm cell proportion at 10 MPa was several times higher than that at 0.1 MPa, although the absolute biofilm cell number at 10 MPa was smaller than

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that at 0.1 MPa (Table 5). This phenomenon was also observed when cells were grown in

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Hungate tubes in the high-pressure incubation chamber (Supplementary Table S3).

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4 Discussion

Acidophilic autotrophs and heterotrophs have been reported to be able to reduce ferric iron by

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coupling to reduced sulfur compound or hydrogen oxidation under aerobic or anaerobic conditions (Hedrich and Johnson, 2013; Johnson and Hallberg, 2008). At. ferrooxidans is the

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most widely studied biomining model organism and it is able to oxidize S0 anaerobically using ferric iron as terminal electron acceptor (Brock and Gustafson, 1976; Marrero et al., 2017; Pronk, 1992). The multiple mechanisms of anaerobic oxidation of S0 coupled to ferric

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reduction by At. ferrooxidans might indicate its good adaptation to various environments

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(Kucera et al., 2016; Norris et al., 2018). At. ferrooxidansT possesses a substantial pressure resistance for both conditions, aerobic (Torma, 1975) and anaerobic conditions (Table 2). At. ferrooxidans was the dominant species in the mixed FIGB cultures grown anaerobically at

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elevated pressure (Fig. 3). A previous study conducted within 48 h indicated that the maximum pressure permitting aerobic growth of At. ferrooxidans on ferrous sulfate was ~30

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MPa (Davidson et al., 1981). The microbial activity of At. ferrooxidans was somewhat affected when subjected to a pressure of 0.69 MPa in the presence of chalcopyrite (Bosecker et al., 1979). These findings indicate that the organism has potential for deep in situ biomining or heap bioleaching (e.g. consumption of ferric iron in anaerobic/microaerobic zones). The substantial growth and activity of FIBG at 10 MPa indicates that these acidophiles could account for considerable microbial activity at 1000 m depth in an anaerobic environment. Further tests of this study showed that a pure culture of At. ferrooxidansT and the FIGB culture remained active at up to 36 MPa in the high-pressure incubation chamber at

ACCEPTED MANUSCRIPT 30 °C for 10 days. In addition, cell growth was recovered after two passages of cultivation at 0.1 MPa (not shown). This implies that the bacteria could survive in the pregnant leach solution in the leaching cycle (Fig. 1). In another study sulfur oxidation by At. ferrooxians and its 14CO2 incorporation was strongly inhibited by the combination of hydrostatic pressure and high concentration of dissolved

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oxygen while iron oxidation was less affected (Davidson et al., 1981). Cell growth of FIGB in the static high-pressure reactor with the glass liner seemed to be slower than in the

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Hungate tubes in the incubation chamber (Fig. 3 and Fig. 4). The amount of CO2 in the aqueous solution and its speciation are controlled by total amount of injected CO2, solution

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volume and pH. The ratio of injected amount of CO2: solution volume in the static high-pressure reactor (~150 mL: 100 mL) was higher than that in the Hungate tubes in the

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incubation chamber (1 mL:20 mL). This might have led to an increased amount of dissolved CO2 in the static high-pressure reactor. Highly pressurized CO2 is inhibitory to cell growth

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(Frerichs et al., 2014).

The iron-oxidizing archaea Ferroplasma spp. have been suggested to play a role in the global

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iron and sulfur cycle (Golyshina, 2011). The organisms could grow anaerobically on yeast

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extract and reduce ferric iron to ferrous iron (Dopson et al., 2004). Ferroplasma spp. interact with other acidophiles and thus impact on the bioleaching efficiency (Maulani et al., 2016; Okibe and Johnson, 2004). F. acidiphilum was present in the FIGB culture at all tested

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conditions and accounted for up to 33% when grown at 10 MPa with potassium tetrathionate in presence of sandstone. It was evident that microbial degradation of tetrathionate occurred

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in the presence of F. acidiphilum DSM 28986 (not shown). These data suggest that F. acidiphilum has potential impact on the sulfur cycle and bioleaching under anaerobic conditions.

Thermophilic Acidianus and Thermoplasma species are able to gain energy anaerobically by sulfur respiration (Segerer et al., 1988; Wheaton et al., 2015). The growth of several thermo-acidophiles at 23-25 MPa demonstrated that these are pressure-resistant (Table 4). Although cellular structures of thermophilic archaea are different from mesophilic or moderate thermophilic leaching bacteria, both mesophiles and thermophiles seem to respond to pressure in a similar manner. Therefore, contributions of thermo-acidophilic archaea -

ACCEPTED MANUSCRIPT especially T. acidophilum - to iron and sulfur metabolism at high temperature environments are relevant for deep in situ biomining. Biofilm lifestyle protects cells from environmental stress like desiccation, nutrient starvation, radiation and/or oxidative stress (Flemming and Wingender, 2010). Acidophiles are known to form biofilms on mineral and S0 surfaces (Bellenberg et al., 2015; Zhang et al., 2016). The

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tendency of cells to divert themselves into biofilm state might indicate a role of biofilms coping with hydrostatic pressure stress. This might be important for the development of the

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decommission process in the in situ deep mining practice (Ballerstedt et al., 2017), e.g. to consider the association of cells with solids under high pressure.

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To summarize, microbial activity of tested acidophiles at high pressure was lower than at atmospheric pressure. The influence of pressure on microbial ferric iron reducing activity was

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species/strain dependent. Various intermediate RISCs were formed during the anaerobic

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oxidation of S0/tetrathionate coupled to ferric iron reduction by acidophiles. Microbial activity in the deep underground has also been described for non-acidophilic microorganisms. For example, in presence of rocks or minerals bacteria and archaea from the

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deep biosphere stimulate the formation of hydrogen and acetate at temperatures above 70 °C

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even under elevated pressure (Parkes et al., 2011). These compounds could also be oxidized and coupled to cell growth by acidophiles. Thus, there are more potential substances in

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addition to sulfur compounds which might stimulate microbial activity in deep in situ leaching processes.

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5 Conclusions

The findings of this study have several implications for deep in situ leaching operations. The BIOMOre concept relies on indirect bioleaching, in which copper sulfides (or other base metal sulfides) are dissolved via chemical oxidation with ferric iron as oxidant at low pH. The ferrous iron is oxidized in a bioreactor containing acidophiles. These acidophiles contained in ferric iron-rich leaching solution are injected into the underground for deep in situ biomining and possess significant biological activities i.e. ferric iron reduction and sulfur oxidation. These potentially influence the leaching process and the subsurface environment by changing e.g. pH, ferric iron availability and ore permeability, since solid elemental sulfur

ACCEPTED MANUSCRIPT and cells may clog pores in the rock. Temperature increase with depth from the earth’s surface, and together with the heat released by exothermic leaching reactions, might restrict the microbial activity (Dixon, 2000; Franzmann et al., 2007). However, our study shows that besides the mesophilic acidophiles such as At. ferrooxidans and Ferroplasma spp., also thermoacidophilic archaea enriched from a deep Kupferschiefer formation are reducing ferric

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iron coupled to sulfur compound oxidation at high temperature and high pressure. Conclusively, microbial iron and sulfur metabolism and cell growth at elevated temperatures

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and pressure have to be considered for deep in situ biomining. Acknowledgement

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This work was supported by the European Union Horizon 2020 project BIOMOre (Grant agreement # 642456). We acknowledge I. Kruckemeyer, P. Grosche, M. Krüger and T. Weger

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for assistance in pressure experiments.

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ACCEPTED MANUSCRIPT Table Legends Table 1. Acidophilic microorganisms used in high pressure tests. Table 2. Comparison of the pressure response of different mesophilic acidophiles. Cells were cultivated on ferric iron (50 mM) and S0 (10 g/L) at 30 °C for three weeks. The amount of reduced iron was measured as ferrous iron in solution (Mean ± standard deviation of three parallel assays is given).

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Table 3. Pressure effects on growth and ferric iron reduction by the thermophilic enrichment culture TK65. Assays with an initial cell number of ~107 cells/mL or no inoculations in

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Hungate tubes were cultivated on S0 ( 0 g/L) anaerobically at 55 ˚C for four days. The

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amount of reduced ferric iron was measured as ferrous iron in solution (Mean ± standard deviation of three parallel assays is given).

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Table 4. Comparison of the pressure response of several thermophilic acidophiles. Cells were cultivated on ferric iron (50 mM) and S0 (10 g/L) at 55 °C and 23-25 MPa for a week. The

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amount of reduced iron was measured as ferrous iron in solution (Mean ± standard deviation of three parallel assays is given).

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Table 5. Comparison of cell population in planktonic and attached communities. Total cell

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numbers of planktonic (cells/100 mL) and attached communities (copies/g S0) were given in 16S rRNA gene copy numbers measured by qPCR. Cells were cultivated in the gold bag

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reactor for 22 days (Mean ± standard deviation of three parallel assays is given).

ACCEPTED MANUSCRIPT Figure Legends Fig. 1. Scheme of BIOMOre process employing in situ leaching and electro-winning for metal recovery from Kupferschiefer deposits, modified according to Johnson, 2014; Schippers and Sand, 1999. Fig. 2. High-pressure incubation chamber (upper; a, b and c) and flexible gold-titanium cell

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(lower; d, e and f) used for generating high-hydraulic pressure. (a) Schematic of the high-pressure incubation chamber; (b) upper: Batch of cultures inoculated in Hungate tubes

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connected with hypodermic needles with sterile syringes, lower: tubes immersed into water in a high-pressure vessel; (c) High pressure vessel operated at 10 MPa in an incubator at 30 °C

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or 50-55 °C. (d) Schematic of the flexible gold-titanium reaction cell. (e) Photos of the gold cells with their titanium closure pieces, the reaction cells are shown before (left) and after

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(right) deformation due to sample withdrawn in an experiment. (f) The rocking high-pressure reactor system containing the gold-titanium cell inside.

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Fig. 3. Pressure effect on growth and ferric iron reduction by the FIGB culture. (a) cell numbers by direct microscopic counting; (b) microbial relative abundance analysis by

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T-RFLP; (c) sum of archaeal and bacterial 16S rRNA gene copy numbers determined by

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qPCR; (d) ferrous iron concentration. In all assays the initial cell number was ~107 cells/mL. Experiments were run in Hungate tubes anaerobically at 30 ˚C for one week. PT, tetrathionate; SS, sandstone.

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Fig. 4. Growth of the FIGB culture and ferrous iron reduction at 0.1 MPa and 10 MPa in the pressure reactor. Changes of ferrous iron and total iron concentration (a), cell numbers and

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pH (b), and tetrathionate (PT) concentration (c) are shown. Cells were cultivated anaerobically at 30 °C. Fig. 5. Growth of the FIGB culture on ferric iron (50 mM) and S0 and at 0.1 MPa and 10 MPa in the flexible gold-titanium cell. Changes of ferrous iron and total iron concentration (left) and cell numbers and pH (right) are shown. Cells were cultivated anaerobically at 30 °C. Graphical abstract

ACCEPTED MANUSCRIPT Table 1. Cultures

Temperature characteristics

TK65 culture

Mixed thermophiles

Thermoplasma (T.) acidophilum (78. 9%)

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Acidianus (A.) brierleyi (20%)

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Sulfolobus (S.) metallicus (1.1%)

Leptospirillum (L.) spp. (82.9%)

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Sulfobacillus (Sb.) thermosulfidooxidans (2.7%)

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Unidentified (12.8%)

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Acidithiobacillus (At.) spp. (1.3%) Ferroplasma (F.) acidiphilum (0.3%)

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Mixed mesophiles

FIGB culture

At. thiooxidans Ram 8T (BGR)

Mesophile

At. thiooxidans DSM 14887T

Mesophile

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Pure cultures

Mesophile

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At. ferrooxidans DSM 14882T

Mesophile/Moderate

F. acidiphilum DSM 28986 thermophile

A. brierleyi DSM 1651T

Thermophile

T. acidophilum DSM 1728T

Thermophile

S. metallicus DSM 6482T

Thermophile

*

The species abundances (numbers shown in the brackets) of mixed cultures FIGB and TK65

were determined by T-RFLP analysis and 16S rRNA gene cloning and sequencing.

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ACCEPTED MANUSCRIPT Table 2. Culture name

Ferrous iron (mM) 0.1 MPa

10 MPa

47.1±2.5

42.2±5.6

At. ferrooxidansT

45.9±0.2

38.5±5.2

At. thiooxidansT

39.4±2.7

18.1±3.1

At. thiooxidans Ram8T

42.8±7.5

F. acidiphilum DSM 28986*

22.3±2.1

Abiotic

0

8.4±3.8

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17.2±1.6

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Additional YE (0.02%) was added to the culture medium.

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FIGB culture

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ACCEPTED MANUSCRIPT Table 3. Growth

Ferrous

Cell count Gene copy

Microbial

conditions

iron (mM)

(cells/mL)

number (copies/mL) community

TK65, 0.1 MPa

27.7±3.0

1.2×109

5.0×109

T. acidophilum

±2.9×108

±8.4×108

(85%),

8.6×108

3.1×109

±1.1×108

±1.9×108

1.9±0.2

0

0

Abiotic, 10 MPa

0.9±0.2

0

0

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Abiotic, 0.1 MPa

T. acidophilum (100%)

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20.0±1.3

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TK65, 10 MPa

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A. brierleyi (3.5%)

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ACCEPTED MANUSCRIPT Table 4. Ferrous iron (mM)

T. acidophilumT

11.0±0.5

A. brierleyiT

5.0±0.8

S. metallicusT

6.7±0.1

TK65 culture

13.0±0.3

Abiotic

1.6±0.1

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Culture name

ACCEPTED MANUSCRIPT Table 5. Planktonic cells

Attached cells

(cells/100 mL)

(copies/g S0)

0.1 MPa

1.9×1010±1.3×1010

1.2×109±1.5×108

10 MPa

3.3×109±6.7×108

6.1×108±1.3×108

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Growth conditions

ACCEPTED MANUSCRIPT Highlights 

First evaluation of anaerobic growth and ferric iron reduction of meso-acidophiles and thermo-acidophiles at high pressure.



Use of different pressure incubation systems to mimic conditions of deep in situ mining environments.

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Meso-acidophiles and thermo-acidophiles are barotolerant, but their growth and ferric

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reduction activity are retarded at elevated pressures.

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

Graphics Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5