This protocol describes microbial experiments under elevated pressures to study in situ biomining processes. The experimental approach employs a rocking high-pressure reactor equipped with a gold-titanium reaction cell containing a microbial culture in an acidic, iron-rich medium.
Laboratory studies investigating subsurface microbial processes, such as metal leaching in deep ore deposits (biomining), share common and challenging obstacles, including the special environmental conditions that need to be replicated, e.g., high pressure and in some cases acidic solutions. The former requires an experimental setup suitable for pressurization up to 100 bar, while the latter demands a fluid container with high chemical resistance against corrosion and unwanted chemical reactions with the container wall. To meet these conditions for an application in the field of in situ biomining, a special flexible gold-titanium reaction cell inside a rocking high-pressure reactor was used in this study. The described system allowed simulation of in situ biomining through sulfur-driven microbial iron reduction in an anoxic, pressure-controlled, highly chemically inert experimental environment. The flexible gold-titanium reaction cell can accommodate up to 100 mL of sample solution, which can be sampled at any given time point while the system maintains the desired pressure. Experiments can be performed on timescales ranging from hours to months. Assembling the high-pressure reactor system is fairly time consuming. Nevertheless, when complex and challenging (microbiological) processes occurring in the earth's deep subsurface in chemically aggressive fluids have to be investigated in the laboratory, the advantages of this system outweigh the disadvantages. The results found that even at high pressure the microbial consortium is active, but at significantly lower metabolic rates.
During the past decade, efforts to minimize the impact of mining on the environment have increased. Open pit mining for the raw material extraction of ores (e.g., copper-rich sulfide ores), impacts the surrounding landscape by the excavation activities and by the large remaining volumes of waste rocks and remains of processed ore after the extraction of precious metals like copper. Extracting copper directly from the ore in the subsurface would significantly reduce these impacts. The technology of in situ biomining is a promising candidate for this process1. This publication describes the use of stimulated microbial activity to extract the precious metals from the ore into an aqueous solution in the subsurface. Thus, a copper-rich solution can be easily pumped back to the surface to further concentrate the metal, for example.
The activity of ore-leaching acidophilic microorganisms has been studied in many laboratories for a diverse array of parameters2,3,4,5,6. However, pressure effects on the microbial activity resulting from the difference between ambient surface lab conditions (near 1 bar) and the subsurface at a depth of 1,000 m with hydrostatic conditions (~100 bar), are not well-documented. Therefore, the effects of pressure on microbial iron reduction have been investigated through different experimental avenues7. Here, the most suitable technique is described in detail.
High-pressure reactors have been used extensively to study reactions at pressures and temperatures occurring in the subsurface of the earth. Such reactors consist of a reactor vessel at the bottom that can contain a fluid sample with a microbial culture. Sitting on top of the reactor vessel, the reactor head offers a diverse array of connections and interfaces for safety measures and monitoring sensors (e.g., temperature or pressure). Most high-pressure reactors are made of stainless steel. This material offers high resilience and good machining properties, but the corrosion resistance of the stainless-steel surface is not adequate for every application. For example, if highly acidic or highly reducing aqueous solutions are investigated, significant reactions of the compounds of interest with the reactor wall may occur. One way to avoid this is to insert a liner into the reactor vessel, for instance a liner made from borosilicate glass7. It is easy to clean and can be sterilized by autoclaving. In addition, it is not attacked by acidic or reducing aqueous solutions. Even though a liner can help to prevent artificial reactions of the solution or microbes in the solution with the stainless-steel reactor wall, several problems remain. For one, if a corrosive gas is formed, such as hydrogen sulfide produced by sulfate-reducing bacteria, this gas might react with the uncovered surface of the reactor head sitting above the liner. Another disadvantage is that it is not possible to withdraw a sample from the reactor while maintaining the pressure.
To overcome these limitations, specialized flexible reaction cells inside the high-pressure reactors have been developed for a variety of applications. A flexible polytetrafluoroethylene (PTFE) cell8 was designed for solubility studies of salts in highly saline brines. However, the limitation of this system is that some gases can easily permeate the PTFE. In addition, this material still has a relatively low temperature stability. Thus, the system was improved by designing a flexible gold bag with a titanium head9 to be placed inside the stainless-steel high-pressure reactor. The gold surface is corrosion-resistant against acidic or reducing solutions and gases. The titanium surface is also highly inert when passivated thoroughly to form a continuous titanium dioxide layer. During sampling from this reaction cell through a connected titanium sampling tube, the gold bag shrinks in volume. The system's internal pressure is maintained by pumping the same volume of water, as is withdrawn by sampling, into the stainless-steel high-pressure reactor accommodating the reaction cell. The sample inside the reaction cell is kept in motion by rocking or tilting the high-pressure reactor by more than 90° during the experiment.
The reaction cell consists of the parts depicted in Figure 1: the gold bag, titanium collar, titanium head, stainless steel washer, titanium compression bolt ring, titanium sampling tube with stainless glands and collars for the high-pressure coned and threaded connections on both sides, and the titanium valve. The gold bag is a cylindrical gold (Au 99.99) cell with a wall thickness of 0.2 mm, an outer diameter of 48 mm, and a length of 120 mm.
All titanium parts are custom-made by the workshop from titanium grade 2 rods. The dimensions of the collar, head, washer, and compression bolt ring are visible in Figure 2. The titanium sampling tube is a capillary of titanium with an outer diameter of 6.25 mm and a wall thickness of 1.8 mm, resulting in an inner diameter of 2.65 mm. It is fixed into the titanium head and the titanium valve by high-pressure coned and threaded connections ensuring a seal of titanium-against-titanium surfaces. The high-pressure titanium valve is equipped with a slow opening stem to allow for very controlled opening or sampling even at high pressure. This system was used in numerous studies10,11,12.
1. Preparation of the medium and inoculation of the microbial culture
2. Preparation of the gold-titanium reaction cell and the high-pressure reactor
3. Filling and assembling the gold-titanium reaction cell under anoxic conditions
4. Assembling the high-pressure reactor with the reaction cell
5. Starting the experiment
6. Sampling the high-pressure reactor in operational mode
7 . Analysis of fluid sample
NOTE: Only the steps for the less common photometric ferrozine assay (i.e., section 7.1) are described here in detail and are mentioned in the video, because the other steps are standard operation procedures in microbiology.
Results of the high-pressure reactor experiment with the special gold-titanium reaction cell show that the microbial mixed culture of acidophiles oxidized sulfur and reduced ferric iron to ferrous iron (Figure 3).
At both 1 bar or 100 bar pressure conditions, the cultures had a lag phase when grown in the gold-titanium reaction cell. After that period, a rapid increase in the ferrous iron concentration from approximately 9 mM to 31 mM occurred in the culture grown at 1 bar. Over the incubation time of 22 days, ~31 mM and 13 mM of ferrous iron were detected in the assays at 1 bar and 100 bar, respectively. This clearly demonstrates that the microbial cells were active at 100 bar, but their ferric iron-reducing activity was significantly lower at elevated pressure. Abiotic control experiments conducted in Hungate tubes and serum bottles did not show ferric iron reduction at 1 bar and 100 bar.
The scanning electron microscopy images (Figure 4) show rod-shaped cells grown in experiments at low and high pressure. No significant change in the cell morphology was observed at 1 bar versus 100 bar. However, cell growth was obviously inhibited by the elevated pressure, as the cell number was 1.3 x 108 cells/mL at 1 bar in comparison to 4.5 x 107 cells/mL at 100 bar7. These data are comparable with the tests done in Hungate tubes7. Thus, the flexible gold-titanium reaction cell itself had no effect on cell growth and was suitable for microbial growth tests.
The results show that bioleaching microorganisms are active even at a high pressure of 100 bar, which is highly relevant for in situ biomining because such conditions occur in deep ore deposits at a depth below 1,000 m7.
Figure 1: Overview of the reaction cell parts. From bottom to top: the gold bag, titanium collar, titanium head, washer, titanium compression bolt ring, titanium sampling tube with stainless glands and collars for the high-pressure coned and threaded connections on both sides, and the titanium valve with an adapter for connecting a Luer Lock syringe. Please click here to view a larger version of this figure.
Figure 2: Dimensional drawings of the titanium parts machined from rods of titanium grade 2. Please click here to view a larger version of this figure.
Figure 3: Changes of the ferrous iron concentrations in the gold-titanium reaction cell with the ferrous iron-oxidizing culture. Cells were cultivated anaerobically at 30 °C. Please click here to view a larger version of this figure.
Figure 4: Morphology of the ferrous iron-oxidizing culture grown at 1 bar and 100 bar. Cells were cultivated anaerobically at 30 °C. Please click here to view a larger version of this figure.
The presented method for high-pressure experiments of microbial reactions within acidic solutions was a powerful tool to simulate deep subsurface geomicrobiological processes in a laboratory environment.
There are numerous manual work steps involved, some of which require special attention. As a general note, no excessive force must be used when assembling the individual parts of the flexible gold-titanium cell and the reactor head (sections 3 and 4). If the manufacturer's specifications (e.g., for maximum pressure, temperature, torque) are ignored, leakage and/or material failure may result.
Cleaning of the gold and titanium parts (section 2.2) is an indispensable work step, not only for this experiment, but especially for experiments involving (in-)organic reactions. Remnants from previous experiments in the gold cell may cause unwanted reactions and therefore biasing of results. When the assembled gold-titanium cell is installed in the reactor head, it is best to work quickly and precisely, because at this time small amounts of oxygen could enter the gold cell. Closing the sampling valve before leaving the glovebox is a good first measure to minimize the exchange between the ambient atmosphere with the interior of the gold cell.
Once the reactor is placed in the rocking device, it is important to set the rocking motion speed to ~170°/min. If the high-pressure reactor moves too fast, rupture of the gold cell may happen due to gravitational effects or the sharp edges of sediment or rock samples when used.
This method can be used in additional research fields. The flexible gold-titanium reaction cell has the potential to be used for a diverse set of scientific investigations9 studying reactions at elevated pressure and temperature and in highly corrosive fluids or gases.
Microorganisms in the deep subsurface at temperatures above 70 °C in the presence of mineral surfaces may stimulate the production of molecular hydrogen or organic acids like acetate even under elevated pressure16. These products, and other compounds, might induce elevated microbial activity during in situ bioleaching processes, in addition to the sulfur compounds investigated in this study.
Applications include the determination of solubility of gases and ions in aqueous fluids, geochemical reactions at conditions of hydrothermal vent systems17, the quantification of isotope fractionation18, geochemical reactions during CO2 sequestration19, abiotic processes during the formation of oil and gas in source rocks20, and microbial reactions at elevated pressures in the subsurface21 as in the present study.
The authors have nothing to disclose.
We thank Robert Rosenbauer (USGS, Menlo Park) in sharing his expertise on the flexible gold-titanium reaction cells, and Georg Scheeder (BGR) for his input during the initial phase of setting up the modified system in Hannover. We would like to thank many scientists (including Katja Heeschen, Andreas Risse, Jens Gröger-Trampe, Theodor Alpermann) using the setup in Hannover in numerous projects that contributed in little improvements along the way and Christian Seeger for developing the rocking device for the high-pressure reactors. We thank Laura Castro (Complutense University of Madrid) for SEM observations. And finally, we would like to express our gratitude to Nils Wölki for producing this high-quality video for the article. This work was supported by the European Union Horizon 2020 project BIOMOre (Grant agreement # 642456).
Acetone | Merck | 100013 | |
CaN2O6 | Fluka | 31218 | |
Conax compression seal fittings | Conax Technologies | PG2-250-B-G | sealant could be selected according to temperatures in experiment |
Copper paste | Caramba | 691301 | |
Copper paste | CRC | 41520 | |
CoSO4x7H2O | Sigma | 10026-24-1 | |
CrKO8S2x12H2O | Roth | 3535.3 | |
CuSO4x5H2O | Riedel de Haen | 31293 | |
Disposable cuvettes | Sigma | z330388 | |
Ethanol absolute | Roth | 9065.3 | |
FE-SEM | JEOL | model no. JSM-6330F | |
Ferrozine | Aldrich | 180017 | |
Fe2(SO4)3x7H2O | Alfa Aesar | 33316 | |
FeSO4x7H2O | Merck | 103965 | |
Gold cell | Hereaus GmbH | manufactured according to dimensions supplied by customer | |
High-pressure reactor | PARR Instruments | model no. 4650 Series | reactors from other vendors could be used, too |
High-pressure syringe pump | Teledyne ISCO | DM-100 | |
HCl | Roth | 6331.3 | |
HNO3 | Fluka | 7006 | |
H3BO3 | Sigma | B6768 | |
KCl | Sigma | P9541 | |
KH2PO4 | Merck | 104873 | |
L-(+)-Ascorbic acid/Vitamin C | Applichem | A1052 | |
Light microscope | Leica DM3000 | ||
MgSO4x7H2O | Merck | 105886 | |
(NH4)2SO4 | Sigma | A4418 | |
NaMoO4x2H2O | Sigma | 331058 | |
NaO3Sex5H2O | Sigma | 00163 | |
NaO3V | Sigma | 590088 | |
Na2SO4 | Merck | 106649 | |
Na2WO4x2H2O | Sigma | 72069 | |
NiSO4x6H2O | Sigma | 31483 | |
Omnifix Luer | BRAUN | 4616057V | |
pH meter | Mettler Toledo | ||
Redox potential meter | WTW | ORP portable meter | |
Safe-Lock Tubes, 2 mL | Eppendorf | 0030120094 | |
Serum bottle | Sigma | 33110-U | |
Spectrophotometer | Thermo Scientific | model no. GENESYS 10S | |
Sterican Hypodermic needle | BRAUN | 4657519 | |
Stoppers | Sigma | 27234 | |
Sulfur powder | Roth | 9304 | |
Thoma Chamber | Hecht-Assistent | ||
Titanium parts of reaction cell | Titan-Halbzeug GmbH | 121-238 | manufactured by workshop at BGR according to dimensions supplied from Titanium grade 2 rods from Titan-Halbzeug GmbH |
Titanium valve | Nova Swiss Technologies | ND-5002 | |
Whatman membrane filters nylon | Sigma | WHA7402004 | |
ZnSO4x7H2O | Sigma | Z4750 |