Using Flexible Gold-Titanium Reaction Cells to Simulate Pressure-Dependent Microbial Activity in the Context of Subsurface Biomining

Biomining describes applied Bioleaching for the extraction of copper and other pressure metals from rocks by dissolving metal sulfides with the help of acidophilic bacteria. For example, copper is coopashefa ore can be extracted with this bacteria by dissolving the sulfides. And copper sulfate rich solution is then generated from which the copper can be easily extracted.

In situ biomining is the extraction of metals with the help of bacteria without the need for excavation and mining activities underground or in an open pit. Instead, an iron-rich acidic solution is pumped down in the rock formation, metal sulfides are getting dissolved and the metal-rich solution is pumped back to the surface from which metals can then be extracted. In this particular experiment, there's a high-pressure reactor cell which simulates the conditions down in the deep formation.

Bacteria are studied and their activity related to iron-reduction coupled to the oxidation of sulfur compounds, and in particular we're interested in to see how active the cells are under high pressure, and how their rates are changing under the pressure. For the experiments, a high-pressure reactor, as the one depicted here, can be used, and can simulate pressure conditions up to 350 bars. The bottom of a high-pressure reactor consists of a reactor vessel, which can contain a fluid sample with microbial culture.

The reactor head offers a diverse array of connections for safety measures and monitoring sensors. For example, temperature or pressure inside the reactor. Most high-pressure reactors are made of stainless steel.

This material offers high resilience and good machining properties. However, for certain applications such as experiments with acidic or highly-reducing aqueous solutions, the corrosion resistance of the stainless-steel surface might not be adequate. One way to avoid this is to insert a liner into the reactor vessel.

Here, a liner made from tefflon. Tefflon has a high corrosion resistance, but it comes with a higher risk of contamination, as it cannot be sterilized by autoclaving and iron faces might precipitate onto its surface. Another liner material that can be used is quartz glass.

It is easy to clean, can be sterilized by autoclaving, and is less affected by acidic or reducing aqueous solutions Even though liner material can help to prevent unwanted reactions of a sample with a stainless-steel reactor wall, several problems remain. If a corrosive gas is formed, for example hydrogen sulfide, this gas might react with the uncovered surface of the reactor head sitting above the liner. Another disadvantage is that is not possible to withdraw sample from the reactor without changing the pressure.

To overcome these limitations, we used a special reaction cell, a flexible gold bag with a titanium head. The gold surface is corrosion-resistant towards acidic or reducing solutions and gases. The titanium surface is highly inert, too.

When to form a continuous titanium dioxide layer, here visible in dark blue. During sampling, the gold bag is shrinking in volume. The volume of the gold bag should not be reduced by more than 50%to prevent formation of sharp kinks and edges.

Parts shown here are the individual pieces of the gold bag experiment's internal setup. One can see, from the bottom to the top, the flexible gold bag reaction cell, the titanium sealing system, consisting of a titanium head, washer, and compression bold ring, and the sampling tube and valve to access the reaction cell during operation mode. Now, we transfer the sample into the gold titanium reaction cell.

Firstly, open and unlock the empty chamber, and load all the inbound material onto the movable tray. Close and lock the front cover. After evacuation of the anti-chamber, wear the glove pair and get closest to the inner cover.

Unlock and open the inner cover to remove the inbound material from the movable tray. Unwrap the clean gold bag and secure its stand. Open the serum bottle which contains 100mL of bacterial culture and elemental sulfur.

Gently shake the serum bottle and transfer the bacterial culture into the gold bag. Insert the titanium head with assembling tube into the titanium cover on the gold bag. Then, slide the washer and the compression bold ring over the titanium sampling tube.

Fasten the six elm screws to the same extent to ensure an even pressure distribution of the titanium hat on the uppermost rim of the gold bag in the titanium cover. This is the sealing surface of the gold titanium reaction cell. Re-install the sampling valve at the top of the titanium tube.

Tighten the connection hand tight. Then, make sure to close the valve. Now, the high-pressure reactor's core piece is fully assembled and can be installed in the reactor hat.

Now, the installation of the high-pressure reactor can take place. This comes with a very short exposure of the open-end of the sampling tube to the surrounding atmosphere, as the sampling valve has to be removed to get the tube through the screw seal in the reactor head. Stop removing the sampling valve and screws from the sampling tube.

Guide the tube through the reactor head, slide the large screw over the tube, and fasten the small cover. Now the reaction cell assembly cannot slide back through the reactor head and both hands are free to re-install the sampling valve. Remove the reactor head from the bench vice to install it onto the reactor vessel.

The reactor head, including the thermal couple, has to be carefully placed on the reactor vessel, not to damage the gold bag or the thermal couple. Finally, the robust cover, fitted with the split ring and compression bolts, is fixed around the reactor head and vessel to appropriately seal the system. The high-pressure reactor is carefully mounted in the rocking device to avoid potential injuries, especially bruising of fingers.

The high-pressure reactor is fixated by two clamps slided over a pair of screws. Washers and screw nuts hold the clamps in place. Connect the control units for the thermal couple and the pressure sensor.

It is important to ensure the sufficient length of the wires for the rocking motion, while preventing its content to heat its surfaces. Finally, slide the heating element over the reactor vessel, and tighten its screw-lock. The water to pressurize the system is taken from a reservoir by a high-pressure pump.

It is transferred through stainless-steel capillaries into the high-pressure reactor. Rocking of the high-pressure reactor guarantees thorough mixing of the reaction cell. For example, of the gas, fluid, and/or solid phases in it.

The slower rocking speed is important to prevent damage of the gold bag by fast-moving solids, or by the formation due to gravity effects on the flexible gold at elevated temperatures. Our system is able to rotate about on the green-and yellow-marked angle on the indicator, which is close to 180 degrees. Experimental parameters are simultaneously logged by a software.

To take a sample, a 5 milliliter syringe is attached to the little lock interface of the sampling valve at the top of the high-pressure reactor. Open the valve carefully. The liquid sample is pushed into the syringe automatically by the over-pressure inside the reaction cell.

Close the valve after the sample volume reaches one milliliter. Detach the syringe. The sample in the syringe is immediately transferred into a two-milliliter tube for further processing.

Determination of the microbial reduction of ferric iron to ferrous iron in the reaction cell is achieved by photometric analysis. A series of ferrous iron standard solutions containing a purple colored concentration dependent ferrous iron complex and indicator, iron ferrocene, serves as a calibration agent. Results of the high-pressure reactor experiment with special gold titanium reaction cell show that the bacteria oxidizes sulfur and ferric iron to ferrous iron and that there is a significant effect on the pressure.

In the figure, the increase of ferrous iron concentrations over the period of 22 days is shown for the experiment carried out at pressures of one bar and 100 bar. Approximately, 31 and 13 millimole of ferrous iron were detected in the assays at one bar and 100 bar, respectively. This clearly demonstrates that microbial cells were active even at 100 bar, but that their ferric iron reducing activity was significantly lower at high-pressure.

The scanning electron microscope image shows rod-shaped cells grow in experiment at low and high pressure. The flexible gold titanium reaction cell developed by Seyfried and coworkers in 1979 has the potential to be used for a diverse array of scientific investigations, and all of them including reactions with corrosive gases and fluids. One application could be determination of the solubility of irons and gases at high pressures and temperatures.

Another one might be the determination of reactions going on in a during the formation of oil and gases. And the third, as in the study here, could be investigating microbial reactions at elevated pressures and temperatures.