Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

The overall goal of this experimental setup is to study the fate of trace metals under sulfate and/or iron-reducing conditions induced by microbial activities. This method can help answer key questions in the biogeochemistry field such as interactions between key biogeochemical cycles like mercury and arsenic. The main advantage of this technique is that due to the continuous water flow, the dynamics of the biogeochemical reactions and lability of trace elements are dissociated and accessible through the sampling ports.

And so, this method can provide insight into the fate of mercury and arsenic as demonstrated here ending to iron oxides. It can also be applied to other trace elements or carrier substrates. Demonstrating the procedure will Hafida Tris, a technician from our laboratory.

To begin this procedure, add 500 milliliters of ultrapure water to a glass reactor equipped with a magnetic stir bar. Add 50 grams of iron(III)chloride hexahydrate while stirring. Manually add approximately 50 milliliters of a 10-molar solution of sodium hydroxide to begin precipitation of ferrihydrite.

Next, adjust the pH to six and continue stirring for one hour to stabilize. For arsenic-spiked hydroxides and oxyhydroxides, prepare 100 milliliters of arsenic trioxide at a concentration of 10 grams per liter. Then, add 70 milliliters of this solution to the iron oxide solution.

Continue stirring for three hours. After this, centrifuge at 2, 000 G for 20 minutes. Remove the supernatant and resuspend the pellet with 50 milliliters of ultrapure water.

Repeat the centrifugation ane resuspension two additional times. Recover and store the humid hydroxides and oxyhydroxides as outlined in the text protocol. Keep four grams of silica gel in 40 milliliters of 7%potassium hydroxide on a hot plate stirring with a magnetic stir bar until dissolved.

Add 60 milliliters of ultrapure water. Cool the solution on ice to about 20 degrees Celsius to avoid the gel setting too quickly. Titrate the silica gel mixture with phosphoric acid 20%to pH 7.5.

Then, quickly mix the liquid silica gel with 320 grams of sterile sand previously mixed with 18.3 grams of either mercury-spiked or arsenic-spiked iron oxides before it solidifies. Using a spatula, break up the gelified mixture. To begin setting up the glass columns, connect the water jackets to a water-cooling system.

Set the system to maintain an average temperature of 20 degrees Celsius. Cut lengths of PTFE tubing with a three-millimeter inner diameter making sure there is sufficient length for the column inlet and outlet. Then, add a layer of damp rock wool to the column from the top.

Add 320 grams of sterile sand. Add the final layer consisting of the previously mixed sterile sand and spiked hydrated amorphous iron oxides fixed in the silica gel matrix. Next, connect the inflow to peristaltic tubing.

Connect the other end of the peristaltic tubing to the water medium supply. Connect an ascendent flow of ultrapure sterile water continuously bubbled by nitrogen gas at low velocity of approximately two milliliters per hour. After this, cover the column with aluminum foil to block out the light.

After two weeks, a black-colored precipitate is observed at the interface between the two layers of sand. This black zone progressively invades the top iron hydroxide and oxyhydroxide-enriched zones with the entire upper layer becoming black by the end of the experiment. After 35 days of continuous running, the measured levels of sulfate decrease followed by a transient increase in the total dissolved iron.

From day 60 onward, the total dissolved arsenic is seen to rise significantly. On day 54, the physical and chemical parameters are profiled. While the pH remains close to pH seven along the column, the redox potential changes significantly with values nearing negative 200 millivolts in the iron-depleted zone where sulfate reduction is occurring and nearing negative millivolts at the top where bacterial and chemical iron reduction is occurring.

Dissolved sulfate concentrations reach to approximately 20 milligrams per liter in the bottom layers and decrease to under one milligram per liter in the iron-rich zone. Sulfate also decreases sharply at the interface between iron-deprived and iron-rich zones indicating a peak of sulfate reduction activity. Meanwhile, arsenic is detected in the upper zone while thioarsenate species are seen closer to the interface and thiosulfate is in the bottom iron-deprived layer.

These concentration profiles indicate a peak of sulfate reduction activity at the interface between iron-deprived and iron-rich layers. Following this procedure, other methods like scanning electron microscopy, Raman spectroscopy or activity assays can be performed in order to answer additional questions such as which new minerals are formed or which bacterial communities have developed. Don’t forget that working with mercury and arsenic can be extremely hazardous and precautions such gloves and fume hoods should be used while performing this procedure.