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

Summary

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Abstract

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg°. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.

Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.

To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Introduction

Understanding and predicting trace element (TE) mobility and biogeochemistry in the environment is essential in order to monitor, develop, and apply appropriate management decisions for polluted sites. This especially applies in the case of toxic TEs such as arsenic (As) and mercury (Hg). The fate and speciation of these TEs in soil or aquifers are closely related to physico-chemical conditions, such as Eh and pH, but also to microbial activities that can play either a direct role on speciation or an indirect role on mobility.

Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). This affects As toxicity, since As(III) is the most toxic form of As, and mobility, since As(III) is more mobile than As(V), which can readily adsorb to iron (oxy)hydroxides or organic matter^1,2. Likewise, bacteria are strongly involved in mercury cycling, either through its methylation, mainly by sulfate and iron reducing bacteria^3,4, forming the neurotoxin monomethyl mercury (readily bioaccumulated in the food chain), or through its reduction to volatile elementary Hg (Hg°)⁵.

Both As and Hg fates are also strongly linked to soil or aquifer composition, since compounds such as organic matter or iron (oxy)hydroxides can influence their sequestration and bioavailability. As(V) adsorbs well to iron (oxy)hydroxides⁶, whereas Hg has a very high affinity for organic matter (OM; mainly for thiol groups) but also for colloidal iron or manganese (oxy)hydroxides in OM depleted environments^7,8,9,10,11.

Bacterial activities can then influence the fate of TEs adsorbed to (oxy)hydroxides or organic matter through the reduction of iron (oxy)hydroxides or the mineralization of organic matter. Direct iron reduction by bacteria is the dominant pathway for iron reduction in sulfur depleted zones^12,13, Fe(III) being used as a terminal electron acceptor, whereas indirectly, Fe(III) can be reduced to Fe(II) by sulfide formed by a bacterial sulfate reduction¹⁴. Moreover, the presence of sulfate can also modify Hg and As speciation through the formation of complexes such as thio-arsenates¹⁵ with As or metacinnabar with Hg.

Thus, a better understanding of the impact of iron and sulfate cycling on the fate of TE, such as Hg and As, could help us to better manage contaminated sites and maintain soil and water quality. Data could also contribute to reinforcing existing metal-mobility models. Microbial Fe(III)-reduction^16,17,18 can cause the desorption of TE. Theoretically, the indirect reduction of iron (oxy)hydroxides by sulfide produced by the microbial reduction of sulfate could also impact TE mobility. However, the extent and kinetics of these reactions are generally studied in batch homogenous systems or batch microcosms^16,18,19,20. The drawback of batch experiments is the lack of dissociation of the occurring phenomena; indeed, activity is based on and limited by the resources present in the batch and only gives a final result of the shifts in speciation and adsorption. Using a column approach enables the renewal of inflowing media and the monitoring of the fate of TE over time and space. These conditions are more realistic when compared to an aquifer, where real phenomena are closely linked to continuous percolation conditions. Moreover, heterogeneous iron (oxy)hydroxide occurrence in aquifer sediments is common^21,23, and the spatial changes in the mineralogical and chemical composition of the solid phases certainly drives microbial activities.

To elucidate the influence of these heterogeneities on geo-microbial phenomena and the fate of iron-associated TE, we developed a laboratory, a continuously-fed column representing a simplified model aquifer. The column is filled to create an iron-depleted zone at the column entrance and an iron-rich zone at the top. Regular sampling ports enable us to study each zone individually as well as interface-associated phenomena. An example of the application of this experimental device for the study of Hg fate and speciation is already available²⁴. Here we give a detailed description of the experimental setup and a second example of its application focused on the behavior of As in contaminated aquifers.

Protocol

1. Experimental Preparation

1. Acid-wash all materials (glass, polytetrafluoroethylene (PFTE)) in contact with samples (5 days in 20% nitric acid (HNO₃) v/v) followed by 5 days in hydrochloric acid (HCl) 10% v/v). Rinse several times with ultra-pure water and dry under a laminar flow hood prior to use.
2. Use polyethylene gloves (or similar) and a fume hood for all steps involving chemicals.

2. Prepare Hg and As Spiked Amorphous Iron Oxides

1. Prepare approximately 20 g of ferryhydrate (Fe(OH)₃): dissolve 50 g of FeCl₃-6H₂O in 500 mL of ultra-pure water (resistivity >18 MΩ cm^-1) under agitation in a glass reactor with a stainless-steel impeller or magnetic stirrer. Initial pH is <2.
2. Manually add a solution of 10 M NaOH to precipitate ferryhydrate.
   NOTE: Approximately 50 mL will be required to precipitate all the iron (oxy)hydroxides. Adjust the pH to 6 and maintain agitation for 1 h to stabilize.
   1. For Hg-spiked (oxy)hydroxides: prepare 10 mL of HgNO₃ at 10 g L^-1 and add 350 µL to the (oxy)hydroxide solution.
      NOTE: This will yield a final Hg content in the wet (oxy)hydroxides of ~4 µg g^-1 (oxy)hydroxides.
   2. For As-spiked (oxy)hydroxides: prepare 100 mL of As₂O₃²⁵ at 10 g L^-1 and add 70 mL to the iron-oxide solution. This will yield a final As(III) content of ~70 mg/g (oxy)hydroxides.
3. Leave under agitation with a stainless-steel impeller or magnetic stirrer for 3 h and then centrifuge for 20 min at 2,000 x g. Remove supernatant and re-suspend the (oxy)hydroxides in 500 mL of ultra-pure water. Repeat the centrifugation and rinsing steps twice. Recover the humid (oxy)hydroxides (solids have a moisture content of 85-90% wt.) and store at 4 °C until use.
4. Sterilize humid Hg or As-spiked iron oxides by gamma radiation, with a minimum absorbed radiation dose of 25 kGy.
5. Control Hg and As (oxy)hydroxides contents
   1. Determine Hg contents of the pellet²⁶.
      NOTE: We found 3.90 ± 0.08 µg Hg g^-1 solid. Thus, the total amount of mercury added to each column in the 18.3 g of iron oxides was 71.4 ± 1.51 µg.
   2. Determine As contents in the pellet. Use hot acid mineralization (8 mL of 5 N HCL for 4 h at 50 °C) and analyze by Atomic Adsorption Spectrometry (AAS).
      NOTE: We found 70 mg As g^-1 solid. Thus, the total amount of As added to the column in the 18.3 g of iron oxides was ~1.3 g.

3. Prepare Silica Gel and Sand Matrix

NOTE: A loose silica gel matrix was used to stop the fine iron oxides migrating from the sand/iron oxide mixture under the water flow. The final gel matrix was 6% silica gel so as not to form a block but just to loosely aggregate the oxides.

1. Prepare a 10% silica gel mixture by heating 4 g of silica gel in 40 mL of a solution of 7% KOH on a hot plate, stirring with a magnetic stir bar until dissolved.
2. Add 60 mL of ultra-pure water then cool the solution to ~20 °C. Quickly titrate with diluted phosphoric acid (20 %) to pH 7.5. Then quickly mix the liquid silica gel with 320 g of sterile sand and the previously added 18.3 g of Hg-spiked or As-spiked iron oxides before it solidifies.
3. Break up the "jellified" mixture by mixing with a spatula and keep sterile before use in step 4.

4. Setup the Column

1. Use glass columns with a water jacket cooling system (internal volume = 400 mL, height = 30 cm, diameter 3.5 cm) and five silica septa set regularly (every 5 cm) along the columns to enable you to sample all along the column.
2. Cut PTFE tubing (PTFE int Ø 3 mm) to ensure sufficient length at the column inlet and outlet. Connect the inflow to peristaltic tubing, which in turn is connected to the water/medium supply.
3. Sterilize all materials (glass, tubing) by autoclaving (1 h at 110 °C).
4. Attach column vertically.
5. Connect the water jacket to a water cooling system to maintain an average temperature of 20 °C.
6. Fill column from the top as follows:
   - A layer of damp rock wool to avoid solid loss;
   - 320 g of sterile sand (Fontainebleau sand, D₅₀ = 209 µm);
   - 320 g of sterile sand mixed with 18.3 g hydrated amorphous iron oxides spiked either with Hg or As (see step 1) and fixed in a 6% silica gel matrix (see step 2).
7. Attach column vertically and connect ascendant flow of continuously N₂ bubbled ultrapure sterile water at low velocity (~2 mL h^-1).
8. Cover the column with aluminum foil to protect from the light.

Figure 1: Sketch and photo of the column setup. Please click here to view a larger version of this figure.

Results

Example 1. Impact of iron reduction of As mobility and speciation

The As column was directly inoculated with groundwater from a site presenting an As concentration higher than the drinking standards (Bracieux, Loire et Cher, France). Groundwater was sampled in sterile bottles, and stored at 5 °C until use. The column was fed from the bottom with this water containing the natural endogenous microbial community a...

Discussion

The experimental column setup proved to be a convenient laboratory device to study anaerobic biogeochemical processes in continuous conditions. Continuous column systems allow working in conditions closer to those of real aquifers than slurry batch systems or microcosms. Continuous systems can simulate the movement of groundwater through aquifer sediments.

The most critical step within the protocol is preparing the TE-iron (oxy)hydroxides and the mixture with silica gel and sand, which needs t...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass columns	Beaucaverre, France	Specific request	columns were composed of 3 separate pieces, the main column core with the cooling jacket and the 5 sampling ports (size GL14 with olive) and a top and bottom piece that fits to the main column body and is held in place with a silicone joint and screw (RIN F 40x38 & SVL 42). note: this design was discussed directly with the company. We recommend to find a local glazier.
Septa PTFE/silicone diameter 20 mm	Sigma-Aldrich	508608
PTFE tubing ID 3 mm	VWR	228-0745
Peristaltic pump	Dominique Dutsher SAS	66493
Peristaltic pump tubing LMT 55	VWR	224-2250	Tygon® LMT 55
Fontainbleau sand D50=209 µm	SIBELCO, France
N2 for bubbling	Air Liquide, France
Gamma irradiation	Ionisos, Dagneux, France
Automatic Mercury Analyzer (AMA 254)	Courtage Analyses, France
Varian SpectrAA 300 Zeeman	Agilent
Name	Company	Catalog Number	Comments
Chemicals
HNO3 Supra pur	VWR	1.00441.1000	Manufacturer: Merck
HCL 30% Supra pur	VWR	1.00318.1000	Manufacturer: Merck
Hg(NO3)2	Merck	516953
As2O3	Merck	202673
FeCl3-6H2O	Merck	207926
silica gel	Sigma-Aldrich	336815-500G