Sediment Collection and Preservation A core section from 43.3–43.9 m depth in Dari Satyabandi village (23.785°N, 90.603°E) in Araihazar, Bangladesh, was used in all experiments after manually homogenizing the sand inside an anaerobic chamber containing 95% N2:5% H2 (Coy Lab. Prod. Inc., AALC). Both ends of the PVC liner containing the core were wax-sealed on site immediately after collection in January 2001 (3). The cores were shipped at ambient temperature and stored at 4°C upon their arrival at Lamont-Doherty Earth Observatory’s core repository in May 2001. When the liner was opened inside the anaerobic chamber in July 2005, the sediment was still orange and moist. The presence of water inside the core after four years of storage in a refrigerated and dry environment indicates that the wax seal was intact. Exposure of the core was therefore limited exposed to atmospheric oxygen slowly diffusing through the wax and the PVC liner.
Shewanella Culture Shewanella sp. strain ANA-3 was previously isolated from an As-treated wooden pier pilling in Woods Hole, Massachusetts (14, 16). The strain was initially grown aerobically and then anaerobically in TME media (20) on an incubator shaker (250 rpm) at 28–30°C for 24 hours and 72 hours respectively. The TME media for aerobic growth was prepared by adding 1.5 g of NH4Cl, 0.6 g of NaH2PO4, 0.1 g of KCl, 0.5 g of yeast extract (50 mL of autoclave sterilized stock solution of 10 g L−1), 20 mmole L−1 Na-lactate (20 ml of autoclaved sterilized stock solution of 1mole L−1) and 10 ml trace metal (modified Wolf’s mineral mix) solution (20) in Milli Q water (18.1 MΩ) to make 1 L of solution. The pH was adjusted to 7 by adding 1 g of Na-HEPES to 1 L of the media. For anaerobic growth of Shewanella, an anaerobic electron acceptor was added to TME media. In this case, 1.56 g of Na2HAsO4.7H2O was added to 500 mL aliquots of initial TME media to reach an As concentration of 10 mmole L−1 (16). The TME media was autoclave sterilized prior to use for aerobic and anaerobic growth. Shewanella sp. ANA-3 strain containing plasmid pBBR1- MCS2 was cultured with 50 μg mL−1 kanamycin (further details provided in Supporting Information).
Incubations The orange sands were incubated anaerobically for 3 months with five different types of amendments. Three sets of controls were incubated without Shewanella: the first, labeled C0, contained only sediment and artificial groundwater; the second (Clac) was amended with lactate (Fisher, syrup 60% w/w) to a concentration of 20 mmole L−1; the third (Clac+kan) was also amended with the same amount of lactate and with 50 μg mL−1 of the antibiotic kanamycin. Incubations inoculated with Shewanella sp. ANA-3 were all amended with 50 μg mL−1 kanamycin, one with 20 mmole L−1 lactate (Slac+kan) and the other without (Skan). For all incubations, approximately 5 g of sand was added to sterile 17-mL culture tubes (Hungate 2047-16125) with 10 mL of artificial groundwater previously purged with ultrapure N2 for 2 hours. During the preparation of culture tubes, tubes with sediment were also purged with ultrapure N2 and capped immediately after addition of artificial groundwater. Artificial groundwater of composition similar to that of groundwater associated with the Pleistocene aquifer in Dari Satyabandi (3) was prepared by adding salts of Na+, Ca2+, K+, Mg2+, Cl−, HCO3 −, PO4 3−and SO4 2− to Milli Q water followed by filtration through a disposable sterile system (Corning, 09-761). The initial pH of 7.9 of the artificial groundwater was higher than for groundwater pumped from the orange aquifer in the village (pH 6.5 ± 0.1) (21). The optical density at 600 nm of 0.05 of the Shewanella cultures indicate that the incubations were inoculated with ~ 5 ×108 cells per 10 mL culture tube (14). The tubes were wrapped in black plastic and set horizontally on an orbital shaker (Cole-Parmer, 51401-15) gently rotating at 50–75 rpm over the entire period. Each set of incubations started with 7 replicates that were sampled sacrificially on day 23 in triplicate, on day 42 in duplicate, and on day 92 in duplicate. The sampling intervals were chosen by monitoring the color change of the sand suspension. After measuring the pH (Orion, model 260A), the supernatant from each sampled tube was filtered in the anaerobic chamber through a 0.23 μm filter.
Sediment Reflectance Changes in the color of the sediment were recorded over the course of the incubations with a Minolta CM2500D diffuse spectral reflectance spectrophotometer through the round bottom of the glass tubes. The difference in reflectance (ΔR) between wavelengths of 530 nm and 520 nm was used as a measure of the conversion of Fe(III) to Fe(II) within the solid phase (22).
As and Fe Concentrations and Speciation Concentrations of As(III) in the filtered but unacidified supernatant were determined immediately after opening a culture tube by differential pulse cathodic stripping voltammetry (DPCSV) (23). The detection limit of this method is 0.2 μg L−1 (3 nmole L−1). Dissolved Fe(II) was also measured immediately in the filtered unacidified supernatant by colorimetry (detection limit of 30 μg L−1) using ferrozine (24). The concentrations of total As and Fe in the filtered supernatant acidified to 1% Optima HCl were analyzed by highresolution inductively-coupled plasma spectrometry (HR ICP-MS) (25). The detection limit of the method for As is 0.1 μg L−1 (0.001 μmole L−1).
To analyze the sediment, one 0.5 g aliquot of wet sediment was leached in 10 mL of 1.2 mole L−1 HCl at 80°C for 1 hour, after which the concentration of Fe(II) and Fe(III) in the leachate was measured with ferrozine (22). Another 0.5 g of wet sediment from each tube was leached for 24 hours in 10 mL of 1 M Na2HPO4 (pH 5) and 0.1 M ascorbic acid at room temperature in the anaerobic chamber. This extraction has been shown to release As strongly bound to goethite, presumably by competitive desorption (26). This procedure, previously applied to Bangladesh sediment by Harvey et al. (27), relies on a phosphate concentration that is 10-fold higher than the one used to extract As from aquifer sands in, for instance, Vietnam (28, 29). Subsequent experiments have shown that the addition of ascorbic acid preserves the oxidation state of adsorbed As without causing significant reductive dissolution of Fe oxides (30). The concentration of As(III) in the phosphate extract was also measured by DPCSV (23) and the concentration of total As by HR ICP-MS.