The dissolution of HgS within the New Idria AMD system occurs solely in the oxic zone. All anoxic microcosms showed a minor spike in Hg due to residual oxygen in the AMD water that was not purged prior to the start of the experiment (Fig. 3). Once the oxygen in the anoxic microcosms was consumed, the Hg concentrations either leveled off or dropped below detection. An increase in Hg released from abiotic and inactivated microcosms incubated in an oxic environment is most likely due to the organic matter contained in solution and mine waste materials (discussed later).
One explanation for the increased Hg concentration in microcosms is the presence of polysulfides, either present in solution or produced by the micro-organisms. Due to the low pH of the system and the lack of an increase in Hg in anoxic microcosms, polysulfides are not considered the driving factor for HgS dissolution in our experiments. Based on literature findings, polysulfides (and other reduced sulfide species) should not be stable in the New Idria AMD system because polysulfide formation and stability require neutral to basic pH as well as anoxic conditions (Paquette & Helz, 1995, 1997; Jay et al., 2000). Although it is well known that biofilms can create microenvironments different than the surrounding environment, the fact that anoxic microcosms did not show any increase in Hg during the HgS dissolution experiments indicates that the AMD microbial community is not capable of producing an anoxic environment required for stable polysulfides or reduced sulfur species in the time frame of our experiments.
An explanation for why HgS(s) is degrading in the microcosms based solely on thermodynamics is not adequate. Both the cinnabar and metacinnabar polymorphs of HgS are quite insoluble, with solubility products of 10−54 and 10−52, respectively (Faure, 1991; Krauskopf & Bird, 1995). Based on thermodynamic calculations of conditions within the microcosms, sulfide concentrations would need to drop to levels below 10−33 m in order to have HgS undersaturated at the New Idria site. A sulfide concentration of 10−33 m is unrealistic for any natural system and would result in supersaturation of the AMD solution with respect to HgS, making the abiotic release of Hg thermodynamically unfavorable. A study by Bura-Nakić et al. shows that even in oxic waters that are at or near O2 saturation, concentrations of total reduced sulfur species are ~10−8 m (Bura-Nakic et al., 2009). Several research groups have published values for HgS stability in the presence of H2S and its deprotonated forms (Paquette & Helz, 1995, 1997). Due to the low pH of the system, the dominant sulfide species will be H2S. Calculated Hg concentrations, using background sulfide concentrations at the New Idria site, necessary for HgS(s) equilibrium would be ~10−12 m, which is significantly lower than the 10−8.5 m detected at the start of the experiment. Nearly, all of these chemical reactions require H2S as a reactant, which would result in HgS becoming more stable as H2S concentrations decrease. Because the initial experimental conditions were already supersaturated with regards to both cinnabar and metacinnabar, a purely abiotic thermodynamic explanation for the increase in Hg concentration in the microcosm is incorrect.
Another possible explanation for enhanced HgS dissolution at New Idria is that the S-oxidizing microbe is oxidizing H2S in solution to sulfate, resulting in a drop in sulfide concentrations and leading to dissolution of HgS. This hypothesis suggests that every S-oxidizing bacterium in the environment would be capable of dissolving HgS. It has been assumed that S-oxidizing bacteria can slowly dissolve HgS, but there is little to no experimental evidence supporting this assumption (Wood, 1974; Madigan et al., 2003). Research by Baldi and Olson using Hg-sensitive and Hg-tolerant Thiobacillus ferrooxidans strains with a mixture of pyrite and cinnabar showed that this Hg-tolerant bacterium was not capable of using cinnabar as the sole energy source (Baldi & Olson, 1987). Although experiments with the Hg-tolerant Thiobacillus ferrooxidans strain in a mixture of pyrite and cinnabar showed that Hg is released into solution as both Hg(II) and Hg0, no Hg was released in experiments with cinnabar only. The release of Hg into solution was potentially attributed to back reactions of pyrite oxidation with HgS, since pyrite oxidation and Hg-release were found to be linked; however, no mechanism for this reaction was proposed (Baldi & Olson, 1987). Although not directly related to the work of Baldi and Olson, Wiatrowski et al. showed that magnetite can reduce Hg(II) to Hg0 in anaerobic systems (Wiatrowski et al., 2009).
The most likely hypothesis for HgS degradation in the New Idria AMD system, and potentially other inoperative Hg mine sites in California, is direct oxidation of the sulfide in the HgS crystals. Binding constants between Hg and S vary greatly depending on the oxidation state of S. As sulfur becomes more oxidized, the binding constants reduce greatly as seen in the following sequence: HS− (1037.71), S2O32− (1029.93), SO32− (1022.85), and SO42− (101.34) (Smith & Martell, 2004). During oxidation of sulfide in HgS to sulfate, Hg should be released into solution from the mineral surface. The presence of sulfate at levels >2600 mg L−1 in the New Idria AMD system (Table 2) clearly demonstrates the oxidation of sulfur. In order to evaluate the role of S-oxidizing bacteria in HgS dissolution at the New Idria AMD site, we isolated a Thiomonas sp. strain from the tailings pond sediment. Because the obtained isolate had a Hg tolerance of >20 mg L−1 Hg, we evaluated the potential of this strain being responsible for the observed dissolution of HgS in the AMD system. However, when testing this Thiomonas sp. strain for HgS dissolution, we did not observe an increase of Hg(II) in the microcosms, suggesting that other members of the microbial community might be responsible for the observed biotic HgS dissolution. Because the media for the isolate experiments was created in the laboratory instead of using the water from the New Idria AMD system, it is highly probable that a constituent in the AMD water not included in the laboratory medium is necessary to facilitate Thiomonas sp. induced HgS dissolution. Another possibility for explaining HgS dissolution is that the process is indirectly coupled to microbial Fe-cycling. The experiment using the AMD microbial community and the settling pond water showed that Fe-cycling was important in those microcosms. The potential of Fe-cycling to help the micro-organisms with the dissolution of HgS is intriguing and requires additional work. One scenario for Fe-cycling impacting HgS dissolution is the creation of Fe(III) by microbial oxidation of Fe(II) in Fe-sulfides by back reaction with the sulfide in HgS, resulting in the oxidation of sulfide and concomitant dissolution of the HgS. Though this scenario has merit, currently there is no evidence for this occurring in the literature.
Regardless of the actual mechanism for HgS dissolution, it is evident that the New Idria microbial consortium has a profound influence on the solubility of HgS, both cinnabar and metacinnabar. Although the bioreactors are far from equilibrium, it is useful to compare the activity quotient for HgS dissolution in the AMD system with that of the idealized solubility constant for both HgS polymorphs. Activities for both sulfide and Hg2+ were calculated using both Geochemist's Workbench and Visual Minteq by using the sulfide concentration (metacinnabar alive, Day 3 sample, 0.1 μm), Hgtot (metacinnabar alive, Day 3 sample, 18.7 μg L−1), and the solution chemistry for the system (Table 2) (Bethke, 2002; Gustafsson, 2009). Using these calculated activities, a LogQ for HgS dissolution of −23.5 was calculated. Comparing activity quotient to the idealized HgS solubility for cinnabar and metacinnabar (LogK = −54 and −52, respectively), the New Idria biofilm is capable of increasing the solubility of HgS by 28.5–30.5 orders of magnitude for the bioreactor experiments.
In contrast with the experimental results described above, which showed that the bacterial consortium in the AMD system is mainly responsible for the greatly enhanced solubility of cinnabar and metacinnabar in the live oxic experiments, release of Hg from the abiotic and killed oxic controls is most likely due to organic matter from solution and the mine waste material used in the experiment. It is possible that outer membrane proteins not denatured during the gamma irradiation were able to dissolve the HgS, but because abiotic and killed controls mirrored each other closely, the most likely cause of HgS dissolution in these microcosms is due to trace amounts of thiol groups associated with the organic matter (Fig. 3). Studies by numerous research groups have shown that organic matter rich in thiol groups such as humic and fulvic acids can actively dissolve HgS (Ravichandran et al., 1998, 1999; Waples et al., 2005). Because cell lysate, which is assumed to be the source of the organics in this system, have thiol containing functional groups, residual organic material in the system likely plays a minor role in HgS dissolution.