Thioarsenate Transformation by Filamentous Microbial Mats Thriving

Article pubs.acs.org/est

Thioarsenate Transformation by Filamentous Microbial Mats Thriving in an Alkaline, Sulfidic Hot Spring Har̈ tig Cornelia†,* and Planer-Friedrich Britta† †

University of Bayreuth, Environmental Geochemistry, Universitaetsstrasse 30, 95440 Bayreuth, Germany S Supporting Information *

ABSTRACT: Thioarsenates dominate arsenic speciation in sulfidic geothermal waters, yet little is known about their fate in the environment. At Conch Spring, an alkaline hot spring in Yellowstone National Park, trithioarsenate transforms to arsenate under increasingly oxidizing conditions along the drainage channel, accompanied by an initial increase, then decrease of monothioarsenate and arsenite. On-site incubation tests were conducted using sterile-filtered water with and without addition of filamentous microbial mats from the drainage channel to distinguish the role of abiotic and biotic processes for arsenic species transformation. Abiotically, trithioarsenate was desulfidized to arsenate coupled to sulfide oxidation. Monothioarsenate, however, was inert. Biotic incubations proved that the intermediate accumulation of arsenite in the drainage channel is microbially catalyzed. In the presence of sulfide, microbially enhanced sulfide oxidation coupled to reduction of arsenate to arsenite could simply enhance abiotic desulfidation of trithioarsenate and potentially also monothioarsenate. However, we were also able to show, in sulfide-free medium, direct microbial transformation of monothioarsenate to arsenate. Some arsenite formed intermediately, which was subsequently also microbially oxidized to arsenate. This study is the first evidence for microbially mediated thioarsenate species transformation by (hyper)thermophilic prokaryotes.

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INTRODUCTION Geothermal waters often contain naturally high concentrations of arsenic. When a geothermal spring with elevated arsenic concentrations emerges at the surface and discharges into its receiving catchment, the fate of arsenic in the discharged fluid is determined by changes in abiotic parameters, such as pH, temperature, and oxygen, as well as by the microbial activity of chemolithotrophic or phototrophic (hyper)thermophilic microorganisms. Abiotic or microbially catalyzed species transformations are well documented for the inorganic oxy-anions arsenite (H3AsO3) and arsenate (H3AsO4) in anoxic and oxic environments.1−5 However, recent studies have shown that under sulfidic conditions, as they exist in geothermal waters, thiol groups can replace one to four of the hydroxyl groups in arsenate, forming (oxy)thioarsenates (H3AsSnO4‑n). In alkaline, sulfidic Yellowstone National Park springs, trithioarsenate was found to be the dominant arsenic species.6−9 Little is known about the fate of thioarsenates upon release from geothermal springs. In a previous study, 6 we described and compared thioarsenate transformation patterns along the drainage channels of seven different alkaline geothermal springs in Yellowstone National Park, U.S. Trithioarsenate eventually transformed to arsenate at the end of the drainage channel, accompanied by an initial increase, then decrease of monothioarsenate and arsenite under increasingly oxidizing conditions along the drainage channel. We interpreted this © 2012 American Chemical Society

sequence as trithioarsenate being desulfidized and reduced to arsenite, which was subsequently oxidized to arsenate. The role of monothioarsenate was unclear. Laboratory studies suggested that monothioarsenate was inert in the presence of oxygen and heat. We did observe, however, that, with limited oxygen supply, the reduced sulfur in trithioarsenate was oxidized (initially to elemental sulfur, which further oxidizes to thiosulfate) while the pentavalent arsenic was reduced and formed arsenite according to the following equation: H3As V S3O + O2 → H3AsIII O3 + 3S0

In synthetic solutions, direct desulfidation of trithioarsenate to arsenate with immediate formation of sulfate was only observed when using strong oxidizing agents according to the following equation: H3As V S3O + 3H2O + 6O2 → H3As V O4 + 3SO4 2 − + 6H+

We thus attributed the trithioarsenate−arsenite conversion observed in the geothermal drainage channels to abiotic oxidation with limited oxygen supply. No evidence was found Received: Revised: Accepted: Published: 4348

November 29, 2011 March 1, 2012 March 1, 2012 March 1, 2012 dx.doi.org/10.1021/es204277j | Environ. Sci. Technol. 2012, 46, 4348−4356

Environmental Science & Technology

Article

flushed three times with sample water. In most cases (specified in detail subsequently), experiments were run twinned: one set contained biota, the other set did not contain biota (to act as a control). Brownish-pink filamentous microbial mats were added to biotic experiments and are referred to as “streamers” in the text. Streamers were collected aseptically with tweezers from sampling point 16 m. The temperature of streamer’s origin water was 78 °C. Routinely, incubation temperatures were in the same range (76−78 °C). To use a reproducible amount of biomass, the wet volume of streamers was measured in a graduated plastic tube. Standard wet biomass volume was 2 mL. In 2011, further experiments (n = 1) were conducted using 0.1, 0.5, 2, 5, and 10 mL streamer volumes. This method is semiquantitative and certainly less accurate than lab-based approaches such as cell cultivation with cell-counting. However, the method proved to be quick and field-practicable, allowing the reproduction of incubation experiments on-site, while ensuring minimal changes in redox-sensitive arsenic species. Incubation bottles were capped and a syringe needle was inserted through the top to create a sampling port. These bottles were placed in the drainage channel for incubation at natural water temperature. A sample volume of 2 mL was taken after 1, 5, 10, 30, 60, and 120 min. All samples were then filtered into 2 mL PP tubes and were immediately flash-frozen on dry ice to preserve the arsenic−sulfur speciation. Seven abiotic incubation experiments (n = 3) were done with water from 0, 2, 5, 7, 11, 16, 22 m distance downstream along the drainage channel at the temperature of their origin (94, 91, 86, 86, 85, 78, 72 °C). The purpose of the abiotic incubations was to determine the effects of different initial arsenic species concentrations and temperatures on abiotic thioarsenate transformation. One experiment (n = 3) carried out at 78 °C compared arsenic species transformations under abiotic and biotic conditions. In selected experiments (n = 3), sterile-filtered water samples were cooled down to 30 °C for 10 min, before starting the incubation experiment to investigate the effect of temperature on thioarsenate transformation in the presence and absence of streamers. Furthermore, the release of arsenic and sulfur species from the streamers was studied incubating 2 mL streamers for 120 min in deionized water at 78 °C (n = 3). In 2011, one abiotic and three biotic experiments with 0.1, 2, and 10 mL streamers were carried out to investigate abiotic versus microbial sulfide oxidation over time; sulfide was measured immediately on-site using the methylene blue method (#8131, Hach DR890). In 2011, sulfide-free medium was used instead of Conch Spring drainage channel water for the incubation experiments. These sulfide-free experiments aimed to exclude any potential chemical reactions in thioarsenate transformation in response to microbial sulfide oxidation. Synthetic low mineral Octopus Spring (OS) water was used as medium that fit the requirements of Thermocrinis ruber. Its exact composition has been previously described.17 The medium was prepared anoxically in the laboratory, but was exposed to air during the field experiments. Three twinned experiments were carried out with and without 2 mL streamer, by adding either (a) 60 μM monothioarsenate, (b) 60 μM arsenite, or (c) 30 μM monothioarsenate plus 30 μM arsenite. The monothioarsenate standard was synthesized following the method published by Suess et al.29 IC-ICP-MS Analysis. Arsenic−sulfur speciation was analyzed by AEC-ICP-MS as described earlier.7,30 Anion-exchange

for direct transformation of thioarsenates to arsenate in the geothermal drainage channels. The subsequent oxidation of arsenite to arsenate coincided with the visible appearance of large amounts of filamentous microbial mats floating in the drainage channels of Ojo Caliente and Conch Spring. In an attempt to gather evidence for microbially catalyzed arsenite oxidation, cells from sterilefiltered water were analyzed by 16s rDNA analysis.6 Members of Aquif icales constituted the most abundant group, represented by Thermocrinis spp. (most closely related to T. ruber) and Sulf urihydrogenibium spp. Less abundant sequences were assigned to Thermus, Geothermobacterium ferrireducens, βProteobacteria, and Cyanobacteria. The observed low diversity, dominated by Aquif icales is typical for hot spring streamer communities in alkaline-chloride systems.10−19 Archaea-related sequences could not be amplified.6 From laboratory studies, members of the Aquif icales are known to use reduced sulfur species, besides hydrogen, as electron donors.17,20−26 Oxygen is often the terminal electron acceptor, along with nitrate or ferrous iron.17,21−23 Some members of Aquif icales are also able to use arsenate as an electron acceptor.24,25 Rapid arsenite oxidation was previously observed in hot springs and reported to be mediated by aerobic arsenite-oxidizing microbes,4,5,27 whereas abiotic arsenite oxidation was found to be negligible.4,28 The role thioarsenates potentially play in bacterial metabolisms is not known. These compounds are interesting as they contain arsenic in its oxidized state and sulfur in its reduced state and could thus serve as both an electron acceptor and an electron donor. The goal of the present study was to better elucidate processes leading to the transformation of thioarsenates within their natural environment, with a special focus on differentiating abiotic and biotic causes.

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MATERIAL AND METHODS Data were collected during three sampling trips to Yellowstone National Park in the summers of 2009, 2010, and 2011. The investigated geothermal feature, Conch Spring (Figure SI1 of the Supporting Information, SI), is an alkaline, sulfidic hot spring.6 The spring is located east of the Firehole River in the Pocket Basin area within the Lower Geyser Basin (LR492, Latitude 44°33.389′, Longitude 110°49.935′). Water Chemistry. The spring and its drainage channel were sampled along one transect at nine different locations. Samples were pumped from the drainage channel with a lowflow peristaltic pump and filtered through 0.2 μm celluloseacetate filters (Membrex, Membra Pure, Mannheim). Samples for arsenic−sulfur speciation were filtered into 60 mL PEbottles, rinsed three times with sample water, and immediately flash-frozen on dry ice in the field. Until analysis, samples were stored below −20 °C.6,7 On-site measurements in 2009 included determination of temperature (Heidolph, EKT 300) and colorimetric sulfide measurements (Hach DR890, methylene blue method #8131). After cooling the water below 50 °C as described elsewhere,6 pH (Hach pH101), dissolved oxygen (Hach LDO), redox potential (WTW SenTix ORP), and electrical conductivity (WTW TetraCon 325) were determined in a flow-through cell until equilibrium was reached (1 to 1.5 h). Incubation Experiments at Conch Spring. All incubation experiments in 2010 were run in triplicate (n = 3). For all experiments, 50 mL sterile-filtered drainage channel water was collected at 16 m distance from the source with a plastic syringe 4349

dx.doi.org/10.1021/es204277j | Environ. Sci. Technol. 2012, 46, 4348−4356


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Figure 1. Trends of (a) arsenic, arsenic−sulfur and (b) sulfur species along the drainage channel of Conch Spring. Shown are average concentrations and their standard deviations based on data collected in 2011, 2010, and 2009 (Table SI1, Table SI2a of the SI) as well as 2007 and 2006 (PlanerFriedrich et al. (2009)). A pronounced reproducible trend is visible in the data, despite relatively high standard deviations due to variations in the initial source concentrations in different years.

Figure 2. The graphs show the arsenic speciation in field incubation experiments in 2010 (n = 3). Sterile-filtered water and streamers derived from sampling point 16 m. Experiments (a) without and (b) with 2 mL streamer biomass incubated at 78 °C (Table SI2 j, k of the SI). Water was cooled down for 10 min and then incubated at 30 °C (c) without and (d) with 2 mL streamer biomass (Table SI2 l, m of the SI). For legend see Figure 1.

Aldrich #563188) were used. Thioarsenates were quantified based on the arsenate calibration curve. Prior to analysis, frozen samples were thawed in a glovebox atmosphere of 95% nitrogen and 5% hydrogen, to prevent oxidation.

chromatography (Dionex ICS-3000 SP) was coupled to an inductively coupled plasma mass spectrometer (XSeries2 from Thermo Fisher). For species separation we used an AG16/ AS16 IonPac column (Dionex) and an alkaline eluent with a gradient of 20−100 mM NaOH and a flow rate of 1.2 mL/min. An oxygen (10%)/helium (90%) mixture served as reaction gas. Arsenic was detected as AsO+ at m/z 91, sulfur as SO+ at m/z 48. For calibration, standard solutions of sodium arsenate pentahydrate (Na2HAsO4*7 H2O, Fluka #71625), sodium (meta)arsenite (NaAsO2, Fluka #71287), ammonium sulfate [(NH4)2SO4, Fluka #09979] and sodium thiosulfate (Na2S2O3,

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RESULTS AND DISCUSSION

Chemistry along the Drainage Channel. In 2009, the pH at Conch Spring was approximately 9.2. Temperatures decreased along the drainage channel (93 to 61 °C), whereas dissolved oxygen (20 to 108 μM) and redox potential (−20 to 100 mV) increased (Figure SI2 of the SI). On the basis of data 4350



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Figure 3. Field incubation experiments applying different quantities of streamer biomass (0 mL (abiotic), 0.1 mL, 0.5 mL, 2 mL, 5 mL, 10 mL) in drainage channel water from 16 m at 78 °C, carried out in 2011. The graphs represent results of single measurements. Sulfide shown in graph (a) was detected with the HACH methylene blue method in the field. Results for (b) thiosulfate, (c) trithioarsenate, (d) monothioarsenate, (e) arsenite, and (f) arsenate were measured with AEC-ICP-MS. No abiotic experiments were done for AEC-ICP-MS speciation, since abiotic effects were already investigated intensively in 2010 (see Figures 2a and Figure SI3 of the SI). Corresponding numeric data are listed in Table SI3 of the SI.

13.9 ± 1.5 μM at the source to a maximum of 24.5 ± 2.1 μM at 29 m, but decreased to 21.2 ± 2.2 μM at 43 m. Abiotic Desulfidation of Thioarsenates to Arsenate. The diagram in Figure 2a (Table SI2j of the SI) shows the results of an abiotic incubation experiment carried out 16 m downstream of the spring (78 °C). Additional abiotic incubation experiments done at six further sampling sites representing temperatures of 94 to 72 °C are shown in Figure SI3 (Table SI2b-h of the SI). Initial trithioarsenate concentrations (range 21.11 ± 1.11 to 7.70 ± 2.44 μM) decreased over time in all these aerobic abiotic incubations. Final concentrations after 120 min were 8.93 ± 0.61 to 0.02 ± 0.002 μM (36 ± 8% of the initial trithioarsenate concentrations). Thus, no significant differences in thioarsenate transformation kinetics within the investigated temperature range of 94 to 72 °C were deducible. Trithioarsenate transformation was also independent of the absence or

collected during 5 different sampling trips, trithioarsenate was the dominant arsenic species at the source (Figure 1a, Table SI1, SI2-a of the SI). From the spring, trithioarsenate concentrations decreased from 15.3 ± 2.6 μM to 1.17 ± 0.22 μM at 43 m downstream. Dithioarsenate concentrations decreased from 2.5 ± 0.6 to 0.9 ± 0.3 μM along the same distance. In contrast, monothioarsenate concentrations initially increased to a maximum of 9.69 ± 1.57 μM at 29 m, then decreased to 7.92 ± 0.63 μM at 43 m. At the farthest downstream sampling point (43 m), arsenate dominated the arsenic speciation with a maximum concentration of 13.81 ± 1.97 μM. Sulfide concentrations decreased from 92 ± 14 μM at the source to 8 ± 2 μM at 43 m downstream, and was oxidized to sulfate which increased from 177 ± 30 μM to 232 ± 41 μM (Figure 1b). Thiosulfate formed as an intermediate species along the drainage. Concentrations showed an increase from 4351



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Sulfur speciation also showed clear differences in abiotic versus biotic incubations with the presence of microorganisms significantly increasing sulfide oxidation kinetics (Figure 3a; Table SI5 of the SI). In biotic experiments with 2 mL streamer biomass, about 42.7 μM of sulfide were consumed within 30 min. In contrast, only 10.9 μM sulfide were oxidized within the same time period abiotically. Sulfide oxidation contributed to intermediate thiosulfate and sulfate production. It explained the differences observed in sulfur speciation between abiotic versus biotic incubations (Table SI2j−k of the SI). Thiosulfate increased within the first 30 min from 20.7 ± 1.6 μM to 24.2 ± 1.3 μM, but then decreased in biotic incubations to 6.4 ± 0.9 μM after 240 min. In the abiotic experiment, thiosulfate concentrations constantly increased over time from 20.7 ± 1.6 to 32.6 ± 1.8 μM. Furthermore, a continuous increase of sulfate was observed over time from 166 ± 1 μM to 204 ± 2 μM in the biotic experiment. The amount of sulfate formation in the abiotic experiment was lower (from 166 ± 1 to 173 ± 2 μM). To quantify the potential release of arsenic and sulfur previously absorbed in the streamers, they were incubated in deionized water at 78 °C. Except for small concentrations of arsenate (max. 0.48 ± 0.08 μM), thiosulfate (max 0.2 ± 0.2 μM) and sulfate (max. Six ±3), there was no release detectable (Table SI2i of the SI). Compared to absolute changes in natural background concentrations of arsenate (1.83 to 15.75 μM), thiosulfate (14.6 to 27.3 μM) and sulfate (128 to 202 μM) in the drainage channel (Table SI2a of the SI), the release was considered negligible for the interpretation of the microbially catalyzed speciation changes reported. Effect of Increasing Biomass. The application of different amounts of streamers in experiments (0.1, 0.5, 2, 5 versus 10 mL) significantly affected transformation kinetics. The more biomass, the faster the transformation of sulfide, tri-, and monothioarsenate (Figure 3a,c,d) and the higher the production of arsenate (Figure 3f). Intermediate thiosulfate formation also increased due to enhanced sulfide oxidation (Figure 3b). At the same time, intermediate formation of arsenite increased due to enhanced decrease of tri- and monothioarsenate (Figure 3e). At first glance, arsenite (Figure 3e) and thiosulfate (Figure 3b) concentrations for 10 mL biomass do not seem to fit the general trend of increasing formation with increasing biomass, as maximum concentrations are reached in incubations with 5 mL biomass. However, it has to be considered that more microbes will also trigger a quicker transformation of these intermediate species. Overall, these findings demonstrate (hyper)thermophilic microbes must be involved in sulfide and arsenite oxidation, as well as thioarsenate transformation. Effect of Temperature on Microbial Activity. To further prove that microbes actively participate in thioarsenate transformation, biotic incubations were carried out at 30 °C. Much slower trithioarsenate transformation kinetics (Figures 2d and Table SI2m of the SI) were observed, when compared to results at 78 °C (Figure 2b). At 78 °C, no trithioarsenate was left after 60 min (0.02 ± 0.002), whereas 3.20 ± 0.59 μM trithioarsenate were still detectable after 120 (+10) min at 30 °C. Mono- and dithioarsenate concentrations did not vary significantly at 30 °C. The starting conditions were not completely identical, as water samples for the 30 °C experiment had to be cooled down for approximately 10 min and some changes in arsenic speciation might have happened during that time. Nonetheless, prokaryotes have a specific optimum temperature range at which they thrive well. It is reasonable

presence of individual other arsenic species such as monothioarsenate, arsenite, or arsenate (Figure SI3 of the SI). Variations in monothioarsenate and dithioarsenate concentrations did not exceed 10% (3 μM) of the average sum of arsenic species (33 ± 1 μM, n = 49, Table SI2b-h of the SI), over the course of the experiment. Thus, both neither formed as intermediate species after abiotic trithioarsenate transformation, nor transformed to any other arsenic species. Arsenite decreased slightly over the same time interval (Figure 2a). The sole product of our abiotic field incubations was arsenate (Figure 2a, Figure SI3 of the SI). We conclude that trithioarsenate decreases by directly forming arsenate abiotically. The most likely mechanism is desulfidation due to H2S-deficiency (eq 1). H3As V S3O + 3H2O → H3As V O4 + 3H2S

(1)

At all sites along the drainage channel (Figure 1b) and in all abiotic incubations (Figures 2a and SI3 of the SI), sulfide is lost due to oxidation to thiosulfate (eq 2) or sulfate (eq 3). S2 − + S0 + 1.5O2 → S2O32 −

(2)

H2S + 2O2 → SO4 2 − + 2H+

(3)

Sulfide oxidation shifts the equilibrium in eq 1 to the right, causing desulfidation of trithioarsenate. Previous studies6,31 have already shown that trithioarsenate is much more sensitive to sulfide loss than monothioarsenate. The present observations confirm that the abiotic desulfidation of trithioarsenate is kinetically faster than that of monothioarsenate. The latter was observed to be inert, at least within the considered time interval of 120 min (Figures 2a and SI3 of the SI). Transformation of Thioarsenates and Sulfide in Biotic Incubations. Clear differences in arsenic speciation were observed for abiotic (Figures 2a and SI3 of the SI) versus biotic (Figure 2b) incubations. In the presence of streamers, trithioarsenate (along with dithioarsenate) disappeared faster (0.17 ± 0.12 μM after 60 min) than in the abiotic control (4.88 ± 0.72 μM after 60 min). Within the first minute, monothioarsenate concentrations increased in biotic incubations. The only corresponding decreasing species is arsenite. We could thus assume that the reaction is formation of monothioarsenate from arsenite in the presence of elemental sulfur. Since we never found formation of monothioarsenate in any of the abiotic incubations (Figures 2a and SI3 of the SI), this oxidative sulfidation is likely to be triggered by microbial activities. A similar reaction might explain the pronounced increase in monothioarsenate concentrations observed from 0 to 16 m in the natural drainage channel (Figure 1a). The temporal resolution of the biotic incubations is, however, too low for further interpretation and no corresponding changes were observed in the sulfur speciation (note: elemental sulfur would have gone unrecognized in our speciation method). After the first minute, monothioarsenate concentrations decreased in biotic incubations from 13.26 ± 0.34 μM to 2.03 ± 0.72 μM (Figure 2b), but did not change significantly in the corresponding abiotic setup (Figure 2a). Both, arsenate and arsenite formed concurrently. But, the increase of the arsenite concentrations was comparatively faster, reaching its maximum after 60 min (14.11 ± 3.03 μM). Arsenate concentrations started to exceed arsenite concentrations after 120 min (15.25 ± 0.83 μM). 4352



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detectable.37 On the basis of observed speciation changes, arsenite oxidation to arsenate by streamers from Conch Spring has previously been postulated.6 However, in that work, it was also claimed that microbially catalyzed arsenate production in near-neutral to alkaline hot springs is not inhibited by the presence of sulfide. The time lag in arsenate production in the current incubation experiments disproves this earlier statement. Direct Microbial Thioarsenate Transformation. An alternative hypothesis for the biotic transformation of thioarsenates could be a so far unknown direct microbial transformation. If that is the case, then thioarsenates could be directly reduced to arsenite coupled to the oxidation of free sulfide and As-bound sulfide to thiosulfate, as shown for trithioarsenate in eq 6.

to assume that (hyper)thermophilic prokaryotes are more active at 78 °C than at 30 °C. With respect to trithioarsenate, the 30 °C biotic incubations (Figure 2d) share some similarity to 78 °C abiotic incubations (Figure 2a). In both cases, about 3 μM trithioarsenate remained after 120 min. But, arsenite transforms slower in 30 °C biotic, vs 78 °C abiotic incubations. Less arsenate (10.64 ± 0.68 μM) is formed after 120 min in 30 °C biotic, compared to 78 °C abiotic incubations (15.57 ± 1.23 μM). At first glance, it thus seems that microbes are not active at all at the low temperature of 30 °C. However, in the abiotic control incubation at 30 °C (Figures 2c and Table SI2l of the SI) more trithioarsenate (5.96 ± 0.31 μM) remained and less arsenate 8.10 ± 5.87 μM formed after 120(+10) min. Arsenite and monothioarsenate did not decrease. We could thus show that high temperatures in general have an accelerating effect on abiotic arsenic species transformation, but that temperature is less relevant than the processes triggered by microbes. Potential Mechanisms of Microbial Thioarsenate Transformation in the Drainage Channel. On the basis of our data, biotic thioarsenate transformation could be explained by two different mechanisms. Indirect Microbial Thioarsenate Transformation. A first obvious hypothesis is that there is no direct microbial thioarsenate transformation. Rather, thioarsenate transformation may only be a consequence of microbially catalyzed transformation of sulfide and arsenate, the products of the abiotic thioarsenate desulfidation (eq 1). Our experiments applying different amounts of biomass demonstrated that more sulfide is oxidized microbially than abiotically (Figure 3a). At the same time, and coupled to the oxidation of sulfide, arsenate could be microbially reduced to arsenite (eq 4): H3As V O4 + S2 − → H3AsIII O3 + S0

H3As V S3O + HS− + OH− + 3.5O2 → H3AsIII O3 + 2S2O32 −

This reaction would explain both, arsenite and thiosulfate formation as observed in our experiments. Even if decoupled from sulfide oxidation, thioarsenate reduction could yield arsenite with formation of elemental sulfur which is then further oxidized to thiosulfate or sulfate, shown for trithioarsenate in eq 7. H3As V S3O + O2 → H3AsIII O3 + 3S0

(7)

An alternative pathway would be thioarsenate desulfidation to arsenate with oxidation of As-bound sulfide to elemental sulfur (eq 8) or to sulfate (eq 9): H3As V S3O + 1.5O2 → H3As V O4 + 3S0

(8)

H3As V S3O + 3H2O + 6O2

(4)

→ H3As V O4 + 3SO4 2 − + 6H+

The observed accumulation of arsenite in our biotic incubations could support this hypothesis (Figure 3e). As a result of both, arsenate reduction and sulfide oxidation, the equilibrium in eq 1 is shifted to the right, accelerating abiotic thioarsenate desulfidation. The reverse process, microbially catalyzed arsenite oxidation to arsenate (eq 5), is observed at a later time point in our incubation experiments (after 60 min in our experiments with 2 mL biomass (Figure 3e)). H3AsIII O3 + 0.5O2 → H3As V O4

(6)

(9)

The observed arsenite accumulation would then have to be explained by microbial arsenate reduction in the presence of sulfide as explained in the previous paragraph (eq 4). Two Aquif icales-strains, namely Sulf urihydrogenibium azorense and S. subterraneum have been demonstrated to use arsenate as electron acceptor, using reduced sulfur species as donors.24 However, nothing similar has been reported for Thermocrinis, which were detected as the most abundant microbial species at Conch Spring.6 Evidence for Direct Transformation of Arsenite and Monothioarsenate in a Sulfide-Free Medium. The hypothesis of direct microbial reduction of thioarsenates to arsenite (eqs 6 and 7) is difficult to test in the presence of sulfide. The influence of microbially catalyzed sulfide oxidation cannot be separated from other microbially triggered processes and might have a significant influence by shifting the equilibrium in (eq 1) to the right. To show that thioarsenates can be transformed directly by microorganisms, we thus focused on a sulfide-free environment. In equilibrium, all thioarsenates would desulfidize in sulfide-deficient environments abiotically (eq 1). However, transformation kinetics decrease from tri-, via di- to monothioarsenate. By selecting monothioarsenate, we hypothesized that abiotic transformation rates could be sufficiently slow to see a clear difference if microorganisms actively transform thioarsenates. We additionally investigated the microbially catalyzed oxidation of arsenite to arsenate in the absence of sulfide. Figure 4 (Table SI4 of the

(5)

Arsenite oxidation only starts when sulfide is completely oxidized (Figure 3a,e), which can be explained by the fact that sulfide is an arsenite oxidase inhibitor.32,33 The involvement of (hyper)thermophilic bacteria in arsenite oxidation was reported earlier along geothermal drainages1,4,5,32,33 and in ex-situ incubations of biomats from Alvord hot spring in Oregon/ U.S., which were dominated by bacteria related to Sulf urihydrogenibium, Thermus and Thermocrinis.34 Within the order Aquif icales, Sulf urihydrogenibium azorense, and S. suberraneum are species capable of using arsenite as a sole electron donor.24 Putative aroA-like arsenite oxidase genes (new nomenclature: aioA35,36) were identified in several Aquif icales species, including Thermocrinis and Sulf urihydrogenibium,28,37,38 which are present at Conch Spring. But, gene expression was shown only for two Hydrogenobacter strains (GV4−1, GV8−4AC-C1) and Sulf urihydrogenibium rodmanii in the presence of thiosulfate as energy source. Growth on arsenite as sole donor was not 4353



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Figure 4. Field incubation experiments in sulfide-free OS-Medium,17 carried out at 78 °C in 2011. Experiments shown in graphs (a) abiotic and (b) biotic were spiked with approximately 60 μM monothioarsenate, in graphs (c) abiotic and (d) biotic with approximately 60 μM arsenite, in graphs (e) abiotic and (f) biotic with 30 μM arsenite plus 30 μM monothioarsenate. Two mL streamer-biomass from sampling point 16 m were added to biotic incubations. The graphs represent results of single measurements. Corresponding numeric data are listed in Table SI4 of the SI. For legend see Figure 1.

abiotic monothioarsenate desulfidation. In the biotic experiments (Figure 4b), monothioarsenate concentrations decreased to 0.63 μM after 240 min, forming arsenite as intermediate (max. 11.06 μM after 30 min) according to eq 10. Arsenate was the final product and presumably both formed directly according to eq 11 and indirectly as shown in eq 5.

SI) shows the results of field tests with sulfide-free OS-medium spiked with either 60 μM arsenite, 60 μM monothioarsenate or with 30 μM of each compound. Spiking arsenite, complete conversion to arsenate was observed in the presence of microorganisms (Figure 4d, Table SI4d of the SI) within 240 min, while arsenite was inert under abiotic conditions (Figure 4c, Table SI4c of the SI). In contrast to biotic assays with natural drainage channel water (Figure 3e, 2b), arsenite was oxidized to arsenate without any initial time delay (Figure 4d), which is probably owed to the absence of arsenite-oxidase inhibiting sulfide.32 We conclude that arsenite was directly oxidized by the microbes. Spiking monothioarsenate, abiotic incubations showed that monothioarsenate was inert over a 240 min time interval, despite the absence of sulfide in solution (Figure 4a, Table SI4a of the SI). In fact, the results are similar to those of abiotic experiments using sulfidic hot spring water (Figure 2a, Figure SI3 of the SI). Hence, the lack of sulfide did not accelerate

H3As V SO3 → H3AsIII O3 + S0

(10)

H3As V SO3 + H2O + 2O2 → H3As V O4 + SO4 2 − + 2H+

(11)

Since abiotic desulfidation can be excluded, the results indicate monothioarsenate was transformed directly by the microbial community (Figure 4b). OS-medium lacks sulfide, hence intermediate arsenite formation cannot be a microbially catalyzed arsenate reduction coupled to sulfide oxidation (eq 4354



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4). The latter must be directly linked to microbial monothioarsenate reduction, likely according to eq 10. Thiosulfate formation was very low (max. < 1.9 μM, Table SI4b of the SI). No sulfate formation trend was detectable, but may be masked by relatively high sulfate background concentrations of the synthetic OS medium (Table SI4b of the SI). Therefore, we assume that the major oxidation product is either elemental sulfur (eq 10), or sulfate (eq 11). In an abiotic assay applying both, monothioarsenate and arsenite, no species variations were detectable within 240 min (Figure 4e, Table SI4f of the SI). In the corresponding biotic assay both transform to arsenate completely (Figure 4f, Table SI4f of the SI). Arsenite concentrations decrease without delay or any intermediate increase. This could be explained by additional arsenite in solution compared to experiments with monothioarsenate spike only. In consequence of this, the balance of eq 10 is shifted to the left, restraining further arsenite formation from monothioarsenate. Hence, only microbially catalyzed monothioarsenate desulfidation to arsenate (eq 11) and arsenite oxidation to arsenate (eq 5) explain the observations. Similar experiments in sulfide-free medium with other thioarsenates were not possible. Dithioarsenate was not available as pure standard. Trithioarsenate is kinetically extremely labile in the absence of sulfide. Rapid abiotic transformation would mask potential biological effects in our simplified incubation setups. At this point, we can thus only speculate that di- and trithioarsenate providing two or three sulfur atoms might be even more efficient electron donors in microbially catalyzed redox processes than monothioarsenate. Future experiments will need to focus on setups capable of proving their direct microbial transformation also in the presence of sulfide. We found a rather complex, partially abiotic, partially microbially catalyzed thioarsenate transformation pattern along Conch Spring’s drainage channel. Abiotically, only arsenate formed during trithioarsenate desulfidation under sulfide-deficient conditions. Monothioarsenate was distinctly less sensitive to abiotic desulfidation and no transformation was observed within up to 240 min in either sulfidic natural water or sulfide-free OS-medium. Microbial oxidation of sulfide will indirectly increase the abiotic trithioarsenate desulfidation (eq 1), therefore masking potential direct microbial transformation of trithioarsenate to arsenite (eqs 6 and 7) or arsenate (eqs 8 and 9) in natural, sulfidic water. Additionally, any arsenate released from trithioarsenate desulfidation could serve as electron acceptor during microbial oxidation of sulfide, forming arsenite (eq 4). Biotic sulfide-free assays, however, clearly showed that the microbial community present at Conch Spring can directly transform arsenite to arsenate (eq 5) and monothioarsenate to arsenite (eq 10) or arsenate (eq 11). The involvement of ((hyper)thermophilic) prokaryotes in thioarsenate transformations has never previously been reported. Systematic growth studies applying individual arsenic and sulfur species as electron donors or acceptors are currently under way in our laboratory. The present study underlines the necessity to consider complexation between arsenic and sulfur in sulfidic environments. It emphasizes the impact that either thioarsenates might have as potential sulfur electron donors and simultaneous as potential arsenic electron acceptors on autotrophic primary producers, or that a sulfur-based autotrophic bacterial metabolism can have on the arsenic chemistry in geothermal environments.

Article

ASSOCIATED CONTENT

S Supporting Information *

Documentation of field site, dissolved oxygen concentrations, redox potential and temperature, all basic numeric data regarding trends of arsenic and sulfur species along the drainage channel and during incubation experiments using natural water, OS-medium, and deionized water. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 921 553644; fax: +49 921 552334; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge generous funding by the German Research Foundation within the Emmy Noether program to B. PlanerFriedrich (Grant No. PL 302/3-1) and thank the Yellowstone National Park Service for sampling permission, Bill Inskeep, Zackary Jay, and Jacob Beam from MSU Bozeman for cooperation in the field, Ulli Seifert, Sebastian Schmitt, Regina Lohmayer, and Rita Schubert for assistance during sampling, Stefan Will for AEC-ICP-MS analyses, and Nathaniel Wilson for proof-reading the manuscript.

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REFERENCES

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