Environ. Sci. Technol. 2007, 41, 2311-2317
Hydrogen Thresholds and Steady-State Concentrations Associated with Microbial Arsenate Respiration A X E L C . H E I M A N N , * ,† CHRISTIAN BLODAU,‡ DIEKE POSTMA,† FLEMMING LARSEN,† PHAM H. VIET,§ PHAM Q. NHAN,⊥ SØREN JESSEN,† MAI T. DUC,§ NGUYEN T. M. HUE,§ AND RASMUS JAKOBSEN† Institute of Environment & Resources, Bygningstorvet, Building 115, Technical University of Denmark, DK-2800 Lyngby, Denmark, Limnological Research Station and Department of Hydrology, University of Bayreuth, D-95440 Bayreuth, Germany, Research Centre for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Vietnam National University, T3 Building, 334 Nguyen Trai Street, Thanh Xuan District, Hanoi, Vietnam, and Hanoi University of Mining and Geology, Hanoi, Vietnam
H2 thresholds for microbial respiration of arsenate (As(V)) were investigated in a pure culture of Sulfurospirillum arsenophilum. H2 was consumed to threshold concentrations of 0.03-0.09 nmol/L with As(V) as terminal electron acceptor, allowing for a Gibbs free-energy yield of 36-41 kJ per mol of reaction. These thresholds are among the lowest measured for anaerobic respirers and fall into the range of denitrifiers or Fe(III)-reducers. In sediments from an arsenic-contaminated aquifer in the Red River flood plain, Vietnam, H2 levels decreased to 0.4-2 nmol/L when As(V) was added under anoxic conditions. When As(V) was depleted, H2 concentrations rebounded by a factor of 10, a level similar to that observed in arsenic-free controls. The sediment-associated microbial population completely reduced millimolar levels of As(V) to arsenite (As(III)) within a few days. The rate of As(V)-reduction was essentially the same in sediments amended with a pure culture of S. arsenophilum. These findings together with a review of observed H2 threshold and steady-state values suggest that microbial As(V)-respirers have a competitive advantage over several other anaerobic respirers through their ability to thrive at low H2 levels.
Introduction High groundwater levels of inorganic arsenic are a threat to the health of millions of people in Bangladesh and West Bengal (1), and pose severe problems for drinking water supplies in many other areas around the world (2, 3). Mobilization of naturally occurring, sediment-bound arsenic, * Corresponding author phone: +45-4525-2172; fax: +45-45932850; e-mail:
[email protected]. † Technical University of Denmark. ‡ University of Bayreuth. § Vietnam National University. ⊥ Hanoi University of Mining and Geology. 10.1021/es062067d CCC: $37.00 Published on Web 03/06/2007
2007 American Chemical Society
can lead to drinking water arsenic levels that are far above the WHO guideline value of 10 µg/L (4). Uptake of arsenic may trigger several types of cancers and many other diseases (5). While arsenic is toxic to humans, various phylogenetically diverse microbes are able to gain energy from the reduction of As(V) (arsenate) to As(III) (arsenite) under anoxic conditions (6-11). This dissimilatory respiration of arsenate likely contributes to the mobilization of arsenic from soils and sediments (12-14), necessitating a good understanding of the role microbes play in the development of high groundwater arsenic levels (15, 16). In addition, microbial respiration of arsenate may significantly contribute to carbon cycling in extreme environments, such as arsenic-rich, alkaline lakes (17, 18). In anaerobic settings, arsenate respiration may compete with other terminal electron-accepting processes (TEAPs) for available electron donors. These substrates are predominantly low-molecular-weight products of fermentation, such as dissolved hydrogen (H2) or acetate (19). Due to its high turnover and low residence time, it is often competition for H2 that controls the competitive advantage of one TEAP over another (20). Consequently, H2 concentrations may assist in evaluating redox conditions of the subsurface in terms of ongoing TEAPs such as the reduction of nitrate, Mn(IV), Fe(III), sulfate, or CO2 (21). The underlying assumption of this approach is that TEAPs with more favorable energetics (e.g., nitrate reduction) can proceed at lower levels of hydrogen than energetically less favorable TEAPs (e.g., methanogenesis via H2/CO2). Tables 1 and 2 list observed H2 concentrations and Gibbs free-energy yields associated with different TEAPs. In this context, it is important to distinguish between H2 thresholds (22) and steady-state concentrations (23). While a threshold marks the level below which H2 consumption ceases, steady-state concentrations are measured during balanced production and consumption of H2. The latter is also sometimes referred to as compensation concentration (24, 25). However, here we use the term steady-state concentration implying steady-state conditions with respect to a balanced H2 production and consumption. The H2 concept was later extended into the partial equilibrium approach which includes in-situ concentrations of all reactants of a given TEAP, thereby allowing for the calculation of in-situ energy yields (26-28). Moreover, H2 levels have recently been employed for delineating microbial respiration of potential groundwater contaminants such as chlorinated solvents (29, 30) or chromate (31). In addition, H2 is a common electron donor for As(V)-respiration (9, 3234). However, to date nothing is known about the capability of As(V)-respiring bacteria to compete for H2, that is, characteristic H2 levels in As(V)-reducing environments or H2 thresholds of the involved bacteria. Filling this gap of knowledge could assist in a better understanding of the competition between arsenate reduction and other TEAPs, and its impact on arsenic mobilization in anoxic soils and sediments. Therefore, we studied H2 thresholds of Sulfurospirillum arsenophilum, a member of the -proteobacteria, isolated from arsenic-contaminated sediments (6) and capable of hydrogenotrophic arsenate respiration (15, 32). We also examined arsenate reduction and associated H2 levels in sediment microcosms from an arsenic-impacted aquifer in the Red River flood plain in Northern Vietnam (35).
Materials and Methods Culture Conditions. Sulfurospirillum arsenophilum (32) was obtained from the German collection of microorganisms and VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Overview of Several H2-Consuming TEAPs and their Threshold and Steady-State H2 Concentrations, Respectively
a
terminal electron acceptor oxidized/reduced
H2, threshold (nmol/L)
source
CO2/acetate CO2/CH4 sulfate/sulfide Fe(III)/Fe(II) nitrate/N2 or ammonia chlorinated ethenes/ethene Cr(VI)/Cr(III) arsenate/arsenite
330-720 19-76 1.8-14 < 0.11-0.31 0.02-0.5 0.05-0.27 < 1.0 0.03-0.09
22 22,45 22,46 47-49 22,49,52 46,49,53 31 this study
H2, steady state (nmol/L)a
source
120-150 7-13 1-1.6 0.03-1.0 < 0.05 0.3-2.5
44 23,44 23,44 23,50,51 23 54-56
0.4-0.7
this study
Comprises levels that are described as characteristic, steady state, or compensation concentrations from different environments.
TABLE 2. Overview of Several H2-Consuming TEAPs and their Gibbs Free Energies under Standard (∆G0) and Hypothetical Environmental (∆Gr) Conditions, Respectively reactions 1/2 O2 + H2 f H2O 2/5 NO3- + H2 + 2/5 H+ f N2 + 6/5 H2O HAsO42- + H2 + 2 H+ f H3AsO3 + H2O 2 FeOOH(a) + H2 + 4 H+ f 2 Fe2+ + 4 H2O 1/4 SO42- + H2 + 1/4 H+ f 1/4 HS- + H2O 1/4 HCO3- + H2 + 1/4 H+ f 1/4 CH4 + 3/4 H2O 1/2 HCO3- + H2 + 1/4 H+ f 1/4 Acetate- + H2O
∆G0 (kJ mol-1)a
∆Gr (kJ mol-1)b
-237.2 -240.1 -162.4 -182.5 -48.0 -43.9 -36.1
-206.7 -186.6 -53.9 -39.8 -9.5 -8.2 +2.4
a Calculated from Gibbs free energies of formation from the elements (57-59); O , H , N , and CH as gaseous species. b At the following 2 2 2 4 conditions: T ) 25 °C, [O2] ) 0.21, [N2] ) 0.78, [CH4] ) [NO3-] ) [Fe2+] ) [SO42-] ) [HS-] ) [Acetate-] ) 10-4, [HAsO42-] ) [H3AsO3] ) 10-5, [HCO3-] ) 10-2, [H+] ) 10-7, [H2] ) 10-5 (corresponding to an aqueous concentration of approximately 8 nmol/L). Square brackets indicate activities (aqueous species) or fugacities (gaseous species).
cell cultures (DSMZ, Braunschweig, Germany), and was grown in mineral salts medium, the composition of which is available as Supporting Information to this article. For culture maintenance, the medium was amended with 2 mL of an N2-flushed sodium arsenate (Na2HAsO4 × 7H2O) stock solution (141 mmol/L), added through a sterile filter (cellulose acetate, 0.2 µm) to produce an approximate initial arsenate concentration of 5 mM (∼280 µmol arsenate per bottle), and the headspace was flushed with a H2/CO2/N2 (15:15:70%) gas mixture. The culture was transferred bimonthly into fresh medium (inoculation with 0.36% v/v) and was stored at 14 °C in the dark, without agitation. This method supported continued growth of S. arsenophilum on H2/arsenate for more than 6 months (as observed with DAPI stain direct counts and determination of arsenic species). Analytical Methods. Arsenate and arsenite were determined using the colorimetric molybdenum blue method of Johnson and Pilson (36), as modified by Anderson and Cook (37). Briefly, filtered samples (0.2 µm, Advantec, MFS) were diluted with Milli-Q water to achieve arsenic concentrations 10 nmol/L (22, 29, 65). This is significant in that it suggests that arsenate respiration can easily outcompete H2-dependent methanogenesis by reducing hydrogen to levels that are insufficient for methane production. However, competition for substrate cannot explain the absence of aceticlastic methanogenesis considering the ample supply of acetate (i.e., millimolar concentrations) of which only a fraction is required to supply sufficient hydrogen for As(V) reduction. Consequently, aceticlastic methanogenesis appeared to be directly inhibited by arsenic levels employed here (0.4-1 mmol/L), most likely as an effect of the produced arsenite (66). In contrast to these acetate-rich systems, As(V)-reduction was associated with even lower H2 levels in acetate-free microcosms (Figure 3). While As(V) was reduced at lower rates as compared to the high-acetate system (especially at later stages of the experiment), H2 levels dropped to about 0.4-0.7 nmol/L, indicating a slower release of hydrogen from organic matter and cell biomass breakdown (again as compared to the high-acetate system in which acetate apparently acted as an additional, viable source of H2). This observation underlines the combined effect of both the thermodynamics of the TEAP and the rate of H2-producing fermentation reactions on steady-state concentrations. The control microcosm without As(V) amendment maintained H2 levels at 11-22 nmol/L, which is a level characteristic of ongoing methanogenesis (Table 1). The observed H2 levels during As(V) reduction fall into the range of Fe(III) reduction (Table 1) corresponding well with the thermodynamics of both processes (Table 2). Previous studied have linked the release and mobilization of arsenic to Fe(III) reduction or Fe(III)-reducing microbes (67-69). In this context, dissolved H2 may be an important factor regulating the competition between and (de)coupling of both processes in arsenicimpacted aquifers. While our findings imply a strong potential for microbial As(V) reduction even at substrate-limiting conditions, its relative importance in terms of total electron flow will not only depend upon the energy gain and characteristic H2 levels, but also on the pool size of the available electron acceptors (e.g., the amount of sedimentassociated Fe(III) and As(V)).
This study is part of the Enhanced Research and Capacity Building (ENRECA) project “Water Resources Research in Vietnam” (VIETAS; http://vietas.er.dtu.dk) funded by the Danish Council for Development Research. Funding was also obtained through the Danish Research Training Council.
Supporting Information Available A description of the growth medium. This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review August 29, 2006. Revised manuscript received January 25, 2007. Accepted January 31, 2007. ES062067D
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