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Appl Environ Microbiol. Oct 1999; 65(10): 4611–4617.

Sulfonates as Terminal Electron Acceptors for Growth of Sulfite-Reducing Bacteria (Desulfitobacterium spp.) and Sulfate-Reducing Bacteria: Effects of Inhibitors of Sulfidogenesis


This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)—Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135–149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.

Bacterial production of hydrogen sulfide occurs in many natural environments as well as in various industrial situations. Examples of the latter include oil recovery and metal grinding, water cooling towers, sewer systems, and paper mill wastewaters (7, 9, 14, 24, 31, 32, 36). Because the sulfide produced has toxic and corrosive (9, 13, 14, 24) properties, much effort and expense have been undertaken to control sulfide generation (7, 31).

The production of sulfide is most often assumed to result from sulfate reduction by sulfate-reducing bacteria (SRB) (1, 14, 29, 31). In addition, sulfidogenesis from the anaerobic metabolism of organosulfur compounds, which may occur presumably at a less significant rate, is carried out by bacteria from a range of different physiological groups (7). Few studies have determined the actual sources of hydrogen sulfide and the types of organisms responsible for its production; among these are those dealing with anaerobic organosulfur (mainly thiol) metabolism resulting in sulfidogenesis (11, 29, 3638). Little is known about sulfidogenesis from the metabolism of other organosulfur compounds. Yet, this is an important topic, especially since appreciable quantities and diverse types of organosulfur compounds are present in many environments (11, 17, 18, 29, 3638). These compounds are produced by various biota or may be accumulated as the result of discharge of chemically synthesized forms (25, 35). One particular group of organosulfur compounds, sulfonates, has been found to occur in appreciable concentrations in forest soils and marine environments (2, 42).

We, and then others, recently described the ability of several anaerobic bacteria to dissimilate sulfonates, with the resultant production of hydrogen sulfide; sulfidogenesis resulted from the use of sulfonates as terminal electron acceptors (TEA) (26) and as a sole source of carbon and energy (22, 25) by SRB. However, sulfidogenesis from sulfonate reduction was not mediated solely by SRB; Laue et al. (23) showed that Bilophila wadsworthia utilized a number of aliphatic sulfonates as TEA for growth, in addition to utilizing sulfite and thiosulfate, but not sulfate (23). We recently found that some members of the genus Desulfitobacterium also use some sulfoaliphatics as TEA for growth (25). Like B. wadsworthia, these bacteria do not reduce sulfate but are able to utilize other inorganic sulfur anions as TEA.

Various types of chemicals have been employed to inhibit sulfate reduction; these included biocides such as glutaraldehyde and hypochlorite (16) as well as compounds with more specific mechanisms of inhibition. Examples of the latter include sulfate analogs, molybdate and tungstate (33), and, more recently, anthraquinone derivatives (7). Molybdate and, to a lesser extent, tungstate have been used mainly in ecological studies to determine the substrates used in situ for sulfate reduction (33); these analogs compete with sulfate for the active site of ATP sulfurylase, resulting in formation of an unstable analog-AMP complex which readily hydrolyzes to AMP and the sulfate analog; the latter is then available to again react with ATP sulfurylase (43). Repetition of these events depletes intracellular ATP, thereby halting growth of the bacteria and, as a result, inhibiting sulfate reduction. The carbon substrates originally consumed for growth and sulfate reduction now accumulate instead and are thus considered to have been significant in situ carbon sources for SRB (33).

Cooling et al. (7) reported that derivatives of anthraquinone are very effective in inhibiting hydrogen-dependent growth of SRB and suggested that they be used in conditions where broadly toxic biocide use is not favorable. Their suggested mechanism of inhibition was uncoupling of ATP synthesis associated with normal electron transfer reactions via anthraquinone-mediated electron transfer reactions. Because SRB are unique in requiring ATP to initiate sulfate reduction, they will be more sensitive to any energy drain (as discussed in reference 7). Specificity of the inhibition was indicated since hydrogen uptake was inhibited by 1,8-dihydroxyanthraquinone (1,8-DHAQ) in sulfate-grown cells but not in sulfite- or fumarate-grown cells.

SRB are metabolically very diverse and are able to utilize different TEA for growth (20, 25). We and others have reported that SRB are able to effect a decrease in activities of the enzymes ATP sulfurylase and adenylylphosphosulfate reductase, early enzymes of the sulfate reduction pathway, during growth with alternate electron acceptors like sulfite (19), nitrate (8), fumarate, or sulfonates (25). We wondered whether the two types of inhibitors—sulfate analogs and anthraquinones—with their different modes of inhibition of dissimilatory sulfate reduction might be similarly effective in inhibiting sulfonate respiration. Accordingly, Desulfitobacterium spp. which reduce sulfonates but not sulfate (and thus are presumed not to utilize ATP sulfurylase in their anaerobic respiration) were compared to SRB with respect to the effects of these inhibitors on anaerobic respiratory growth.



The chemicals used were of analytical or reagent grade and were purchased from Fisher Scientific (Pittsburgh, Pa.), Fluka, and Sigma (St. Louis, Mo.). Gases were purchased from Northeast Airgas.

Cultures and cultivation.

Cultures of Desulfitobacterium hafniense (DSM 10664) and Desulfitobacterium sp. strain PCE 1 (DSM 10344) were kindly provided by Jan Gerritse (then of the University of Groningen, Groningen, The Netherlands), those of Desulfitobacterium dehalogenans (DSM 9161) were provided by Juergen Wiegel (University of Georgia, Athens), those of Desulfitobacterium chlororespirans (DSM 11544) and Desulfitobacterium sp. strain Viet 1 were provided by Frank Löffler (Michigan State University, East Lansing), those of Desulfomicrobium norvegicum (DSM 1741) (formerly Desulfomicrobium baculatum) were provided by Derek Lovley (University of Massachusetts, Amherst), and those of Desulfovibrio desulfuricans IC1 (DSM 12129) (26) were from our collection. Desulfitobacterium spp. were grown in a slightly modified medium used for growth of strain IC1 (26); a final concentration of 0.01% (wt/vol) yeast extract (Difco) was added, and the final amount of sulfide used as a reductant was 0.4 mM. The medium used for strain IC1 and Desulfomicrobium norvegicum has been described elsewhere (26). Cultures were grown at 28°C.

Analytical techniques.

Organic acids were quantified by high-pressure liquid chromatography as described elsewhere (26). Culture density (optical density at 650 nm [OD650]) was determined by inserting a Balch tube used for growing cells directly into a Spectronic 20 spectrophotometer. On occasion, cell numbers were determined by using a Petroff-Hausser counting chamber. Protein concentration was determined by a modified Lowry method (27); sulfide was detected and quantified by the method of Cline (6).

Growth studies in the presence of inhibitors.

Cells were grown in Balch tubes containing different concentrations of substrates and inhibitors (molybdate, tungstate, or 1,8-DHAQ). An uninoculated tube with no inhibitor was used as the reference (control).

A set of standards made with molybdate (5 mM) and increasing concentrations of sulfide (5, 10, and 15 mM) were employed to assess any background change in absorbance for the color produced as a result of the sulfide reaction with molybdate.

1,8-DHAQ was added as a solution in acetone (final concentration of acetone in culture was 12 mM) as described elsewhere (7); the final concentration of 1,8-DHAQ was 10 μM.

Desulfitobacterium spp. were maintained in 20 mM lactate–5 mM sulfite and used as the inoculum for inhibitor studies. SRB were maintained in 20 mM lactate–10 mM sulfonate (isethionate or cysteate) and used as the inoculum for such studies.

ATP depletion studies.

Cells in late exponential or early stationary phase were collected; washed once with cold, degassed, and prereduced (2 mM dithiothreitol) 10 mM Tris-HCl buffer (pH 7.2); and resuspended in anoxic, reduced SRB minimal medium (26) lacking both carbon and energy sources. A sulfate analog (molybdate or tungstate) was added to a final concentration of 5 mM, and at a selected time point, 0.3 ml of cells was withdrawn and divided into three 0.1-ml portions. Extralight reagent (0.1 ml; Analytical Luminescence Laboratory, San Diego, Calif.) was added to the cell suspension to release ATP. Then, 0.05 ml of this mixture was transferred into a polystyrene cuvette for measurement in a semiautomatic luminometer (Monolight 2010, model Lumac; Berthold Analytic, Nashua, N.H.). ATP was quantified by using an ATP Bioluminesce assay kit (Sigma). A standard curve for ATP (10−12 to 10−5 g/ml) was prepared under these same experimental conditions as well in the presence of molybdate (5 mM) or tungstate (5 mM) to ensure that these did not affect the luciferase reaction; these sulfate analogs caused no change in the standard curves.

PAGE analysis.

Bacterial cells in late exponential to early stationary phase were centrifuged and washed twice in 10 mM Tris-HCl buffer (pH 7.2). Cells were then centrifuged at 10,000 × g, resuspended in a lesser volume of buffer, and broken in a French pressure cell (15,000 lb/in2). The extract was prepared by centrifugation (10,000 × g; 15 min), and the pellet was discarded. This supernatant was used for polyacrylamide gel electrophoresis (PAGE) analysis by the method of Laemmli (21). Molecular weight standards were purchased from Sigma.


Experiments were done in triplicate to establish reproducibility.


Sulfonate utilization by Desulfitobacterium spp.

Both Desulfitobacterium sp. strain PCE 1 and Desulfitobacterium dehalogenans grew with 2-hydroxyethanesulfonate (isethionate) and alanine-3-sulfonate (cysteate) as TEA (Table (Table1);1); Desulfitobacterium hafniense grew only with isethionate. Depending on the sulfonates tested, growth usually was evident 2 to 3 days after inoculation. Other sulfonates tested as TEA but not able to support growth of any of the Desulfitobacterium spp. were methanesulfonate, taurine, coenzyme M, sulfosuccinate, and 2,3- and 4-sulfobenzoates. Desulfitobacterium chlororespirans and Desulfitobacterium sp. strain Viet 1 did not grow with any of the sulfonates tested (data not shown). None of the sulfonates tested served as a fermentable energy source to support growth of Desulfitobacterium spp.

Effects of sulfate analogs on growth of sulfidogenic bacteriaa

One of the end products of isethionate’s reduction by Desulfitobacterium hafniense was acetate; the final concentration of acetate was more than could be accounted for from lactate oxidation alone; the increase in acetate (and sulfide) was proportional to the amount of isethionate initially provided (data not shown).

Effects of inhibitors on growth of Desulfovibrio desulfuricans IC1 and Desulfitobacterium spp. (i) Inhibitory effects of sulfate analogs.

Both sulfate analogs, molybdate and tungstate, inhibited growth with sulfate by the SRB strain IC1 (Table (Table1).1). Molybdate completely inhibited growth with sulfite, isethionate, and cysteate, while the inhibitory effects of tungstate on growth were only partial (Table (Table1).1). A lag of about 2 to 10 days (depending on the TEA metabolized) longer than that of the control (no addition of tungstate) was always observed in the presence of tungstate, and final growth yields were between one-half and two-thirds those of cultures lacking inhibitor (Table (Table11).

Molybdate and tungstate, however, had different effects on growth of the Desulfitobacterium spp.; neither analog affected growth with sulfite (Table (Table1);1); no difference in lag was observed between cultures with and those without inhibitors. The higher optical density values (Table (Table1)1) observed during growth with molybdate were not due to an increase in cell numbers. Direct cell counts established that cell numbers during growth with molybdate were never more than those in controls. For example, cell numbers of Desulfitobacterium hafniense with isethionate as TEA (Table (Table1)1) were 2.2 × 108 cells per ml (control), 2.1 × 108 cells per ml (molybdate), and 3.1 × 108 cells per ml (tungstate) while the corresponding OD650 values were 0.27, 0.45, and 0.37, respectively. Additionally, the average cell size (3.8 by 0.9 μm) during growth on molybdate was larger than that in its absence (3.5 by 0.6 μm). Differences in cell size in the presence of tungstate were not seen. This phenomenon was observed regularly for the other Desulfitobacterium spp. tested (data not shown). In the presence of the analogs, a lag (about 3 to 5 days longer than that of control) was noted during growth with sulfonates.

A differential inhibitory effect of molybdate was noted in that it inhibited growth with isethionate, but not with cysteate, for both Desulfitobacterium dehalogenans and strain PCE 1. Tungstate partially inhibited growth with cysteate for these same bacteria. This analog had no inhibitory effect on Desulfitobacterium hafniense, with either sulfite or isethionate (Table (Table11).

The color of the culture medium changed to dark orange due to the formation of Mo-S complexes (4, 40) when Desulfitobacterium hafniense grew in the presence of molybdate (Fig. (Fig.1B).1B). Uninoculated tubes with 10 mM sulfide and 5 mM molybdate also exhibited the same dark orange color (Fig. (Fig.1A1A and E). Uninoculated control tubes with the same concentration of molybdate (5 mM) added to increasing concentrations of sulfide (5, 10, and 15 mM) resulted in increases in the color’s intensity. However, the most intense color (15 mM) resulted in an increase of OD650 of only 0.02 compared to that of a blank without molybdate. For strain IC1 cultures with molybdate, no growth resulted and the dark orange color was not observed (Fig. (Fig.1F).1F).

FIG. 1
Effects of sulfate analogs on growth of sulfidogenic bacteria. (A and E) Uninoculated tubes with 10 mM sodium sulfide and 5 mM molybdate; (B to D) Desulfitobacterium hafniense grown with 20 mM lactate and 10 mM isethionate in the presence of 5 mM molybdate, ...

In the presence of tungstate, growth of both Desulfitobacterium hafniense and strain IC1 resulted in the culture medium turning yellowish (Fig. (Fig.1C1C and G).

(ii) Inhibitory effects of 1,8-DHAQ.

When formate was the energy source for sulfate or sulfite reduction with strain IC1, 1,8-DHAQ (10 μM) completely inhibited growth on sulfate and partially inhibited growth on sulfite (Table (Table2).2). However, when isethionate instead was the TEA, the inhibitor was only partially effective: growth occurred but cell yields were only slightly more than one-half that in the absence of 1,8-DHAQ. Growth yields with isethionate in the presence of the inhibitor were mostly identical to formate-dependent sulfate reduction without 1,8-DHAQ (Table (Table2).2). Lactate-dependent sulfate reduction still occurred in the presence of 1,8-DHAQ; cell yields were about two-thirds that of control without inhibitor (Table (Table2).2). When Desulfitobacterium hafniense was grown with lactate and sulfite (data not shown) or isethionate in the presence of 1,8-DHAQ, a long lag of 22 to 25 days (longer than that of control with no addition of 1,8-DHAQ) occurred before growth; final growth yields were about one-half that of control without inhibitor (Table (Table2).2).

Effects of 1,8-DHAQ on growth of sulfidogenic bacteriaa

As 1,8-DHAQ was added as a solution in acetone, we tested the effects of acetone on growth of strain IC1 and the Desulfitobacterium spp.; acetone (10 mM) neither inhibited growth nor served as a carbon and energy source.

Effects of sulfate analogs on intracellular ATP content in cell suspensions of Desulfovibrio desulfuricans IC1 and Desulfitobacterium spp.

Both molybdate and tungstate effected similar decreases in ATP content in cell suspensions of strain IC1 (Table (Table3)3) grown with sulfate or isethionate as TEA.

Effects of sulfate analogs on depletion of ATP in cell suspensions of sulfidogenic bacteriaa

Cell suspensions of Desulfitobacterium sp. strain PCE 1 exhibited no decrease in ATP content in the presence of molybdate (Table (Table3).3). Cellular ATP content actually increased slightly in sulfite-grown cells. In contrast, tungstate caused a significant and reproducible decrease in ATP content in cells grown with either sulfite or cysteate (Table (Table33).

In the presence of tungstate, cell suspensions of Desulfitobacterium hafniense also exhibited a significant decrease in ATP content in cells grown with either sulfite or isethionate (Table (Table3)3) compared to the effects of molybdate on ATP depletion.

PAGE profiles of Desulfitobacterium spp. and SRB grown with various TEA.

A polypeptide of approximately 97 kDa was observed with extracts of Desulfitobacterium hafniense, strain PCE 1, and the SRB Desulfomicrobium norvegicum as well as strain IC1 grown with isethionate as TEA (Fig. (Fig.2).2). This polypeptide was not detected in the bacteria grown on TEA other than isethionate.

FIG. 2
PAGE profiles of sulfidogenic bacteria grown with various TEA; the carbon and energy source for growth of all bacteria was lactate. Each lane contained approximately 20 to 25 μg of protein (see Materials and Methods). Polypeptides were stained ...


Sulfonate reduction by sulfite- and sulfate-reducing bacteria.

As established earlier (22, 26) for SRB, sulfite-reducing bacteria (B. wadsworthia [23] and Desulfitobacterium spp. [this study]) are also able to utilize sulfonates as TEA for growth and release the sulfonate-sulfur as sulfide. In the case of isethionate reduction by Desulfitobacterium hafniense, isethionate was metabolized to the end products acetate and sulfide (data not shown), as had been reported previously for SRB strain IC1 (26).

As was the case for strain IC1 and Desulfomicrobium norvegicum (Fig. (Fig.2),2), PAGE analysis of extracts of Desulfitobacterium spp. revealed the presence of at least one distinctive polypeptide band (ca. 97 kDa) seen only when isethionate was employed as the TEA for anaerobic respiratory growth. Acetate and sulfide production from isethionate metabolism, along with the finding that this polypeptide is produced only in cells grown with isethionate (but not other TEA, including cysteate) as TEA, is a basis of our proposal that the pathways for isethionate’s metabolism and the proteins involved in these two different bacterial groups will be similar.

Effects of sulfate analogs on growth with various TEA by Desulfovibrio desulfuricans IC1 and Desulfitobacterium spp. (i) Molybdate.

Considering molybdate’s assumed mode of action (see above), its inhibitory effect on the growth of strain IC1 with the range of TEA tested was surprising, especially since the level of ATP sulfurylase was markedly lower in cells grown with sulfite and sulfonate-sulfur than it was in cells grown with sulfate (25). This inhibition (Table (Table1)1) and the decrease in ATP content (Table (Table3)3) we take to mean that the levels of ATP sulfurylase with, for example, isethionate as TEA still remain sufficient to effect the depletion of ATP and stop growth.

The inhibition of growth of two Desulfitobacterium spp. employing isethionate as TEA was equally surprising, based on the expectation that ATP sulfurylase is absent in these bacteria and the demonstration that there were no dramatic decreases in cellular ATP content of Desulfitobacterium sp. strain PCE 1 and Desulfitobacterium hafniense (grown with either sulfite or sulfonate) upon treatment with this inhibitor. It is quite unlikely that inhibition of growth with isethionate reflected formation of Mo-S complexes that resulted in sulfur being made unavailable for assimilatory purposes, as yeast extract had been included in the medium (see Materials and Methods).

Taken together, these results suggest that molybdate must exert mechanisms of inhibitory action other than those of a competitive inhibitor of ATP sulfurylase or sulfate permease (28). If so, the unexpected effect on strain IC1 grown with TEA other than sulfate may also have an additional explanation(s).

The increase in size of Desulfitobacterium spp. grown in the presence of molybdate but not tungstate was unexpected and has not been reported before. Since controls established that the orange coloration of the Mo-S complexes did not have a significant effect on optical density, the increase (Table (Table1)1) very likely is due to the augmentation in size of the Desulfitobacterium spp. grown with molybdate.

(ii) Tungstate.

In contrast to molybdate, this analog inhibited growth of strain IC1 when sulfate was TEA but was only partially effective with sulfite or the sulfonates (OD650 values were at least one-half those of cultures without tungstate). This is probably the result of lowered but constitutive ATP sulfurylase production in sulfite- or sulfonate-grown cells (as noted below) causing a steady loss of intracellular ATP occurring in concert with ATP production from sulfonate respiration. Thus, the maximum energy yield obtainable from the reduction of sulfonate is not achieved by strain IC1 in the presence of tungstate. Consistent with previous results (39), cell suspension studies (Table (Table3)3) showed that tungstate was equally as effective as molybdate in depleting intracellular ATP of strain IC1.

Growth of Desulfitobacterium spp. with cysteate (but not other TEA) was partially inhibited. Stimulation in growth of all three Desulfitobacterium spp. by tungstate was noted only during growth on isethionate, but not sulfite, as TEA. When resting cell suspensions were treated with tungstate, a substantial reduction of ATP content ensued even for combinations where growth inhibition was not seen. This phenomenon was also observed for a freshwater denitrifying culture as well (39) and thus is probably an effect not specific to sulfidogenic bacteria. The explanation(s) for these apparent discrepancies is not clear and is beyond the scope of the present study.

The results from the studies with sulfate analogs are consistent with past suggestions that the Mo-S complex itself could act as an inhibitor (as discussed in references 4 and 33). Tungstate does not form these complexes (33) and, unlike molybdate, did not cause the unexpected inhibition of growth with isethionate by two Desulfitobacterium spp. In addition, since tungstate was specific in its ability to completely inhibit growth of the SRB strain IC1 only with sulfate but not sulfite, isethionate, or cysteate, this property may prove useful in ecological studies concerning sulfonate reduction, as will be discussed below.

Effects of an anthraquinone derivative on anaerobic respiration.

As anticipated from the report of Cooling et al. (7), we found that growth of strain IC1 was inhibited by 1,8-DHAQ during formate-dependent respiration with sulfate but not sulfite. When, however, isethionate was substituted as TEA, growth was only partially inhibited. Although this pattern is similar to that seen with tungstate, most likely it is for different reasons. Tungstate affects growth by causing a degradation of intracellular ATP via ATP sulfurylase activity, while 1,8-DHAQ is presumed to act by partially uncoupling ATP synthesis from electron transport (7).

On the other hand, lactate-dependent sulfate reduction by strain IC1 could occur even in the presence of the inhibitor, probably because the additional ATP obtained from substrate-level phosphorylation helped to alleviate a shortage caused by the uncoupling effects of the inhibitor (there is no substrate-level ATP generated from formate oxidation to carbon dioxide). This, again, is consistent with the results of Cooling et al. (7), who reported that pyruvate alleviated 1,8-DHAQ inhibition of hydrogen-dependent sulfate respiration by providing substrate-level ATP generated by the phosphoroclastic reaction and needed for sulfate activation.

With excess formate as the electron donor, the cell yields from isethionate reduction were approximately twice those resulting from the reduction of an equimolar amount of sulfate (Table (Table2).2). This difference in cell yield could reflect an energy-yielding reduction of the carbon-sulfur bond of isethionate (25) coupled with the lack of a requirement for expenditure of ATP in sulfate activation. Thus, it is likely that the energy yield from isethionate metabolism is high enough to permit strain IC1 to overcome the uncoupling effects of 1,8-DHAQ on electron transport.

1,8-DHAQ caused a great lag and a twofold reduction in final growth yields of Desulfitobacterium hafniense grown with sulfite (data not shown) or isethionate (Table (Table2),2), suggesting that it is only partially effective in inhibiting growth of this organism on sulfonates and other sulfur-containing TEAs.

The long lags observed in the presence of both the sulfate analogs and 1,8-DHAQ suggest that the cells had to adjust to the energy-draining effects (and perhaps other unknown inhibitory effects) of the inhibitors before growth could occur. These have not been reported before, most likely a reflection that there have been few studies of the effects of these inhibitors on growth of SRB with different TEA.

Environmental and ecological significance.

The results reported here, along with earlier ones (22, 23, 25, 26), extend the range of sulfide-sulfur sources beyond those classically considered as involved in anaerobic respiration. The presence of sulfonates in different habitats and the ability of SRB and sulfite-reducing bacteria to reduce sulfonate-sulfur to sulfide indicate, with high probability, that inorganic forms of sulfur are not the sole source of biogenic sulfide in anaerobic respiratory events. Examples include linear alkybenzenesulfonate discharge from laundromats (10) and those present in industrial fluids (15). SRB present in cutting fluids have been found able to grow and produce sulfide by metabolizing petroleum sulfonates (15); the metal sulfonates (30) often included as lubricants in cutting fluids may be another source of sulfide.

The fact that some compounds used to inhibit sulfate reduction are less effective in inhibiting sulfide formation as a result of sulfonate reduction may have implications for the use of such inhibitors both in the prevention of biofouling and biodeterioration and in ecological studies. Further assessment of the prospect, for example, that SRB may carry out sulfonate reduction if an inhibitor of sulfate reduction is present is needed; the results from our inhibitor studies show that the SRB strain IC1 could grow with isethionate and cysteate in the presence of tungstate but not molybdate. Therefore, we suggest that tungstate, in addition to molybdate, be used in ecological studies to discriminate between sulfate versus sulfonate and organosulfur reduction by SRB and other sulfidogenic bacteria.

The demonstration that Desulfitobacterium spp., isolated for their ability to utilize organohalogens as TEA (3, 5, 12, 34), are also able to carry out sulfonate respiration makes important an examination of whether sulfonate respiration might occur in preference to organohalogen respiration, in analogy to the reduction of sulfate, sulfite, or thiosulfate in preference to the reduction of organohalogens by Desulfomonile tiedjei (41).

It seems clear, then, that numerous aspects of bacterial anaerobic sulfonate utilization may have significant consequences for our knowledge in the areas of enzymology, ecological studies, and the use of inhibitors to prevent biofouling.



Another bacterium, Desulforhopalus singaporensis, with a decreased activity of adenylyl phosphosulfate reducatase activity (and presumably of ATP sulfurylase as well), failed to grow when either molybdate or tungstate was present during taurine fermentation (T. J. Lie, M. L. Clawson, W. Godchaux, and E. R. Leadbetter, Appl. Environ. Microbiol. 65:3328–3334, 1999).


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