APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1994, p. 3011-3019 Vol. 60, No. 8 0099-2240/94/$04.00+0 Copyright X 1994, American Society for Microbiology Isolation, Growth, and Metabolism of an Obligately Anaerobic, Selenate-Respiring Bacterium, Strain SES-3 RONALD S. OREMLAND,* JODI SWITZER BLUM, CHARLES W. CULBERTSON, PIETER T. VISSCHER, LAURENCE G. MILLER, PHILLIP DOWDLE, AND FRANCES E. STROHMAIER U.S. Geological Survey, Menlo Park, Califomia 94025 Received 6 April 1994/Accepted 24 May 1994

A gram-negative, strictly anaerobic, motile vibrio was isolated from a selenate-respiring enrichment culture. The isolate, designated strain SES-3, grew by coupling the oxidation of lactate to acetate plus CO2 with the concomitant reduction of selenate to selenite or of nitrate to ammonium. No growth was observed on sulfate or selenite, but cell suspensions readily reduced selenite to elemental selenium (Seo). Hence, SES-3 can carry out a complete reduction of selenate to Seo. Washed cell suspensions of selenate-grown cells did not reduce nitrate, and nitrate-grown cells did not reduce selenate, indicating that these reductions are achieved by separate inducible enzyme systems. However, both nitrate-grown and selenate-grown cells have a constitutive ability to reduce selenite or nitrite. The oxidation of ['4C] lactate to '4Co2 coupled to the reduction of selenate or nitrate by cell suspensions was inhibited by CCCP (carbonyl cyanide m-chlorophenylhydrazone), cyanide, and azide. High concentrations of selenite (5 mM) were readily reduced to Seo by selenate-grown cells, but selenite appeared to block the synthesis of pyruvate dehydrogenase. Tracer experiments with [75Se] selenite indicated that cell suspensions could achieve a rapid and quantitative reduction of selenite to Seo. This reduction was totally inhibited by sulfite, partially inhibited by selenate or nitrite, but unaffected by sulfate or nitrate. Cell suspensions could reduce thiosulfate, but not sulfite, to sulfide. These results suggest that reduction of selenite to Seo may proceed, in part, by some of the components of a dissimilatory system for sulfur oxyanions.

The bacterial reduction of selenate ions to elemental sele- and strain SES-1 constituted the only reports of DSeR in pure nium (Se') represents an important aspect of the selenium culture. We now report on the characteristics of a novel cycle by which this toxic element is sequestered into sediments freshwater isolate, designated strain SES-3, a strictly anaerobic (30). The phenomenon is widespread in nature and includes vibrio which grows by DSeR coupled with the oxidation of contaminated as well as pristine sediments, and activity in lactate to acetate plus carbon dioxide. assayed sediment samples occurs without a noticeable lag (32, 40). Selenate reduction to Seo represents an important quan- MATERIALS AND METHODS titative sink for selenium oxyanions in shallow, lotic environ- ments, such as waste treatment ponds (31), and may be Isolation and cultivation. Strain SES-3 was isolated from an exploited for bioremediation purposes (9, 20, 27, 28). Although acetate-oxidizing, selenate-respiring enrichment (39) recov- much is known about assimilation of selenium in its role as an ered from Massie Slough, a freshwater marsh in the Stillwater enzyme cofactor (38), relatively little is known about bacteria Wildlife Management Area of western Nevada. Ambient which are capable of reducing selenate in sufficient quantities selenate concentrations in Massie Slough waters were about 51 to contribute to the biogeochemical cycling of this element. nM (32). In surveys of different sediment types, Massie Slough Two facultative anaerobes which have the ability to bio- material had the highest potential DSeR rates (40) and the chemically reduce selenate to Seo have been isolated. These most rapid rates of in situ selenate turnover (32). The enrich- include a strain of Pseudomonas stutzeri (14) and a novel ment was streaked onto 2% agar plates composed of the same species, Thauera selenatis (21, 22). P. stutzeri seems to reduce mineral salts medium (20 mM acetate plus 20 mM selenate). selenium oxyanions solely for the purpose of detoxification, All manipulations were performed in an anaerobic glove box. while in T. selenatis, dissimilatory selenate reduction (DSeR) After the enrichment was streaked, the plates were sealed in an to selenite is a mode of anaerobic respiration capable of anaerobic jar and incubated at room temperature. After a few sustaining growth (19). With regard to strict anaerobes, after weeks, red colonies were abundant, and isolated colonies were adaptation to selenium oxyanions, Wolinella succinogenes can picked and transferred into crimp-seal 25-ml culture tubes precipitate Seo from selenate or selenite but cannot use containing 10 ml of liquid medium. The tubes were sealed selenium oxyanions for respiratory growth (44). A strictly under either 4:1 N2-C02 or 4:1 H2-CO2. No growth was anaerobic, gram-negative coccus (strain SES-1) was isolated detected in the tubes with acetate under N2-C02; however, from estuarine sediments which grew via DSeR to Seo by using growth, evident as turbidity and the formation of red elemental acetate as the electron donor (30). Unfortunately, SES-1 was Seo, occurred in the tubes with H2. The H2 requirement may lost before it could be studied definitively. Thus, T. selenatis have been due to the initial selective pressure caused by the presence of -1% H2 in the sealed petri-incubator jars. We determined that the isolate could grow on acetate plus H2, on without * Corresponding author. Mailing address: U.S. Geological Survey, pyruvate or lactate under N2-C02, but not on acetate ms 465, 345 Middlefield Rd., Menlo Park, CA 94025. Phone: (415) H2. For convenience, we worked with a lactate-based medium, 329-4482. Fax: (415) 329-4463. Electronic mail address: roremlan@ and all subsequent growth experiments were conducted with rcamnl.wr.usgs.gov. lactate as the electron donor. The medium contained the 3011 3012 OREMLAND ET AL. APPL. ENVIRON. MICROBIOL. following: K2HPO4 (0.225), KH2PO4 (0.225), NaCl (0.46), lactate and 5 mM Na selenite (or 1 mM nitrite). The effect of (NH4)2SO4 (0.225), MgSO4* 7H20 (0.117), yeast extract (1.0), various electron acceptors (sulfate, selenate, sulfite, thiosul- Na lactate (2.24), Na2SeO4 (3.78) or NaNO3 (1.7), NaHCO3 fate, nitrate, and nitrite; 5 mM each) upon reduction of 2.5 (4.2), Na2S * 9H20, (0.1), and cysteine-HCl (0.1) (all in grams mM selenite was monitored in a tracer experiment with per liter); SL10 trace element solution (1.0 ml) (47); Na2WO4 [75Se]selenite (Amersham Inc., Arlington Heights, Ill.; specific (3 pug/liter); and vitamin solution (10 ml). With the exception activity, 757 mCi mmol-1). Cell suspensions of selenate-grown for quantification of growth on nitrate, ammonium sulfate was cells (30 or 70 ml) received 1.5 or 3.0 ,uCi, respectively, of the included routinely when cells were grown with nitrate as the radioisotope, and loss of [75Se]selenite from solution and the electron acceptor. The vitamin solution contained the follow- precipitation of 75Se0 was determined by expressing 0.5 ml of ing (in milligrams per liter): p-aminobenzoic acid (5), biotin the suspension through disposable 0.2-,im-pore-size filters (5), folic acid (2), pyridoxine-HCl (1), riboflavin (5), thiamine (13-mm diameter; Alltech Inc.). The filters were rinsed by (5), nicotinic acid (5), pantothenic acid (5), thiotic acid (5), and injection with an additional 0.5 ml of unlabeled buffer, and the vitamin B12 (0.1). The basal salts were dispensed into serum filters and the filtrate were counted separately (see below). bottles (usually with 30 ml in 59-ml bottles but, if proportion- Nitrate-grown cells were tested in a similar fashion for their ally scaled up, with as much as 900 ml in special 2-liter serum ability to reduce [75Se]selenite. The ability of selenate- or bottles) or into 10 ml of medium in 25-ml culture tubes, crimp nitrate-grown cells to oxidize [U-14C]lactate (New England sealed under N2-CO2, and autoclaved. After the bottles were Nuclear, Boston, Mass.; specific activity, 177 mCi mmol-') to cooled, the vitamins, lactate, electron acceptors, reducing 14Co2 with various electron acceptors (NOf3, SeO42-, MnO2, agents, and bicarbonate solutions were added by injection from FeOOH, HAs042-, fumarate, S032-; 5 mmol liter-') or with anaerobic, filter-sterilized stock solutions. The final pH was various ionophores or inhibitors was examined. Cell suspen- 7.3. Cultures were incubated statically at 30°C. Gentle shaking sions were incubated with 1 mM Na lactate plus 2.2 ,uCi of was found to inhibit growth of the enrichment (38a), and [14C]lactate for time periods ranging from 2 to 19.25 h (see therefore, the pure cultures were not shaken. Subsamples of text) at 30°C with slow rotary shaking (-100 rpm). Activity was the liquid and gas phases were taken by syringe (after hand stopped by injection of 1 ml of 6 N HCl, and bottles were swirling to achieve uniform dispersement) for determination of shaken (250 rpm) for -20 h after which the headspace was cell density and the concentrations of substrates and interme- analyzed for 14Co2 (see below). Carbonyl cyanide m-chloro- diates. The volume withdrawn was replaced with N2-CO2. phenylhydrazone (CCCP) and 2,4-dinitrophenol were added Washed cell suspensions. Two liters of late-log-phase cells from 10 mM stock solutions prepared in acetone. Corrections grown with either selenate or nitrate as the electron acceptor were made for inhibition caused by acetone or ethanol addi- were dispensed into 10 200-ml centrifuge bottles and harvested tion without the inhibitors. Azide and cyanide were added by centrifugation at 10,000 x g for 20 min at 4°C. After the from 1 M and 50 mM stock solutions in water. Final concen- bottles were decanted, the pellets were washed by being trations were as follows: azide, 10 mM; cyanide, 500 jiM; resuspended in 50 to 100 ml of buffer solution composed of (in 2,4-dinitrophenol, 100 ,uM; and CCCP, 100 ,uM. To monitor grams per liter): K2HPO4, 0.338; KH2PO4, 0.338; NaCl, 0.46; the pathway of nitrate reduction, cell suspensions (100 ml) MgCl2 - 6H20, 0.117; and CaCl2 - 2H20, 0.06 (with cysteine- were given 25 mM Na lactate and 25 mM NaNO3 and sulfide [0.1 each] as a reductant [pH 7.3]). Reducing agent was subsampled over an incubation period for nitrate, nitrite, eliminated when testing for the ability of selenate-grown cells ammonium, and nitrous oxide. In another experiment, 15% to reduce sulfite or thiosulfate to sulfide. The resuspended cells acetylene was added to the headspace of nitrate-grown cell (volume, 200 to 400 ml) were combined into four bottles and suspensions, prepared as described above, to screen for en- recentrifuged. In total, the cells received two washings. The hanced production of N20 by inhibition of nitrous oxide final pellets were rinsed without disruption with 10 ml of buffer reductase (1, 33). Results were compared with those of a to remove all traces of ions carried over from the washings, and control incubated without acetylene. the pellets were combined and resuspended in 200 to 300 ml of Analyses. Selenium and nitrogen oxyanions were deter- buffer to achieve the final cell suspension. A subsample was mined by ion chromatography (29). Lactate and acetate were taken to determine cell density by direct counts (see below) or, determined by high-performance liquid chromatography in the case of some nitrate-grown cells, were filtered for dry (HPLC) (3), a method which could also resolve and quantify weight determinations, and cells per milliliter were calculated selenate, selenite, sulfite, and thiosulfate. To prevent oxidation from standard curves of cells per milliliter versus dry weight or in samples, we preserved the sulfite in 10% acetonitrile (13, absorbance. Aliquots of the cell suspension (various volumes) 41). Ammonium was determined by the phenol-hypochlorite were then transferred to serum bottles and crimp sealed with method (37). The combination of gas chromatography and gas thick black butyl rubber stoppers. All manipulations were proportional counting was used for analysis of 14C02 (4). performed in an anaerobic glove box. The sealed bottles were Quantification of 75Se was done on a Packard Minimax 5000 removed from the glove box, and the headspace was flushed series auto-gamma counter, and that of 14C was done upon a with 02-free N2 for 10 min (flow rate, -80 ml min-'). Beckman LS 6000 scintillation counter. Separation of ["4C]lac- Experiments were started by the injection of substrates, elec- tate and [14C]acetate was achieved on the HPLC, and eluted tron acceptors, inhibitors, and radioisotopes (see below). Cell fractions were collected and counted by liquid scintillation (4). suspensions were incubated at 30°C with slow rotary shaking Nitrous oxide was measured by 63Ni-electron capture detector (100 rpm) and subsampled by syringe, and the volume with- gas chromatography of headspace samples (33). A dimension- drawn was replaced by N2. less Henry's Law constant of 1.7 for N20 in distilled water at To determine if selenate-grown cells could reduce nitrate or 30°C (2) was applied to the partitioning equation (42) to if nitrate-grown cells could reduce selenate, 30 ml of cell calculate the total quantity of N20 in the assay bottles (gas suspension was injected with 20 mM Na lactate and received phase, 27 ml; liquid phase, 30 ml). Bacterial biomass was either 5 mM selenate or 10 mM nitrate. Controls containing quantified by performing acridine orange direct counts (11). 200 ,g of chloramphenicol ml-' were run in parallel. To Molar growth yields (YMd were based on a calculated conver- determine if selenate-grown cells could reduce selenite (or sion factor of 1.9 x 10-1 mg cell-' achieved by comparison of nitrite), cell suspensions (30 ml) were injected with 20 mM direct counts with dry weight data. Sulfide was determined by VOL. 60, 1994 AN OBLIGATELY ANAEROBIC, SELENATE-RESPIRING BACTERIUM 3013

FIG. 1. Electron micrograph of SES-3. a potentiometric procedure employing an ion-specific micro- titatively, the Se' represented only about 5% of the selenate electrode (45). reduced, with the bulk evident as selenite. Cell densities Reagents. Manganese dioxide and FeOOH were prepared increased to 6 x 108 cells ml-' (Fig. 2B), with a doubling time by the methods of Lovley and Phillips (16). All other chemicals of 4.5 h. No growth occurred in medium lacking selenate, with were of standard reagent-grade, commercial-supply quality. air or sulfate as the electron acceptor, or in selenate medium Scanning electron micrographs. The procedures outlined by without lactate (not shown). In contrast to the results with Smith et al. (36) were employed. selenate, growth with nitrate as the electron acceptor was more rapid and extensive (Fig. 3). Strain SES-3 completely con- RESULTS sumed the lactate, generated an equivalent quantity of acetate, and yielded a consumption ratio of nitrate to lactate of -1.1 Morphological characteristics. Strain SES-3 is a motile (Fig. 3A). Nitrate loss was followed by the transient appear- vibrio which stains gram negative. Electron microscopy re- ance of nitrite and then by the accumulation of ammonium vealed cells to be about 1.5 ,um in length and about 0.3 ,um in (Fig. 3B). The final levels of ammonium plus nitrite accounted diameter (Fig. 1). Strain SES-3 is similar in appearance to for 88% of the nitrate consumed, with the missing quantity Desulfovibnio sp. presumably retained for cellular N biosynthesis. The doubling Growth. SES-3 grew with lactate as the electron donor and time on nitrate was 3 h, and final cell densities were 109 cells selenate as the electron acceptor (Fig. 2). There was a stoi- ml-,. chiometric balance between the amount of lactate oxidized and Growth did not occur when selenite was the electron accep- acetate formed (Fig. 2A) and between the quantities of tor (data not shown). Inoculation of selenate-grown cells into selenate consumed and selenite produced (Fig. 2B). However, medium containing 10 mM selenite demonstrated no increase only about half of the lactate was consumed, with a consump- in cell density or consumption of either lactate or selenite over tion ratio of selenate to lactate of -1.85. The inoculated a 72-h period (not shown). We hypothesized that this selenite medium turned a dense orange-red color with time as a result concentration was toxic and could not sustain growth, but of the formation of the amorphous Se' precipitate, but quan- perhaps growth could occur at lower levels. We attempted to 3014 OREMLAND ET AL. APPL. ENVIRON. MICROBIOL.

30 35 . I

25 30 ACETATE_ _.0o 2 4 0- -o- _ E < ~~~~A E LACTATE / aNLCTATE 25 T 20 I-.-. UJ 'U

0 is5 0- - :- 0 15 UJc 0 10 - < 10 / A 5 / ACETATE 5 d / _ I1 10 x 0 40 80 120 160 200 0 10 20 30 40 50 60 TIME (HOURS) TIME (HOURS)

30

-i 2

X -i z 0.. CO _. -i S1 co 0 0 C.) O 0 0I co z

4r- 160_200 0 40 120 200 so 160 0 10 20 30 40 50 60 TIME (HOURS) TIME (HOURS) FIG. 2. Growth and metabolism of SES-3 on lactate with selenate FIG. 3. Growth and metabolism of SES-3 on lactate with nitrate as as the electron acceptor. (A) Consumption of lactate (-) and produc- the electron acceptor. (A) Consumption of lactate (0) and production tion of acetate (0); (B) consumption of selenate (A), production of of acetate (0); (B) consumption of nitrate (U); production of nitrite selenite (A), and cell density (+). (O) and ammonium ([1), and cell density (+).

detect increases in cell density in medium containing 0.5, 1.0, cells rapidly reduced 5 mM selenate, and selenite was a and 5.0 mM selenite compared with a control without electron transient intermediate (Fig. 4). A quantitative recovery of Seo acceptor. However, even these levels of selenite proved inhib- (as measured by dry weight) was observed by 20 h, after which itory to growth. Initial cell densities were 6 x 106 to 9 X 106 time Seo slowly declined (not shown). Suspensions of nitrate- cells ml-'. After 24 h, some growth occurred (7 x 107 cells grown cells achieved a complete reduction of nitrate, with ml- 1) in selenite-free medium, while cultures with 5.0, 1.0, and nitrite as a transient intermediate, and quantitative recovery as 0.5 mM selenite had 2 x 107, 3 x 107, and 3 x 107 cells ml-1, ammonium (Fig. 5). Nitrous oxide accumulated in the head- respectively. None of the cultures demonstrated significant space over time; however, the total amount of N20 (gas plus changes from these densities over the subsequent 144 h of liquid phases) accounted for only 1.5% of the total nitrate. incubation. Therefore, the growth observed in the control was Addition of acetylene to the headspace of cell suspensions did likely due to carryover of selenate, and selenite proved inhib- not enhance the production of N20. After 24 h of incubation, itory to this small amount of growth. We did not detect any a 10-ml suspension having 100 ,umol of nitrate at the outstart significant loss of selenite over 72 h of incubation. and incubated with acetylene had accumulated 1.0 ,umol of Growth cultures of SES-3 labeled with [U-'4C]lactate pro- N20. However, cells without acetylene had a comparable duced 14C02 as well as [14C]acetate (not shown). After 19.25 h amount of N20 at 24 h (i.e., 1.3 ,umol). of incubation, nitrate-grown cells oxidized 96% of the ['4C]lac- Nitrate-grown cells rapidly reduced 10 mM nitrate but had tate, with 38% of the initially added 14C recovered as 14Co2 no capacity to reduce selenate (Fig. 6). Likewise, selenate- and 58% recovered as ['4C]acetate. In selenate-grown cultures, grown cells were able to reduce selenate but had no capacity to after 19.25 h, 50% of the lactate was oxidized, with 13% of the reduce nitrate. Selenate-grown cells were able to reduce initial 14C recovered as 14C02 and 37% recovered as ['4C]ac- nitrate only after prolonged exposure (-120 h), and this etate. activity was inhibited by chloramphenicol (not shown). In Washed cell suspensions. Suspensions of selenate-grown contrast, both selenate- and nitrate-grown cells had the con- VOL. 60, 1994 AN OBLIGATELY ANAEROBIC, SELENATE-RESPIRING BACTERIUM 3015

12 12

8 I- E 4 4 0

6' 54

2 z 0~~~~~~~~~6. 0I 0 10 20 30 40 50 TIME (hours)

0 5 10 30 35 FIG. 6. Constitutive metabolism of oxyanions by washed cell sus- pensions of nitrate- or selenate-grown cells. Consumption of nitrate TIME (HOURS) (*) or selenate (A) by nitrate-grown cells and consumption of nitrate (O) or selenate (A) by selenate-grown cells are shown. Cell suspension FIG. 4. Consumption of selenate (A), and production of selenite densities for selenate- and nitrate-grown cells were 4.0 x 108 and 3.9 (A) during incubation of selenate-grown cell suspensions with 10 mM x 109 cells ml-', respectively. lactate. Cell density was 8.5 x 108 cell ml-'.

,uM sulfite over a 24-h period. However, autoclaved controls stitutive ability to reduce their corresponding opposite reduced also demonstrated a similar loss of sulfite with time (not intermediate (i.e., nitrite or selenite). Replicate suspensions of shown), which suggests a chemical rather than biological selenate-grown cells completely removed 1 mM nitrite within 5 removal. Cells incubated with sulfate did not consume sulfate h (linear rate, -0.26 ,umol ml-l h'), while there was no loss or produce sulfide. We detected comparable removals of of nitrite from an autoclaved control (not shown). Nitrate- thiosulfate and sulfite in nitrate-grown cells and detected by grown cells were readily able to reduce [75Se]selenite (see smell (but did not quantify) the presence of sulfide in cells below). incubated with thiosulfate. Incubation of selenate-grown cells with 10 mM selenite Washed-cell experiments with [14C]lactate. The ability of resulted in the initial accumulation of acetate, followed by the nitrate- or selenate-grown cells to employ a variety of alternate accumulation of pyruvate (not shown). After 8 h of incubation, electron acceptors for the oxidation of lactate to 4CO2 is cells had consumed 7 mM lactate and produced 4 mM acetate, shown in Table 1. Both selenate- and nitrate-grown cells used an action that was coupled to the removal of 5 mM selenite. their corresponding electron acceptors (but not each other) After 33 h, lactate consumption increased to 12 mM with no and were equally able to use fumarate, arsenate, and Fe3+. The further production of acetate but with the appearance of 2 mM results for MnO2 were equivocal in that while this electron pyruvate. Pyruvate ultimately reached 4 mM after 56 h. In acceptor clearly stimulated 14C02 production over the endo- contrast, cells incubated with 5 mM selenate accumulated only genous rate, an equal amount of activity was detected in acetate. Similar pyruvate accumulation was also evident in cells autoclaved cells. Apparently, lactate is chemically oxidized by incubated with 5 mM selenite or with 10 mM selenite plus 5 stimulated mM selenate (not shown). MnO2. Oxygen 4CO2 production by nitrate-grown Selenate-grown cells were able to slowly reduce thiosulfate to sulfide in a 1:2 ratio. After 19 h of incubation, a drop in thiosulfate from 1,003 to 884 ,uM was detected; while sulfide TABLE 1. Oxidation of [U-"'C]lactate to 14CO2 by washed levels increased from 0 to 234 ,uM. We also observed consump- suspensions of selenate- and nitrate-grown cells incubated tion of sulfite but did not detect formation of sulfide. In in the presence of various electron acceptorsa duplicate cell suspensions, we observed a linear loss of -200 % "'CO2 recovered Electron acceptor Selenate- rown Nitrate-grown cells cellsc 8.0 120 Selenate 68 12 Selenate (boiled)d 2 NDe aE 6.0 *\e * , .. 90 Nitrate 32 65 Fumarate 65 60 FeOOH 65 68 Z4.0t \,,,i 60 Arsenate 73 69 0 MnO2 67 61 ~~~~~~~~~~zMnO2 (boiled) 55 58 0.Od2.0 z ' ,/' < '\ 30 Oxygen ND 90 Sulfite 7 2 None 27 12 0 6 12 18 24 30 a Electron acceptor concentration, 5 mmol liter-'; lactate concentration, I mM; incubation time, 19 h. Time (h) b Cell density, 1.2 x 109 cells ml-'. c Cell density, 3.9 x 109 cells ml- I. FIG. 5. Consumption of nitrate (0) and production of nitrite (-), d No 14C02 was detected in boiled nitrate-grown cells in the presence of nitrous oxide (K), and ammonium (V) in cell suspensions of nitrate- nitrate, fumarate, FeOOH, or arsenate. grown cells. Cell density was 1.9 x 109 cells ml-'. eND, not determined. 3016 OREMLAND ET AL. APPL. ENVIRON. MICROBIOL.

40 40

E 30 E 30 C 0. "OC O 0 20 a):tL 20 tn 10 10 1

0 0 1 2 3 4 5 0 1 2 3 4 5

Hours Hours

aI 4 < -o O °- -e- -oEW 3!0 E tX Q. c 0. C X 0s D0 0 ao 0 2 c#)o C#) tl°3 0

i.la . . 1 2 3 4 5 0 1 2 3 4 5

Hours Hours FIG. 7. Reduction of 75SeO32- (0) and production of 75Se0 (A) in selenate-grown cell suspensions containing 20 mM lactate as the electron donor and incubated with 2.5 mM selenite (A), 2.5 mM selenite plus 5.0 mM sulfate (B), 2.5 mM selenite plus 5 mM selenate (C), or 2.5 mM selenite plus 5 mM sulfite (D). Cell density was 3.5 x 109 cells ml-'. cells (selenate-grown cells were not tested). Sulfite strongly of [75Se]selenite to 75Se0 was linear and complete by 1.8 h inhibited activity for cells grown with either electron acceptor, (rate, 0.29 ,mol ml-' h-1), while duplicate suspensions con- and recovery of "4Co2 was less than that for cells suspended taining sulfite exhibited a 43% slower rate of selenite reduction without electron acceptor. (0.17 ,umol ml-' h-1) (data not shown). The effect of other Azide, CCCP, 2,4-dinitrophenol, and cyanide inhibited oxyanions (all 5 mM) on the reduction of 2.5 mM selenite (not '4Co2 production from ['4C]lactate in selenate-grown cells by shown) was as follows (percent inhibition): thiosulfate, 12%; 81, 86, 83, and 81%, respectively (not shown). In comparison, nitrate, 0%; and nitrite, 70%. the respective inhibitions for nitrate-grown cells were 47, 40, 9, Nitrate-grown cells were also able to reduce 0.5 mM selenite and 47%. The difference in cell densities for the two experi- to Seo, and reduction was complete in 3.3 h (data not shown). ments (selenate, 5.9 x 108 cells per ml incubated for 3.5 h; The rate of reduction was linear (-40 nmol ml-' h-l) and was nitrate, 3.7 x 109 cells per ml for 2.0 h) explains the higher not affected by 1.0 mM nitrite. However, 1.0 mM sulfite caused sensitivity of the selenate-grown cells. Uninhibited controls a total inhibition of selenite reduction, and of Seo formation, (selenate or nitrate) oxidized .88% of the [14C]lactate in the for the duration of the experiment (3.3 h). same amount of time, while cells incubated without electron acceptor demonstrated 91% inhibition (selenate-grown cells) DISCUSSION and 95% inhibition (nitrate-grown cells). Washed-cell experiments with [75Se]selenite. Removal of Strain SES-3 grew in the presence of selenate (Fig. 2) and [75Se]selenite in the presence of 2.5 mM selenite was linear nitrate (Fig. 3), but it did not grow in controls incubated and complete by 4.25 h (Fig. 7A). Loss of soluble counts was without an electron acceptor. Thus, the biochemical reductions completely balanced by the accumulation of 75Se0 counts on of selenate to selenite (Fig. 2B) and of nitrate to ammonium the filter. Cells incubated with 5 mM sulfate plus 2.5 mM (Fig. 3B) were dissimilatory and capable of sustaining growth. selenite displayed the same kinetics as cells with only selenite Further proof that these oxyanions were used for anaerobic (Fig. 7B), while a partial inhibition (-54%) occurred with respiration was the sensitivity that both selenate- and nitrate- selenate (Fig. 7C) because of its action as a competitive sink grown cells displayed to inhibitors of electron transport and for electrons. In contrast, near-total (97%) inhibition of selen- ionophores. Thus, the cytochrome oxidase inhibitors azide and ite reduction was achieved with 5 mM sulfite (Fig. 7D). cyanide, as well as the protonophore inhibitor CCCP, all Because of the observed inhibitory effects of this sulfite achieved substantial diminishment of lactate oxidation to CO2 concentration on the metabolism of whole cells (Table 1), we with either selenate or nitrate serving as the electron acceptor. repeated this experiment with only 0.5 mM selenite with or Growth of SES-3 on selenate (Fig. 2) was slower and without 1.0 mM sulfite. In duplicate suspensions, the reduction attained lower cell yields than corresponding growth on nitrate VOL. 60, 1994 AN OBLIGATELY ANAEROBIC, SELENATE-RESPIRING BACTERIUM 3017

TABLE 2. Thermodynamic calculations of the AGf0 reaction values for the observed respiratory reactions of strain SES-3 Respiratory reaction AG0of lactate)a(ki/mol 1. Lactate- + 2SeO42- Acetate- + 2SeO32- + HCO + H+...... -343.1 -> 2. Lactate- + SeO32- + H+ -529.5Ac etate- + Se + HCO3- + H20 ...... 3. Lactate- + 2SeO42- + H+ - 3 Acetate- + 2See + 3HC03- + 2H20.46...... -467.4 4. Lactate- + SeO32- + H2 + 2H+ - Pyruvate- + Se + 3H20...... -164.9 5. Lactate- + 2NO3- -> Acetate- + 2NO2- + HCO3- + H ...... -231.3 6. 3 Lactate- + 2NO2- + 2H2O + H+ - 3 Acetate- + 2NH4+ + 3HCO3...... -249.5 - + 7. 4 Lactate- + 2NO3- + 2H20 Acetate-4 2N H4++ 4HCO3 ...... -245.0 a The published values of AGfP for the reactants were obtained from Thauer et al. (43) and Woods and Garrels (48).

(Fig. 3). The observation of better growth on nitrate than millimolar concentrations, an approach which precludes batch selenate cannot be explained on thermodynamic grounds cultures and may only be tractable with chemostats. (Table 2) since reduction of selenate to selenite (Table 2, Strain SES-3 has similarities with and differences from T reaction 1) yields more energy than the corresponding reduc- selenatis. With regard to differences, SES-3 is a strict anaerobe tion of nitrate to nitrite (Table 2, reaction 5). Indeed, we whereas T. selenatis is facultative and can grow aerobically. calculated a higher Ym value for selenate (11.5 g of cells per Growth of SES-3 on nitrate proceeds via dissimilatory reduc- mol of lactate) than for nitrate (7.1 g of cells per mol of tion of nitrate to ammonium whereas in T. selenatis, growth is lactate). Likewise, growth on selenate with complete reduction by denitrification (20). However, full evidence for denitrifica- to Se' (Table 2, reaction 3) yields nearly twice the amount of tion in T. selenatis is lacking and primarily consists of the free energy per mole of lactate oxidized than does growth by detection of N20 rather than a complete nitrogen balance. dissimilatory reduction of nitrate to ammonium (Table 2, Indeed, SES-3 produces traces of N20, as do other bacteria reaction 7). Furthermore, the inability of cells growing on which achieve dissimilatory reduction of nitrate to ammonium, selenate to achieve a significant reduction beyond selenite is presumably via reduction of nitrite (12). Since SES-3 did not paradoxical because nitrate-grown suspensions quantitatively display enhanced N2O production in the presence of acetylene, reduce nitrate to ammonium (Fig. 5), and selenate-grown it lacks N20 reductase (1), where it appeared to be present in suspensions achieved quantitative reductions of selenate or SES-1 (30). Another difference was that T. selenatis uses selenite to Seo (Fig. 4 and 7). Similarly, T. selenatis grows by acetate as an electron donor for growth, while SES-3 uses reduction of selenate to selenite but does not grow on selenite lactate. With regard to similarities, selenate and nitrate are (19, 21) even though extracts can reduce selenite to Se0 (6). reduced by separate enzyme systems in SES-3 (Fig. 6), which is The answer to these paradoxes probably lies in the high also true for T selenatis (34) as well as for Fe3+ and nitrate toxicity of selenite to growing cells (8, 26). We observed the reduction by Geobacter metallireducens (10). accumulation of pyruvate rather than acetate during prolonged Reduction of selenite to Seo in T. selenatis is achieved by a exposure of cell suspensions to selenite (see Results). The periplasmic nitrite reductase (6). In SES-3, both selenate- and energy available from this reaction (Table 2, reaction 4) is nitrate-grown cells had the constitutive ability to reduce selen- considerably less than in selenate or selenite reduction (Table ite as well as nitrite. Reduction of selenite by a nitrite 2, reactions 1, 2, and 3) and additionally requires the metab- reductase could be possible in SES-3 because, in selenate- olism of H2 to balance the equation. We hypothesize that the grown cells, nitrite caused a 70% inhibition of 7SeO32- high concentrations of selenite (0.5 to 10 mM) employed in our reduction to 75Se0 (see Results). However, the inhibition batch culture experiments inhibited synthesis of additional observed with sulfite for both selenate- and nitrate-grown cells pyruvate dehydrogenase during prolonged incubation. (Fig. 7 and see text) suggests that an alternative pathway of From an environmental perspective, it must be borne in selenite reduction exists. Furthermore, the ability of nitrate- mind that these physiological experiments challenge the cells grown SES-3 to reduce selenite was unaffected by nitrite, which with concentrations of selenate or selenite that are 3 to 4 indicates reduction by another mechanism. The inhibition by orders of magnitude higher than what is encountered in the sulfite suggests involvement of an alternate enzyme system (in field, even within seriously selenium-contaminated locales. For addition to nitrite), perhaps one specific for sulfur oxyanions. example, selenite concentrations in ponds from the former We observed the production of low levels of sulfide when Kesterson Wildlife Refuge were 0.4 to 0.5 p.M, while those in selenate-grown cells were incubated with thiosulfate (see Re- contaminated groundwaters ranged from 0.008 to 0.09 ,uM sults), and we detected, but did not quantify, sulfide production (46). Even for the extreme case of an agricultural evaporation from thiosulfate in nitrate-grown cells. Thus, a constitutive pond in the western San Joaquin Valley, the selenate concen- enzyme system which is capable of reduction of thiosulfate is tration was only 38 ,uM and that of selenite was 2 ,uM (30, 31). present in nitrate- and selenate-grown cells. We have made Attempts to follow growth and metabolism by batch cultures, preliminary observations of growth of SES-3 taken through cell suspensions, and cell extracts requires work at millimolar several transfers in a high-Fe2" medium with thiosulfate as the concentrations, but extrapolation of these results to natural sole electron acceptor. Growth is associated with formation of systems can be a risky leap of faith and can distort one's sulfide as evidenced by formation of an FeS precipitate (la). perspective. Thus, the observation that rapid 75SeO42- reduc- Collectively, these results suggest that reduction of selenite in tion to 75Se0 is readily detected in many diverse sediments and SES-3 may proceed, at least in part, by components of an soils (31, 32, 40) argues that DSeR via reaction 3 actually enzyme system for the dissimilation of sulfur oxyanions. The occurs in nature because selenite is well below the level at inability of cell suspensions to form sulfide from sulfite is which it would constrain growth of these organisms. Actual perplexing. One possibility is that SES-3 lacks trithionate proof that SES-3 can grow on selenite via reaction 2 would reductase, which would preclude formation of sulfide from require experiments conducted at micromolar rather than sulfite but not from thiosulfate. Additionally, we hypothesize 3018 OREMLAND ET AL. APPL. ENVIRON. MICROBIOL. that SES-3 lacks ATP-sulfurylase and adenosine phospho- Fed. 63:799-805. sulfate reductase, which would explain its inability to either 10. Gorby, Y. A., and D. R Lovley. 1991. Electron transport in the dissimilatory iron reducer, GS-15. Appl. Environ. Microbiol. 57: grow on or reduce sulfate. 867-870. Incubation of cell suspensions with alternative electron 11. Hobbie, J. E., R. L. Daley, and S. Jaspar. 1977. Use of Nuclepore acceptors showed that formation of 14Co2 from ['4C]lactate filters for counting bacteria for fluorescence microscopy. Appl. can be coupled to fumarate, arsenate, oxygen, and FeOOH in Environ. Microbiol. 33:1225-1228. addition to nitrate and selenate (Table 1). Although the Mn4+ 12. Kaspar, H. F., and J. M. Tiedje. 1981. Dissimilatory reduction of results were clouded by chemical oxidation, we have observed nitrate and nitrite in the bovine rumen: nitrous oxide production growth of SES-3 with Mn4+ or Fe3+ as electron acceptors (lb), and effect of acetylene. Appl. Environ. Microbiol. 41:705-709. which suggests that respiratory reduction of these metals by 13. Lawrence, J. F., and R. K. Chadha. 1987. Headspace liquid SES-3 is similar to that which occurs in G. metallireducens and chromatographic technique for the determination of sulfite in 18, 25). The ability of nitrate- food. J. Chromatogr. 398:355-359. Shewanella putrefaciens (17, 14. Lortie, L., W. D. Gould, S. Rajan, R. G. L. McCready, and K.-J. grown SES-3 to couple lactate oxidation (but not growth) with Cheng. 1992. Reduction of selenate and selenite to elemental respiration of oxygen is not unusual for a strict anaerobe and selenium by a Pseudomonas stutzeri isolate. Appl. Environ. Micro- has been reported in sulfate-reducing bacteria (5, 7). Indeed, biol. 58:4042-4044. many species of sulfate reducers can also reduce nitrate to 15. Lovley, D. R 1993. Dissimilatory metal reduction. Annu. Rev. ammonium (23, 24) and some can achieve growth via dissim- Microbiol. 47:263-290. ilatory reduction of nitrate to ammonium (35). Preliminary 15a.Lovley, D. R Personal communication. results with nitrate- or selenate-grown SES-3 indicate that it 16. Lovley, D. R., and E. J. Phillips. 1986. Organic matter mineraliza- has a c551-type cytochrome protein profile (i.e., by sodium tion with the reduction of ferric iron in anaerobic sediments. Appl. gel electrophoresis) similar to Environ. Microbiol. 51:683-689. dodecyl sulfate-polyacrylamide 17. Lovley, D. R., and E. J. P. Phillips. 1988. Novel mode of microbial that of Desulfovibrio desulfuricans (40a). It is tempting to energy metabolism: organic carbon oxidation coupled to dissimi- speculate that these physiological results collectively suggest latory reduction of iron or manganese. Appl. Environ. Microbiol. that SES-3 may be located in the delta subclass of proteobac- 54:1472-1480. teria along with G. metallireducens and D. desulfuricans (15), 18. Lovley, D. R., E. J. P. Phillips, and D. J. Lonergan. 1989. which would further distinguish it from T. selenatis, which is Hydrogen and formate oxidation coupled to dissimilatory reduc- located in the beta subclass (22). Obviously, sequencing the tion of iron or maganese by Alteromonas putrefaciens. Appl. 16S rRNA segment of the SES-3 genome is needed to deter- Environ. Microbiol. 55:700-706. mine its phylogeny and whether or not such speculation is 19. Macy, J. M., and S. Lawson. 1993. Cell yield (YM) of Thauera SES-3 can be distinguished physiologically selenatis grown anaerobically with acetate plus selenate or nitrate. valid. However, Arch. Microbiol. 160:295-298. from these two delta-subclass organisms in that G. metallire- 20. Macy, J. M., S. Lawson, and H. DeMoll-Decker. 1993. Bioreme- ducens does not reduce selenate (1Sa), while D. desulfovibrio diation of selenium oxyanions in San Joaquin drainage water using will reduce traces of selenate to selenide but cannot grow by Thauera selenatis in a biological reactor system. Appl. Microbiol. DSeR and is inhibited by 1 mM selenate (49). Biotechnol. 40:588-594. 21. Macy, J. M., T. A. Michel, and D. G. Kirsch. 1989. Selenate ACKNOWLEDGMENTS reduction by a Pseudomonas species: a new mode of anaerobic respiration. FEMS Microbiol. Lett. 61:195-198. We thank D. Lovley, R. Conrad, and J. Stolz for their constructive 22. Macy, J. M., S. Rech, G. Auling, M. Dorsch, E. Stackebrandt, and comments on earlier drafts of the manuscript. L. I. Sly. 1993. Thauera selenatis gen. nov., sp. nov., a member of the beta subclass of Proteobacteria with a novel type of anaerobic REFERENCES respiration. Int. J. Syst. Bacteriol. 43:135-142. 1. Balderston, W. L., B. Sherr, and W. J. Payne. 1976. Blockage by 23. McCready, R. G. L., W. D. Gould, and F. D. Cook. 1983. acetylene of nitrous oxide reduction in Pseudomonas perfectoma- Respiratory nitrate reduction by Desulfovibrio sp. Arch. Microbiol. rinus. Appl. Environ. Microbiol. 31:504-508. 135:182-185. la.Blum, J. S. Unpublished observations. 24. Mitchell, G. J., J. G. Jones, and J. A. Cole. 1986. Distribution and lb.Blum, J. S., E. A. Phillips, R. Oremland, and D. R. Lovley. regulation of nitrate and nitrite reduction by Desulfovibrio and Unpublished data. Desulfotomaculum species. Arch. Microbiol. 144:35-40. 2. Braker, W., and A. L. Mossman. 1980. Matheson gas data book, 25. Myers, C. R., and K. H. Nealson. 1988. Bacterial manganese 6th ed. Matheson Co., Lindhurst, N.J. reduction and growth with manganese oxide as the sole electron 3. Culbertson, C. W., F. E. Strohmaier, and R. S. Oremland. 1988. acceptor. Science 240:1319-1321. Acetylene as a substrate in the development of primordial bacte- 26. Oremland, R. S. 1994. Biogeochemical transformations of sele- rial communities. Origins Life Evol. Biosphere 18:397-407. nium in anoxic environments, p. 389-419. In W. T. Frankenberger, 4. Culbertson, C. W., A. J. B. Zehnder, and R. S. Oremland. 1981. Jr., and S. N. Benson (ed.), Selenium in the environment. Marcel Anaerobic oxidation of acetylene by estuarine sediments and Dekker, Inc., New York. enrichment cultures. Appl. Environ. Microbiol. 41:396-403. 27. Oremland, R. S. December 1993. Selenate removal from waste- 5. Dannenberg, S., M. Kroder, W. Dilling, and H. Cypionka. 1992. water. U.S. patent 5,271,831. Oxidation of H2, organic compounds and inorganic sulfur com- 28. Oremland, R. S. April 1991. Selenate removal from wastewater. pounds coupled to reduction of 02 or nitrate by sulfate-reducing U.S. patent 5,009,786. bacteria. Arch. Microbiol. 158:93-99. 29. Oremland, R. S., and C. W. Culbertson. 1992. Evaluation of 6. DeMoll-Decker, H., and J. M. Macy. 1993. The periplasmic nitrite methyl fluoride and dimethyl ether as inhibitors of aerobic meth- reductase of Thauera selenatis may catalyse the reduction of ane oxidation. Appl. Environ. Microbiol. 58:2983-2992. selenite to elemental selenium. Arch. Microbiol. 160:241-247. 30. Oremland, R S., J. T. Hollibaugh, A. S. Maest, T. S. Presser, L. G. 7. Dilling, W., and H. Cypionka. 1990. Aerobic respiration in sulfate- Miller, and C. W. Culbertson. 1989. Selenate reduction to elemen- reducing bacteria. FEMS Microbiol. Lett. 71:123-127. tal selenium by anaerobic bacteria in sediments and culture: 8. Doran, J. W. 1982. Microorganisms and the biological cycling of biogeochemical significance of a novel, sulfate-independent respi- selenium. Adv. Microb. Ecol. 6:17-32. ration. Appl. Environ. Microbiol. 55:2333-2343. 9. Gerhardt, M. B., F. B. Green, R. D. Newman, T. J. Lundquist, 31. Oremland, R. S., N. A. Steinberg, A. S. Maest, L. G. Miller, and R. B. Tresan, and W. J. Oswald. 1991. Removal of selenium using J. T. Hollibaugh. 1990. Measurement of in situ rates of selenate a novel algal-bacterial processes. Res. J. Water Pollut. Control removal by dissimilatory bacterial reduction in sediments. Envi- VOL. 60, 1994 AN OBLIGATELY ANAEROBIC, SELENATE-RESPIRING BACTERIUM 3019

ron. Sci. Technol. 24:1157-1164. chromatography method for the analysis of polythionates. LC-GC 32. Oremland, R. S., N. A. Steinberg, T. S. Presser, and L. G. Miller. 11:874-878. 1991. In situ bacterial selenate reduction in the agricultural 42. Swarzenbach, R. P., P. M. Gschwend, and D. M. Imboden. 1993. drainage systems of western Nevada. Appl. Environ. Microbiol. Environmental organic chemistry. John Wiley & Sons, Inc., New 57:615-617. York. 33. Oremland, R. S., C. Umberger, C. W. Culbertson, and R. L. Smith. 43. Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy 1984. Denitrification in San Francisco Bay intertidal sediments. conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. Appl. Environ. Microbiol. 47:1106-1112. 41:100-180. 34. Rech, S. A., and J. M. Macy. 1992. The terminal reductases for 44. Tomei, F. A., L. L. Barton, C. L. Lemanski, and T. G. Zocco. 1992. selenate and nitrate respiration in Thauera selenatis are two Reduction of selenate and selenite to elemental selenium by distinct enzymes. J. Bacteriol. 174:7316-7320. Wolinella 35. Seitz, H. J., and H. Cypionka. 1986. Chemolithotrophic growth of succinogenes. Can. J. Microbiol. 38:1328-1333. Desulfovibrio desulfuricans with hydrogen coupled to ammonifica- 45. Visscher, P. T., J. Beukema, and H. van Gemerden. 1991. In situ tion of nitrate or nitrite. Arch. Microbiol. 146:63-67. characterization of sediments: measurements of oxygen and sul- 36. Smith, R. L., F. E. Strohmaier, and R. S. Oremland. 1985. fide profiles with a novel combined needle electrode. Limnol. Isolation of anaerobic oxalate degrading bacteria from freshwater Oceanog. 36:1476-1480. lake sediments. Arch. Microbiol. 141:8-13. 46. White, A. F., S. M. Benson, A. W. Yee, H. A. Wollenberg, Jr., and 37. Solorzano, L. 1969. Determination of ammonia in natural waters S. Flexser. 1991. Groundwater contamination at the Kesterson by the phenol hypochlorite method. Limnol. Oceanogr. 14:799- Resevoir, California. 2. Geochemical parameters influencing sele- 801. nium mobility. Water Resources Res. 27:1085-1098. 38. Stadtman, T. C. 1990. Selenium biochemistry. Annu. Rev. Bio- 47. Widdel, F., G.-W. Kohring, and F. Mayer. 1983. Studies on the chem. 59:111-127. dissimilatory sulfate that decompose fatty acids. III. Characteriza- 38a.Steinberg, N. Personal communication. tion of the filamentous gliding Desulfonema limicola gen. nov., sp. 39. Steinberg, N. A., J. S. Blum, L. Hochstein, and R. S. Oremland. nov., and Desulfonema magnum sp. nov. Arch. Microbiol. 134:286- 1992. Nitrate is a preferred electron acceptor for growth of 294. freshwater selenate-respiring bacteria. Appl. Environ. Microbiol. 48. Woods, T. L., and R. M. Garrels. 1987. Thermodynamic values at 58:426-428. low temperature for natural inorganic materials. Oxford Univer- 40. Steinberg, N. A., and R. S. Oremland. 1990. Dissimilatory selenate sity Press, New York. reduction potentials in a diversity of sediment types. Appl. Envi- 49. Zehr, J. P., and R. S. Oremland. 1987. Reduction of selenate to ron. Microbiol. 56:3550-3557. selenide by sulfate-respiring bacteria: experiments with cell sus- 40a.Stoltz, J. Personal communication. pensions and estuarine sediments. Appl. Environ. Microbiol. 41. Strege, M. A., and A. L. Lagu. 1993. A high performance liquid 53:1365-1369.