APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2001, p. 2499–2506 0099-2240/01/$04.00⫹0 DOI: 10.1128/AEM.67.6.2499–2506.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 67, No. 6
Kinetics of Perchlorate- and Chlorate-Respiring Bacteria BRUCE E. LOGAN,1* HUSEN ZHANG,1 PETER MULVANEY,1 MICHAEL G. MILNER,2,3 IAN M. HEAD,2,3 AND RICHARD F. UNZ1 Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park, Pennsylvania,1 and Fossil Fuels & Environmental Geochemistry,2 and Centre for Molecular Ecology,3 University of Newcastle, Newcastle upon Tyne, United Kingdom Received 30 November 2000/Accepted 22 March 2001
Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated “Dechlorosoma” sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 hⴚ1, respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 hⴚ1, respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 hⴚ1, respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 hⴚ1, respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 hⴚ1) and exceeded that of strain KJ on perchlorate (0.14 hⴚ1). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 ⴞ 0.07 [n ⴝ 7]), chlorate (0.44 ⴞ 0.05 [n ⴝ 7]), and perchlorate (0.50 ⴞ 0.08 [n ⴝ 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors. Observed maximum bacterial growth rates on chlorate and perchlorate have been reported to be in the range of 0.012 to 0.28 h⫺1 (5, 12, 19, 27, 40). In only one case have growth rates of perchlorate- and chlorate-reducing bacterial isolates on different terminal electron acceptors been previously compared. The growth rate of isolate GR1 was 0.1 h⫺1 on chlorate or perchlorate under acetate-oxidizing conditions (40). In the presence of both nitrate and chlorate, the GR1 growth rate was reduced to 0.08 h⫺1, suggesting that growth of GR1 was slower on nitrate than on chlorate. There are no chlorate or perchlorate degradation kinetic data for bacterial isolates other than maximum growth rates, although there is a need for such data in modeling perchloratedegrading bioreactors (18). Kinetic constants have been reported for mixed cultures under chlorate-reducing conditions for three different electron donors (19) but not for isolates or for other electron acceptors. The purpose of this study was therefore to obtain, for the first time, growth rates of perchlorate-respiring bacteria using different electron acceptors. Such data will be useful for analyzing and designing biological treatment systems for treating perchlorate-contaminated water containing dissolved oxygen and other alternate electron acceptors (3, 16, 18, 41).
Perchlorate has recently become a national drinking water concern due to high perchlorate concentrations in ground and surface waters (1, 4, 34, 35). In California, 30 of 110 water supply wells tested had perchlorate concentrations above the action level of 0.018 mg/liter established by the California Department of Health Services (1). In Suffolk County, N.Y., nearly 50% of the wells tested contained perchlorate concentrations of up to 0.040 mg/liter (1). Perchlorate has been added to the U.S. Environmental Protection Agency’s candidate contaminant list and is the subject of ongoing toxicity studies to determine a safe dose for drinking water regulations (4, 35, 36). It has been known since 1928 that certain bacteria are capable of chlorate (ClO3⫺) reduction (2), but the ability of bacteria to use perchlorate (ClO4⫺) as a terminal electron acceptor was not reported until 1976 (17) and thereafter (42). Enzymatic reduction of chlorate to chlorite (ClO2⫺) by nitrate reductase occurs as a competitive reaction between nitrate and chlorate in certain denitrifying bacteria (33). However, chlorate reduction by most denitrifiers is not an energy-yielding process, and very few denitrifying bacteria are capable of using perchlorate or chlorate as a terminal electron acceptor (6, 12, 18, 23). Bacteria capable of perchlorate and chlorate reduction are, however, widely distributed in the environment (6, 39, 43), even though naturally occurring sources of perchlorate so far appear to be limited to Chilean mineral deposits rich in nitrate (30, 35).
MATERIALS AND METHODS Media. All media were prepared using ultrapure water (Milli-Q system; Millipore Corp., New Bedford, Mass.) and research-grade chemicals in the amounts (per liter) indicated below. Medium MS contained 1 g of K2HPO4, 0.5 g of NaH2PO4, 0.5 g of NH4H2PO4, and 0.1 g of MgSO4 䡠 7H2O. Medium VG was prepared as previously described (28). Slight modifications of medium VG resulted in the following media: medium B (1 g of K2HPO4, 50 mg of MgSO4 䡠 H2O, 3 mg of EDTA, 4 mg of FeSO4 䡠 H2O, 0.4 mg of NaMoO4 䡠 H2O, 0.1 mg
* Corresponding author. Mailing address: Dept. of Civil and Environmental Engineering, 212 Sackett Bldg., The Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-7908. Fax: (814) 863-7304. E-mail:
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of NiCl2 䡠 6H2O, 1 mg of NaSeO3 䡠 5H2O, and 0.6 mg of H3BO3), medium H (0.1 mg [each] of NiCl2 䡠 6H2O and Na2SeO3 䡠 5H2O), and medium G (medium B with 1.55 g of K2HPO4). Sodium salts of acetic (NaC2H3O2), lactic (NaC3H5O3), chloric (NaClO3), or perchloric (NaClO4) acid were added at 500 mg/liter unless stated otherwise. Bacterial isolation procedures. All enrichment culture and isolation procedures were conducted in an anaerobic glove box (Coy Scientific Products, Grass Lake, Mich.) at 24°C. Isolates were obtained from enrichment cultures developed with inocula of primary digester sludge (D-8 and all PD isolates) or effluent from an acetate-fed, perchlorate-degrading packed bed bioreactor (isolate KJ) (20). Sludge samples were collected in clean 500-ml Nalgene containers from the Pennsylvania State University wastewater treatment plant on two different dates. Isolates PDX and PDY were obtained from enrichment cultures developed in medium MS. Isolates PDA, PDB, PDC, PDD, PDE, and KJ were recovered from enrichment cultures produced with medium VG. Enrichment cultures consisted of 100 ml of inoculated medium contained in 130-ml serum bottles fitted with butyl rubber stoppers and crimped with an aluminum seal. Cultures became turbid in 7 to 14 days and were transferred at least four times (1% by volume) to fresh medium. Inocula from enrichments were streaked or spread on plating medium and incubated. Select colonies were picked, transferred to liquid medium, and incubated, and the resultant cultures were streaked on solid medium. Four successive transfers involving alternate liquid and plate culturing were made before isolated organisms were considered axenic. Purity was confirmed by microscopic examination. Batch growth kinetics. Growth experiments were conducted using cultures acclimated to the appropriate electron acceptor and donor. In experiments using chlorate or perchlorate, cells were harvested during late-log-phase growth (optical density at 600 nm [OD600], 0.3 to 0.4), washed once at 8,000 ⫻ g for 10 min, and resuspended in medium MS to the same OD under anaerobic conditions in a glove box. This cell suspension (0.75 ml) was transferred to test tubes (28 ml) prepared under anaerobic conditions and containing 14.25 ml of medium G with different concentrations of acetate and 500 mg of perchlorate/liter. Abiotic controls (no inoculum) were prepared at the same time. Tubes were sealed with butyl rubber stoppers and removed from the glove box, and OD600 readings were taken. In aerobic growth experiments, cultures were prepared as described above except that cells were grown in medium G in 500-ml flasks on a shaker table (model 3520; Lab-line Instrument Inc., Melrose Park, Ill.) and all transfers (15 ml of washed cell suspensions into 28-ml tubes) were done under aerobic conditions in a laminar flow hood. Tubes were shaken on their sides at 150 rpm. Oxygen utilization was calculated from oxygen depletion in the tube headspace using methods described elsewhere (21). Kinetic constants were calculated assuming Monod kinetics. A nonlinear regression analysis (SigmaPlot; SPSS Inc., Chicago, Ill.) was used to obtain the maximum growth rate (m) and the half-saturation constant (Ks). Chemostat growth kinetics. The growth rates of one isolate were measured in continuous-culture experiments on lactate and chlorate or perchlorate using a chemostat (1.5 liters; The VirTis Co. Inc., Gardiner, N.Y.) as previously described (19). The reactor medium was inoculated with a cell suspension and operated in batch mode until the culture became turbid. The reactor was then switched to continuous-flow mode by pumping in medium at a constant flow rate (Q). Anoxic conditions were maintained by continuous nitrogen gas sparging. Samples were obtained directly from the reactor and analyzed for OD and concentrations of perchlorate and lactate. The reactor was turned over at least three detention times () before changing pumping rates. Growth rates were calculated at steady state, where ⫽ 1/ ⫽Q/V and V is the liquid volume in the reactor (1.2 liters). Uptake kinetics. Perchlorate uptake kinetics were determined using washed cell suspensions (OD600 ⫽ 0.2) prepared anaerobically in medium B and transferred into a nitrogen-free medium B (in 50-ml flasks) to preclude cell growth during the experiment. Phosphate concentrations were reduced by 1.5 orders of magnitude to minimize interferences with perchlorate measurements. Perchlorate was added to the medium at different concentrations (0.1 to 500 mg/liter) and measured over time (typically 30 to 90 min) on filtered (⬍0.2-m pore diameter) 5-ml samples using either an ion-specific probe (⬎1 mg of perchlorate/ liter) or ion chromatography (⬍1 mg/liter). A positive control for growth in unmodified medium was included in the experiment to confirm that the inoculum was viable. Constant cell mass was verified by OD measurements. Analytical techniques. Cell suspensions were monitored by OD600. Protein was measured by the Bradford Coomassie blue method (total protein assay; Pierce, Rockford, Ill.). Yields were determined from the dry weight (DW) of cells (triplicate or quadruplicate samples; Mettler Toledo UMT2, Greifensee, Swit-
APPL. ENVIRON. MICROBIOL. zerland) using membrane filters (25 mm, 0.2-m pore diameter; Osmonics Corp., Minnetonka, Minn.). Unless stated otherwise, perchlorate concentrations were determined with an ion chromatograph (DX500; Dionex, Sunnyvale, Calif.) equipped with an AS11 column and guard column, a self-regenerating suppressor, and an autosampler (14). Lactate, acetate, chlorate, and chloride anions were measured using the ion chromatograph and 10 mM NaOH eluent, and 100 mM NaOH eluent was used in connection with perchlorate ion measurement. Perchlorate concentrations in excess of 5 mg/liter were measured occasionally with an ion-selective probe fitted with a double-junction reference electrode (model 93-81 and model 90-02; Orion, Cambridge, Mass.). Minimum perchlorate detection limits for the probe and ion chromatograph were 1 and 0.004 mg/liter, respectively. Gas production in nitrate-amended cultures in an inverted Durham tube was taken as presumptive evidence of denitrification. The presence of chlorite dismutase in aerobically grown cells was demonstrated by the evolution of dissolved oxygen after the addition of chlorite (50 mg/liter) (6, 28). Dissolved oxygen was measured using a three-place dissolved oxygen device (YSI 5300 Standard Oxygen Monitor and Probe; Yellow Springs Instrument Co., Yellow Springs, Ohio). 16S ribosomal DNA sequencing. DNAs of all isolates (except PDA and PDB) were extracted from cells contained in 10 ml of liquid culture following centrifugation (9,000 ⫻ g) and washing of the pellet (in triplicate) with 5 ml of sterile 10 mM Tris-HCl–1 mM EDTA (TE) buffer (pH 8.0). Cell masses of PDA and PDB were obtained directly from colonies on agar medium. The washed pellet was suspended in 0.1 ml of TE buffer, lysed in an additional 0.2 ml of TE buffer containing 3% (wt/vol) sodium dodecyl sulfate, and extracted three times in TE-buffered phenol and chloroform, respectively. Nucleic acids were precipitated overnight at ⫺20°C from the aqueous phase of the lysate following addition of 2 volumes of ice-cold ethanol (98%, vol/vol) and recovered by centrifugation (13,400 ⫻ g). DNA was dissolved in 75 l of sterile TE buffer, and the 16S ribosomal DNAs were amplified by conventional PCR employing primers PA and pH⬘ (8) and Dynazyme DNA polymerase (Flowgen). PCR products were purified and sequenced as described elsewhere (13). Phylogenetic analysis. Approximately 500 bp of sequence was initially obtained from strains KJ, KJ3, KJ4, PDX, PDA, PDB, and JM. The sequences from KJ, KJ3, and KJ4 were identical to each other, and those from PDA and PDB were identical to the 16S rRNA sequence of the type strain of Pseudomonas stutzeri (CCUG 11256T). Therefore, almost-full-length 16S rRNA sequences (ca. 1,500 bp) were determined only for strains KJ, PDX, and JM. A subset of 16S rRNA sequences, obtained from the Ribosomal Database Project (22) and GenBank and showing the highest similarity to the sequences determined here, were used in phylogenetic analyses. The sequences were aligned using the genetic data environment sequence editor (31). An alignment of 37 bacterial rRNA sequences from the  subdivision of the class Proteobacteria, including sequences from four recently described “Dechlorosoma ” spp. and five “Dechloromonas” spp. (6), was used for phylogenetic analyses. The final alignment consisted of 1,307 unambiguously aligned positions corresponding to positions 33 to 68 and 104 to 1376 (Escherichia coli numbering) of the 16S rRNA molecule and was used in all phylogenetic analyses. Distance analyses (15) performed with the TREECON package (37) were employed to form trees from distance matrices by the neighbor-joining method (29). Parsimony and maximum-likelihood analyses (9) were accomplished with DNAPARS (10) and fast DNAml (26), respectively, and sequence data were subjected to bootstrap resampling (100 replicates) employing TREECON (distance analysis) and SEQBOOT (parsimony analysis). Consensus trees were constructed with the CONSENSE program from the PHYLIP package (10). Genus and species names that do not appear on the approved list of bacterial names or updates of the list are in quotation marks. Nucleotide sequence accession numbers. The sequences determined in this study have been deposited in the GenBank database with accession numbers AF323489 to AF32393.
RESULTS Isolates. A total of 10 chlorate-reducing isolates were obtained by the alternating serial tube-to-plate transfer method: 6 from primary digester wastewater on lactate (isolates PDA, PDB, PDC, PDD, PDE, and PDX), 1 from an activated-sludge aeration basin on lactate (D-8), and 3 from a perchloratedegrading bioreactor on acetate (KJ, KJ3, and KJ4). All isolates were gram negative, used lactate and acetate as electron donors, and used chlorate and oxygen as electron acceptors. Only isolates PDA and PDB could not grow with perchlorate
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FIG. 1. Growth of KJ on 100 mg of acetate/liter and three different electron acceptors measured by solution absorbance at 600 nm (open and closed symbols). The growth rate is calculated from the slope of the line using only the open symbols.
as the electron acceptor. Several isolates (PDX, KJ, KJ3, KJ4, and D-8) were tested for nitrate reduction, and all produced gas with nitrate as the sole electron acceptor and lactate as the electron donor. The lag time for growth of these perchlorategrown cultures on nitrate (3 to 4 days) was less than the lag on chlorate, perchlorate, and oxygen (7 to 14 days) (25). No growth or sulfide production was observed with sulfate as the electron acceptor following a 30-day anoxic incubation period (data not shown). Two of the isolates, KJ and PDX, were selected for more extensive testing based on factors such as growth rate and source. Log-phase cell dimensions (in micrometers) were 1.6 ⫾ 0.13 by 0.74 ⫾ 0.05 for KJ and 2.1 ⫾ 0.14 by 0.55 ⫾ 0.05 for PDX. Both isolates evolved dissolved oxygen when spiked with chlorite, indicating presumptive evidence of a chlorite dismutase (data not shown). Both isolates grew using ethanol and Tween 20 under aerobic and anoxic (perchlorate and chlorate) conditions. Growth kinetics. Batch cultures of isolates KJ and PDX, growing on acetate as the electron donor and oxygen, chlorate, or perchlorate as the electron acceptor, were used in connection with growth rate determinations. Only data representing the linear portion of early exponential growth were used to calculate growth rates (Fig. 1). The maximum observed growth rates of KJ were similar on oxygen or chlorate (0.26 and 27 h⫺1) but were much lower on perchlorate (0.14 h⫺1) (Fig. 2). Growth rates at lower acetate concentrations (⬍100 mg/liter) decreased with the three electron acceptors in the order of oxygen ⬎ chlorate ⬎ perchlorate. Growth data were fitted by nonlinear regression analysis to obtain half-saturation constants of 14, 60, and 470 mg/liter with oxygen, chlorate, and perchlorate, respectively, as electron acceptors (Table 1). The growth rates of isolate PDX with chlorate or perchlorate as the electron acceptor were similar over the range of acetate concentrations measured (Fig. 3), and half-saturation constants (given as Ks for all electron donors) were not significantly different (Table 1). The highest observed growth rates of isolate PDX (0.21 to 0.28 h⫺1) (Fig. 3) were similar to those
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FIG. 2. Growth rates of isolate KJ using acetate as the sole electron donor and oxygen, chlorate, or perchlorate as the electron acceptor. Regression lines are based on the open symbols. The closed symbols are from a separate experiment. Error bars indicate standard errors of the slopes used to calculate the growth rates.
of KJ (0.26 to 0.27 h⫺1) (Fig. 2) on acetate with oxygen and chlorate as electron acceptors (Table 1). The maximum observed growth rate of isolate PDX on perchlorate (0.21 h⫺1 at ⬃500 mg of acetate/liter) (Fig. 3), however, was 50% greater than that of isolate KJ (0.14 h⫺1) (Fig. 3). The growth rates of isolate PDX were also determined on lactate (Fig. 4). Using data from both batch and chemostat tests, the maximum observed growth rate was found to be
TABLE 1. Summary of the maximum observed growth rates in batch culture and kinetic parameters for growth on the indicated electron donors of chlorate-reducing isolates grown under aerobic or anaerobic conditions Isolate
Electron donor
Electron acceptor
Maximum observed (h⫺1)
m (h⫺1)a
Ks (mg/liter)a
KJ
Acetate Oxygen Chlorate Perchlorate
0.27 0.26 0.14
0.25 ⫾ 0.00 0.27 ⫾ 0.03 0.20 ⫾ 0.07c
14 ⫾ 1 60 ⫾ 25b 470 ⫾ 290d
PDX
Acetate Oxygen Chlorate Perchlorate Lactate Chlorate
0.28 0.21 0.21 0.15
0.28 ⫾ 0.01 0.27 ⫾ 0.02 0.24 ⫾ 0.03 0.13 ⫾ 0.01
2.7 ⫾ 2.1e 75 ⫾ 16 45 ⫾ 19b 10 ⫾ 4c
PDA
Acetate Oxygen Chlorate Perchlorate
0.64 0.18 NGg
—f — —
— — —
PDB
Acetate Oxygen Chlorate Perchlorate
0.41 0.26 NG
— — —
— — —
a The maximum growth rate and half-saturation constants, m and Ks, were obtained by a nonlinear regression analysis using data shown in Fig 2 through 4 and are significant at a P value of 0.01 except as noted. b P ⬍ 0.10. c P ⬍ 0.05. d P ⫽ 0.14. e P ⫽ 0.26. f —, not tested. g NG, no growth.
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FIG. 5. Perchlorate uptake rates (milligrams of perchlorate per milligram of protein per hour) for isolates KJ and PDX. FIG. 3. Growth rates of isolate PDX using acetate as the sole electron donor and oxygen, chlorate, or perchlorate as the electron acceptor. Notation is as in Fig. 2.
much lower on lactate (0.15 h⫺1) than on acetate (0.21 h⫺1). The half-saturation constant Ks (10 ⫾ 4 mg/liter) for lactate with isolate PDX was very much lower than that for acetate (75 ⫾ 16 mg/liter). Neither PDX or KJ ferments lactate, which is consistent with results obtained for chlorate-respiring isolates by others (6). The maximum observed growth rates of the two non-perchlorate-utilizing isolates, PDA and PDB, were much higher with oxygen (0.64 and 0.41 h⫺1, respectively) than with chlorate (0.18 and 0.21 h⫺1, respectively) as the electron acceptor. However, growth rates of these organisms were in the same range as growth rates measured for isolates KJ (0.26 h⫺1) and PDX (0.21 h⫺1) under chlorate-reducing conditions (Table 1).
Perchlorate uptake kinetics. Perchlorate uptake at ⬎50 mg/ liter by nongrowing cultures of KJ was faster than that by nongrowing cultures of PDX, although perchlorate uptake kinetics for both isolates did not change at perchlorate concentrations of greater than 100 mg/liter (Fig. 5). The maximum rate constant determined for perchlorate uptake by KJ cultures (Vm ⫽ 0.055 ⫾ 0.004 mg of ClO4⫺/mg of protein/h) was 3.2 times that measured for PDX cultures (Vm ⫽ 0.017 ⫾ 0.002 mg of ClO4⫺/mg of protein/h). Half-saturation constants (given as Km for all electron acceptors) for perchlorate uptake were similar for the two strains, with Km ⫽ 33 ⫾ 9 mg/liter for KJ and Km ⫽ 12 ⫾ 4 mg/liter for PDX. Biomass yields. The biomass yields (grams [DW] per gram of acetate) measured for KJ were not significantly different (P ⱕ 0.05) for oxygen (0.46 ⫾ 0.07 [n ⫽ 7]), chlorate (0.44 ⫾ 0.05 [n ⫽ 7]), and perchlorate 0.50 ⫾ 0.08 [n ⫽ 7]) (Table 2). Therefore, the results for all three electron acceptors were grouped together to produce an overall yield of 0.47 ⫾ 0.07 g (DW)/g of acetate (n ⫽ 21). Phylogeny. Comparative 16S rRNA sequence analysis indicated that strains KJ and PDX were most closely related to “Dechlorosoma suillum” strain PS and “Dechlorosoma” sp.
TABLE 2. Comparison of cell yields in the presence of various electron acceptors of isolate KJ versus those reported by others Culture
KJa GR1 AB1 Mixed
FIG. 4. Growth rates of PDX on lactate and chlorate determined in batch and chemostat experiments. Vertical error bars indicate standard errors of the slopes used to calculate the growth rates in batch experiments; horizontal error bars are standard deviations of lactate concentration.
Cell yield (g [DW]/g of acetate) with the following electron acceptor: Oxygen
Chlorate
Perchlorate
0.46 ⫾ 0.07 0.27 ⫾ 0.01 0.13 ⫾ 0.04 — —
0.44 ⫾ 0.05 0.28 ⫾ 0.01 0.10 ⫾ 0.04 0.30–0.61c 0.12 ⫾ 0.06
0.50 ⫾ 0.08 0.24 ⫾ 0.01 —b — —
Reference
This study 28 27 23 19
a Cell yields for isolate KJ are not significantly different (p ⬎ 0.05) for the three different electron acceptors. b —, not tested. c Converted from grams of volatile suspended solids (VSS) per equivalent of available electrons to grams (DW) per gram of acetate by assuming that 0.85 g of VSS ⫽ 1 g (DW) and that there are eight equivalents of available electrons per mole of acetate.
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FIG. 6. Phylogenetic relationships between -proteobacterial (per)chlorate-reducing bacteria and representative members of the  Proteobacteria. The tree is a distance tree produced using the Jukes-Cantor correction for multiple substitutions at a single site. Essentially the same topology was obtained with parsimony and maximum-likelihood analyses. However, the branching order within the major groups recovered could be variable, as indicated by low bootstrap values at some nodes. The numbers at nodes represent the bootstrap support for the groupings appearing to the right of the node. Values for distance analysis are given above the node, and those for parsimony analysis are given below the node. The scale bar represents 2% divergence.
strains Iso1, Iso2, and SDGM in the  subdivision of the class Proteobacteria (Fig. 6). The 16S rRNA sequence identity of both KJ and PDX when compared with the sequences from these “Dechlorosoma” spp. was greater than 99.5%. All three methods of phylogenetic inference used placed KJ and PDX
with the genus “Dechlorosoma,” and this was strongly supported by high bootstrap support in distance and parsimony analysis (100% support in both analyses). Furthermore, analysis of shorter lengths of 16S rRNA sequence indicated that isolates KJ3 and KJ4, which were obtained from the same
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TABLE 3. Maximum reported growth rates of previously described chlorate- and perchlorate-respiring isolates or mixed cultures Culture
Electron acceptor
Electron donor
Observed maximum growth rate, (h⫺1)
Reference
GR1
Chlorate Oxygen Oxygen ⫹ nitrate
Acetate
0.10 0.23 0.077
40
AB1
Chlorate
Acetate
0.012
27
Perclace
Perchlorate
Acetate
0.07
12
CKB
Chlorate
Acetate
0.28
5
Mixed
Chlorate
Acetate GGa Phenol
0.085 0.20 0.035
19
a
Glucose-glutamic acid (50:50 mixture).
sample using the same medium as for KJ, had 16S rRNA sequences identical to that of isolate KJ (data not shown). In conjunction with the phenotypic data for the isolates, this indicated that KJ, KJ3, KJ4, and PDX were “Dechlorosoma” spp. However, because 16S rRNA sequence analysis does not permit discrimination at the species level when sequence identities are greater than 97.5% (32), it is not possible based on these data alone to assign isolates KJ and PDX to new species. Nevertheless, it is known that different bacterial species can share very high 16S rRNA sequence identity despite evidence from other data (e.g., DNA-DNA hybridization and phenotypic data) that they represent distinct species (11). Further analyses are therefore required to determine if strains KJ and PDX represent new species of “Dechlorosoma.” Strain JM was isolated using acetate as the sole source of carbon and energy from a packed-bed perchlorate-reducing bioreactor with hydrogen as an electron donor (24). Isolate JM is also a member of the  subdivision of the class Proteobacteria but was recovered with the related genus “Dechloromonas” (Fig. 6). This bacterium shared less than 97.5% 16S rRNA sequence identity with previously described “Dechloromonas” spp. (6) and most probably represents a new species of “Dechloromonas.” Analysis of ca. 500 bp of 16S rRNA sequence from isolates PDA and PDB demonstrated that the 16S rRNA sequences were identical to each other and to the 16S rRNA sequence of P. stutzeri CCUG 11256T from the ␥ subdivision of the class Proteobacteria. (Per)chlorate-reducing isolates related to the denitrifying bacterium P. stutzeri have previously been isolated (6, 7). DISCUSSION The growth data obtained in the present study for several perchlorate-degrading isolates suggest that these microorganisms have high growth rates and high cell yields. The maximum observed growth rates using chlorate and perchlorate were higher than those reported for three other chlorate-reducing isolates (GR1, AB1, and perclace) but were comparable to that calculated for isolate CKB (Table 3). Half-saturation constants (Ks) for the growth of isolates KJ
and PDX on acetate varied widely depending on the electron acceptor. For both isolates, Ks values were calculated to be above 45 mg of acetate/liter (up to 470 mg of acetate/liter) with chlorate and perchlorate as electron acceptors, and observed growth rates of less than half the maximum rate confirm this value (Fig. 2 and 3). Although calculated Ks values for aerobic conditions are less accurate than those for anaerobic conditions, aerobic Ks values were clearly lower than those obtained under chlorate- and perchlorate-reducing conditions. Aerobic growth rates of PDX were essentially constant over the total acetate concentration range of 20 to 400 mg/liter. As a result, the Ks value obtained was insignificant and therefore too low to be measured in these batch growth tests. For KJ, measured aerobic growth rates were 58% of the maximum rate, providing a significant (P ⬍ 0.05) Ks value of 14 ⫾ 1 mg of acetate/liter under aerobic conditions. Half-saturation constants for perchlorate uptake (Km) were slightly larger for KJ than for PDX (33 ⫾ 9 and 12 ⫾ 4 mg of perchlorate/liter, respectively). Based on these results, we conclude that perchlorate reduction would follow first-order kinetics under typical environmental conditions of perchlorate concentrations in the parts-per-billion range. Biomass yields for acetate were not significantly different with oxygen, chlorate, and perchlorate as electron acceptors. The biomass yield measured here for KJ using chlorate (0.45 ⫾ 05 mg [DW]/mg of acetate) was within the range reported by Malmqvist et al. (23) for a chlorate-degrading mixed culture (0.30 to 0.61 mg [DW]/mg of acetate) but greater than those reported for another isolate, GR1, and a mixed culture on chlorate (Table 2). However, biomass yields for isolate GR1 were also found not to be a function of the electron acceptor (oxygen, chlorate, or perchlorate) (Table 2). A much lower growth yield (0.012 g [DW]/g of acetate) was previously found for the growth of a mixed culture on acetate and chlorate (19) than reported here for KJ. A likely explanation is that oxygen was chemically consumed before it could be used by the mixed culture. Dissolved oxygen is generated by the disproportionation of chlorite during the breakdown of chlorate (6, 40). Oxygen does not accumulate in solution and can be consumed by most chlorate-respiring bacteria (6, 40). In the mixed-culture chemostat experiments reported by Logan et al. (19), dissolved oxygen was scavenged by iron sulfide added to maintain low redox conditions in the reactor. Chemical scavenging of oxygen by iron sulfide may have reduced or eliminated the potential for chlorate-respiring microorganisms to utilize oxygen generated by chlorite dismutase, thereby reducing measured biomass yields. Two isolates (PDA and PDB) were found to respire chlorate but not perchlorate. This finding was unexpected based on previous reports that all chlorate-respiring bacteria could grow with perchlorate as the electron acceptor (6, 12, 18). van Ginkel et al. (38) obtained a chlorate-reducing enzyme from isolate GR1 that was found to reduce perchlorate, reinforcing the idea that a single enzyme facilitates both chlorate and perchlorate reduction. The failure of isolates PDA and PDB to grow using perchlorate provides suggestive evidence that there is more than one type of respiratory enzyme that can react with chlorate. In addition, a recent study (43) found that there were consistently higher numbers of chlorate-respiring than perchlorate-respiring bacteria in several soil, water, and wastewa-
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ter samples. This implies not only that there are differences in respiratory enzymes for perchlorate and chlorate respiration but that chlorate reducers are more abundant than perchlorate reducers in the natural environment. 16S rRNA sequencing suggests that most chlorate- and perchlorate-respiring isolates can be classified within the chloratereducing genera “Dechloromonas” and “Dechlorosoma” of the  class of Proteobacteria. This is consistent with previous observations that the majority of (per)chlorate-reducing bacteria isolated from diverse environments belong to these genera (7). Direct analysis of environmental samples using molecular probes specific for these genera also indicated that they are widespread and has prompted the suggestion that they are the most prevalent (per)chlorate reducers in nature (7). The ␥-Proteobacteria isolates obtained in this study (PDA and PDB) were most closely related to the (per)chlorate-reducing isolate PK and the denitrifying bacterium P. stutzeri, suggesting that there may be a link between the ability to use (per)chlorate as a terminal electron acceptor and the ability to denitrify. Nevertheless, PDA and PDB were unusual among chloratereducing isolates in that they could not grow with perchlorate as a terminal electron acceptor, whereas even closely related organisms, e.g., isolate PK, can reduce chlorate and perchlorate (6, 7). The apparent differences between microorganisms with respect to the presence and absence of perchlorate-reducing capability in chlorate-reducing bacteria warrant further biochemical investigations on the respiratory enzymes of these unusual microorganisms. ACKNOWLEDGMENTS This research was supported in part by the National Science Foundation (grant BES9714575), the American Water Works Association Research Foundation (AWWARF grant no. 2557), and a gift from Regenesis Corp. We thank Arlene Rowan for her assistance in obtaining partial 16S rRNA sequence data for isolates PDA and PDB. REFERENCES 1. American Water Works Association Research Foundation. 1997. Final report of the Perchlorate Research Issue Group workshop, September 30 to October 2. American Water Works Association Research Foundation, Denver, Colo. 2. Aslander, A. 1928. Experiments on the eradication of Canada thistle, Cirsium arvense, with chlorates and other herbicides. J. Agric. Res. 36:915–928. 3. Attaway, H., and M. Smith. 1993. Reduction of perchlorate by an anaerobic enrichment culture. J. Ind. Microbiol. 12:408–412. 4. Betts, K. S. 1999. A wide variety of bugs can break down perchlorate. Environ. Sci. Technol. 33:515.A. 5. Bruce, R. A., L. A. Achenbach, and J. D. Coates. 1999. Reduction of (per) chlorate by a novel organism isolated from a paper mill waste. Environ. Microbiol. 1:319–328. 6. Coates, J. R., U. Michaelidou, R. A. Bruce, S. M. O’Connor, J. N. Crespi, and L. A. Achenbach. 1999. Ubiquity and diversity of dissimilatory (per)chlorate reducing bacteria. Appl. Environ. Microbiol. 65:5234–5241. 7. Coates, J. R., U. Michaelidou, S. M. O’Connor, R. A. Bruce, and L. A. Achenbach. 2000. The diverse microbiology of (per)chlorate reduction, p. 257–270. In E. T. Urbansky (ed.), Perchlorate in the environment. Kluwer Academic/Plenum Publishers, New York, N.Y. 8. Edwards, U., T. Rogall, H. Blo ¨cker, M. Emde, and E. C. Bo¨ttger. 1989. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res. 17:7843–7853. 9. Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17:368–376. 10. Felsenstein, J. 1989. PHYLIP—phylogeny inference package. Cladistics 5:164–166. 11. Fox, G. E., J. D. Wisotzkey, and P. Jurtshuk, Jr. 1992. How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int. J. Syst. Bacteriol. 42:166–170.
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A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol. 44:846–849. 33. Stouthamer, A. H. 1988. Bioenergetics and yields with electron acceptors other than oxygen, p. 345–437. In L. E. Erikson and D. Y. C. Fung (ed.), Handbook on anaerobic fermentations. Dekker, New York, N.Y. 34. Urbansky, E. T. 1998. Review and discussion of perchlorate chemistry as related to analysis and remediation. Bioremed. J. 2:81–95. 35. Urbansky, E. T. (ed.). 2000. Perchlorate in the environment. Kluwer Academic/Plenum Publishers, New York, N.Y. 36. Urbansky, E. T., and M. R. Shock. 1999. Issues in managing the risks associated with perchlorate in drinking water. J. Environ. Management 56: 79–95. 37. van de Peer, Y., and R. de Wachter. 1994. TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput. Applic. Biosci. 10:569–570. 38. van Ginkel, C. G., A. G. M. Kroon, G. B. Rikken, and S. W. M. Kengen. 1998. Microbial conversion of perchlorate, chlorate and chlorite, p. 92–95. In Proceedings of the National Ground Water Association Southwest Focused Ground Water Conference: Discussing the Issue of MTBE and Perchlorate in the Ground Water. National Ground Water Association, Columbus, Ohio. 39. van Ginkel, G. C., C. M. Plugge, and C. A. Stroo. 1995. Reduction of chlorate
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APPL. ENVIRON. MICROBIOL. reactor. J. Ind. Microbiol. Biotechnol. 20:126–131. 42. Wallace, W. H., T. Ward, A. Breen, and H. Attaway. 1996. Identification of an anaerobic bacterium which reduces perchlorate and chlorate as Wolinella succinogenes. J. Ind. Microbiol 16:68–72. 43. Wu J. 2000. Degradation of pollutants in soil columns under chlorate-reducing conditions. M.S. thesis. The Pennsylvania State University, University Park.
