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
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 h
1,
respectively) were only slightly higher than the anoxic growth rates
obtained by these isolates during growth with chlorate (0.26 and 0.21 h1, 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 h1,
respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h1, respectively). The maximum growth rates of
PDX on perchlorate and chlorate were identical (0.21 h1)
and exceeded that of strain KJ on perchlorate (0.14 h1).
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 ± 0.07 [n = 7]), chlorate
(0.44 ± 0.05 [n = 7]), and perchlorate
(0.50 ± 0.08 [n = 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.
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INTRODUCTION |
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).
Observed maximum bacterial growth rates on chlorate and perchlorate
have been reported to be in the range of 0.012 to 0.28 h1 (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 h1 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 h1, 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 perchlorate-degrading 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).
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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 · 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 · H2O, 3 mg of EDTA, 4 mg of FeSO4 · H2O, 0.4 mg of
NaMoO4 · H2O, 0.1 mg
of NiCl2 · 6H2O, 1 mg of NaSeO3 · 5H2O, and 0.6 mg of
H3BO3), medium H (0.1 mg
[each] of NiCl2 · 6H2O and
Na2SeO3 · 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 (µm) 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 µ = 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-µm 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, Switzerland)
using membrane filters (25 mm, 0.2-µm 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 µl 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 perchlorate-degrading 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 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 perchlorate-grown 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).
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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.
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The maximum observed growth rates of KJ were similar on oxygen or
chlorate (0.26 and 27 h1) but were much lower
on perchlorate (0.14 h1) (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).
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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.
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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
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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 h1) (Fig. 3) were similar to those of KJ (0.26 to 0.27 h1) (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 h1 at ~500 mg of acetate/liter) (Fig. 3),
however, was 50% greater than that of isolate KJ (0.14 h1) (Fig. 3).
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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.
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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 much
lower on lactate (0.15 h1) than on acetate
(0.21 h1). 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).
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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.
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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 h1, respectively)
than with chlorate (0.18 and 0.21 h1,
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 h1) and PDX (0.21 h1) 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.
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FIG. 5.
Perchlorate uptake rates (milligrams of perchlorate per
milligram of protein per hour) for isolates KJ and PDX.
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Biomass yields.
The biomass yields (grams [DW] per gram of
acetate) measured for KJ were not significantly different
(P 
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).
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TABLE 2.
Comparison of cell yields in the presence of various
electron acceptors of isolate KJ versus those reported by others
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Phylogeny.
Comparative 16S rRNA sequence analysis indicated
that strains KJ and PDX were most closely related to
"Dechlorosoma suillum" strain PS and
"Dechlorosoma" sp. 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 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."
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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.
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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).
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TABLE 3.
Maximum reported growth rates of previously described
chlorate- and perchlorate-respiring isolates or mixed cultures
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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 wastewater 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
chlorate-reducing 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 chlorate-reducing 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.
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.
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Abstract
Introduction
