Perchlorate and chlorate degradation by two organisms isolated from
wastewater:
Microbial identification and kinetics
Prata, F. P., Costa C., Reis M. A. M., Lemos, P. C.
The biological removal of perchlorate (ClO4-) and chlorate (ClO3-) can be viewed as a very promising
water treatment technology. The process is based on the ability of specific bacteria to use (per)chlorate as
an electron acceptor in the absence of oxygen. The present research work was focused on the isolation
and kinetic characterization of perchlorate reducing bacteria. The enrichment process started with a
sludge sample taken from an anaerobic digester of a domestic wastewater treatment plant (Beirolas,
Portugal). Two perchlorate-reducing bacteria (per1) and (per2) were isolated using different selection
methods, platting and liquid transfer respectively. The purity of the isolates was confirmed by genetic
characterization of 16S rDNA. The BLAST search showed that the microorganims shared a 99%
sequence similarities to the 16S rDNA of Dechlorospirillum sp. DB (per1) and Dechlorosoma sp. PCC
(per2). Batch tests were performed under anaerobic conditions with acetate as the electron donor and
perchlorate and/or chlorate as electron acceptor. During perchlorate reduction by Dechlorospirillum sp.
DB it was observed transient accumulation of chlorate. The isolates showed different behaviour
concerning perchlorate and chlorate reduction. Chlorate was preferentially reduced when both electron
acceptors were present, being perchlorate reduced after completely depletion of chlorate. The former
performance was observed in both bacteria.
Keywords: Bioremediation, perchlorate/chlorate reduction, competitive inhibition, Dechlorospirillum sp.
DB, Dechloromona sp. PCC.
1
INTRODUCTION
Perchlorate (ClO4-) and chlorate (ClO3-) have been produced on a large scale by the chemical industry for
use in a wide range of applications. The improper storage and/or disposal of these oxyanions have led to
harmful concentrations in surface and groundwater, as they are extremely soluble and not significantly
broken down in the environment. These characteristics make them persistent and problematic
environmental pollutants of drinking waters. The biological removal of theses anions can be viewed as a
very promising water treatment technology. ClO4- is a strong oxidizing agent, mostly used as ammonium
perchlorate (NH4ClO4) in the manufacturing of solid rocket fuel, missiles and explosives for various
military munitions and also in industrial products (e.g., fireworks, air bag inflators and paint). The presence
of perchlorate in water supplies and soils is also linked to the earlier use of Chilean nitrate as fertilizer
(Urbansky et al., 2001), and recently its natural formation was reported (Dasgupta et al. 2005). Perchlorate
competitively blocks thyroid iodine uptake and inhibit normal thyroid hormone production, which may lead
to metabolic problems in adults and anomalous development in children (Greer et al., 2002).
ClO3- is also a potential chlorine oxyanion pollutant, which has been used as an herbicide in agriculture, and
it is used for the on-site generation of the bleaching agent chlorine dioxide (ClO2) in the paper and pulp
industry (Rosemarin et al., 1990). It can also be formed through the ozonation of drinking waters treated
with chlorine (Siddiqui, 1996). ClO3- and also chlorite (ClO2-) added to drinking water can cause haemolytic
anaemia in rats (Condie, 1986), due to oxidative damage of red blood cells.
Perchlorate is on drinking water contaminant candidate list, but in the meantime the US Environmental
Protection Agency (EPA) has established an official reference dose (RfD) corresponding to a drinking water
level of 24.5 ppb od ClO4-. Chlorate has no legislation yet.
The high oxidation state of (per)chlorate makes them ideal electron acceptors for microbial metabolism, and
the microbial reduction of these compounds is energetically favourable. Many perchlorate-reducing bacteria
(PRB) have now been isolate. Perchlorate-reducing strains reported in the literature include: Vibrio
dechloraticans Cuznesove B-1168 (Korenkov et al., 1976), Wolinella succinogenes HAP-1 (Wallace et al.,
1996), Dechlorosoma suillum (Chaudhuri et al., 2002) and isolates GR-1 (Rikken et al., 1996), perclace
(Herman et al., 1998), and CKB (Bruce et al., 1999). These PRB are mainly Gram-negative, nonfermentative facultative anaerobes. Analysis of the 16S rDNA demonstrated that these organisms are
phylogenetically diverse with members in the α-, β-, γ-, and ε-subclasses of the Proteobacteria (Coates et al.,
1999; Wallace et al., 1996). However, the majority resides in the β-subclass of the Proteobacteria, being
members of the genus Dechloromonas or Dechlorosoma (Coates et al., 1999).
In 1996, Rikken and his colleagues proposed a three-step mechanism of perchlorate reduction (Equation 1):
ClO4- ClO3- ClO2- O2 + Cl-
(1)
The perchlorate reduction pathway consist in three steps, the first two steps via two electrons transfers by
(per)chlorate reductase, which sequentially reduces perchlorate to chlorate, then chlorate to chlorite (Kengen
et al., 1999; Bender et al., 2005). The third step with chlorite dismutase, which transforms chlorite into
2
chloride and oxygen by disproportionation, does not consume electrons and therefore does not directly
produce energy for the cells (van Ginkel et al., 1996; Bender et al., 2002). Chlorate accumulation has only
been reported for a mixed culture growing on nitrate and perchlorate (Nerenberg et al., 2002) and for a pure
culture growing on perchlorate (Nerenberg et al., 2006). Chlorite dismutase and (per)chlorate reductase are
the only enzymes in the perchlorate reduction pathway that have been isolated and characterized (Coates et
al., 1999; Kengen et al., 1999;). However, it is still not clear if only a single enzyme is used by PRB for
perchlorate and chlorate reduction, or if there are separate enzymes used for perchlorate and chlorate
reduction. Evidence for different enzymes was provided indirectly by the fact that not all chlorate-reducing
bacteria (CRB) were capable of respiring perchlorate, although this question will require further research in
order to be solved. Up to now, at least three enzymes that can reduce chlorate have been purified and
characterized. A chlorate reductase C was purified from the denitrifying strain Proteus mirabilis (Oltmann et
al., 1976), as well as from Pseudomonas chloritidismutans (Wolterink et al., 2003) and Pseudomonas sp.
PDA (Steinberg et al., 2005).
Improved understanding of the biological perchlorate reduction kinetics and its biochemical mechanisms will
lead to better biological remediation processes. Since there are hardly any perchlorate degradation kinetic
data available, the present work was focused on isolation of possible new perchlorate reducing bacteria as
well as its kinetic characterization.
MATERIALS AND METHODS
Media. For the enrichment process it was used Basal medium amended with 1 mL/L of mineral solution SL10. For growth kinetics of Dechlorospirillum sp. DB, to the Basal medium was added 1 mL/L of Medium KL
and for growth kinetics of Dechlorosoma sp. PCC, the Basal medium was amended with 1 mL/L of Medium
SLA (Table 1, page 4).
All media were prepared using ultrapure water (Milli Q system) and research grade chemicals in the
amounts indicated (per liter).
Sodium acetate was used in 1:2 molar ratio to sodium perchlorate and/or sodium chlorate. Solid agar plates
were prepared by adding 15 g/L agar on the media previous described.
Bacterial isolation procedures. A sludge sample was collected at the anaerobic digester of a wastewater
treatment plant (Beirolas, Portugal). To start the enrichment a primary inoculum was obtained by dilution of
the sludge sample. The medium used for the enrichment process was Basal medium + SL-10. The culture
became turbid in 7 to 14 days and continuous transfers (10% by volume) were made during a period of two
months. The enrichment process was performed in 50 mL glass flasks under anaerobic conditions. For
further enrichment, ClO4- concentration was increased from 5 to 10 mM. Subsequently, two different
selection methods were applied to reach pure cultures. In the first one, a sample of the enriched culture was
serially diluted to 10-3 and spread onto agar plates. Select colonies were picked and then re-grown in fresh
medium. The second method applied consisted in the continuous transfers (10% by volume) of the enriched
3
liquid culture at exponential phase to fresh medium during a period of 20 days. The enrichments were
followed by microscopic examination and the purity of the isolates obtained was further confirmed by
molecular methods.
Table 1 – Media and reagents used for enrichment and isolation.
Reagent
Basal
Medium
K2HPO4
1.55
NaH2PO4.H2O
0.85
NH4Cl
0.25
MgSO4.7H2O
0.1
SL-10
HCl (37%)
10 ml
Na2SeO3
0.0017
Na2SeO3.5H2O
FeCl2.4H2O
Medium
KL
Medium
SLA
0.15
0.1
1.5
FeSO4.7H2O
18
4
Na2MoO4.2H2O
0.036
0.4
0.3
NiCl2.6H2O
0.024
0.1
0.1
EDTA
3
H3BO3
ZnCl2
0.6
0.07
5
1
MnCl2.4H2O
0.1
0.7
CoCl2.6H2O
0.19
2.5
CuCl2.2H2O
0.002
0.1
16S ribosomal DNA extraction and sequencing. DNA extraction of the isolates was performed using
FastDNA® SPIN Kit (for soil). Some changes were performed in order to adjust the kit to our sample. The
extraction was confirmed by gel electrophoresis. The 16S ribosomal DNAs were amplified by conventional
PCR. The amplification program included initial denaturation at 94oC for 5 minutes followed by three steps
repeated 30 times. Step 1: 94oC for 30 seconds; step 2: 48oC for 30 seconds; and step 3: 72oC for 2 minutes.
The final elongation was at 72oC for 5 minutes. The primers 27f and 1492r, and Taq polymerase (Invritogen)
were used in this amplification. The PCR products were purified and sequenced by BaseClear, DNA
sequencing services, The Netherlands.
Phylogenetic analysis. For establishing the identity of the isolates by 16S rDNA nucleotide-nucleotide
sequence homology, the BLAST (Basic Local Alignment Search Tool) network service at the National
Center for Biotechnological Information (NCBI) was used. (http://www.ncbi.nih.gov, 2007).
4
Batch growth kinetics. Isolate (per1) was grown on basal medium + medium KL and (per2) was grown on
basal medium + medium SLA. The batch tests were performed in a reactor filled with 0.5 L of the
appropriate medium with electron acceptor and donor under anaerobic conditions. The growth kinetics was
conduct at controlled temperature (37oC) and the redox potential was measured with a redox electrode insitu. The pH measurements were made ex-situ and samples were taken (5 mL) in sterilized conditions, at
regular time intervals for further analysis.
Analytical techniques. Culture growth was monitored by optical density at 600nm (OD600nm) with a
spectrophotometer and converted to dry weight (DW) using a calibration curve. The DW determination was
made using the method described elsewhere (Olsson and Nielsen, 1997).
The anions concentration was analyzed by HPLC. The concentration of perchlorate was determined by ion
chromatography equipped with a Ion Pac AS16 column and a AG16 guard column (4mm, Dionex), a selfregenerating suppressor (SRS Ultra II), and an autosampler. The samples were analyzed with a 50mM NaOH
mobile phase at a flow rate of 1 ml min-1. The injection loop volume was 30µl. The chlorate, chlorite,
chloride and acetate were determined with the same ion chromatography system described before while an
Ion Pac AS9 column and a AG9 guard column was used this time. The eluent used was 9 mM Na2CO3 at a
flow rate of 1ml min-1. The injection loop volume was 30µl and the suppressor controller was set at 50 mA
for all the analysis.
RESULTS AND DISCUSSION
Microbiologic characterization of the isolates. With the first selection method (platting) it was obtained a
spirillum-shaped enriched culture (per1). The individual cells were highly motile and occasionally growing
as clusters. The purity was confirmed by genetic characterization of 16S rDNA. The BLAST search showed
that the first isolate (per1) shared a 99% sequence similarities to the 16S rDNA of Dechlorospirillum sp. DB.
Dechlorospirillum sp. DB has its complete sequence of 16S rDNA with the accession number AY530551
(Bender et al., 2004). Phylogenetically belongs to the α-subclasse of Proteobacteria and was first isolated
during a cld (chlorite dismutase) primer development (J. Coates, unpublished data). The chlorite dismutase
gene was also sequenced (Bender et al., 2004), but no kinetic parameters were determined so far. Through
the second isolation method (liquid) it was achieved a rod-shaped enriched culture (per2). The cells were in
its majority non-motile and clusters were not observed. Concerning the genetic characterization by 16S
rDNA of the second isolate (per2), the sequencing showed that the microorganim shared a 99% sequence
similarities to the 16S rDNA of Dechlorosoma sp. PCC. This bacteria have the 16S rDNA partially
sequenced with the accession number AY126453 (Nerenberg et al., 2002), but no other publication related
with this bacteria is available at the moment. Phylogenetically belongs to the β-subclasse of Proteobacteria.
5
Growth and kinetic parameters. The growth kinetic of Dechlorospirillum sp. DB and Dechlorosoma sp.
PCC were performed in batch tests under anaerobic conditions with acetate as the electron donor and
perchlorate and/or chlorate as electron acceptor. Both bacteria were grown in medium amended with 20mM
of CH3COO- as electron donor and 10 mM of ClO4- as final electron acceptor (Figure 1).
Dechlorospirillum sp. DB
25
Dechlorosoma sp. PCC
25
-ClO4
0.4
-CH3COO
0.35
ClO4
-ClO3
20
CH3COO
20
0.2
10
0.15
Acet, ClO4- (mM)
0.25
15
ClO3- (mM)
Acet, ClO4- (mM)
0.3
15
10
0.1
5
5
0.05
0
0
0
0
1
2
3
4
5
6
7
8
9
10
11
0
12
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Time (h)
Tim e (h)
Figure 1 – Perchlorate reduction by Dechlorospirillum sp. DB and Dechlorosoma sp. PCC. Chlorate formation
and subsequent degradation was observed during the reduction of 10mM of ClO4- by Dechlorospirillum sp. DB. Note
the different concentration scale for ClO3-.
It was observed with Dechlorospirillum sp. DB accumulation and subsequent degradation of the intermediate
chlorate. Around 3.4% on a molar basis of perchlorate concentration was accumulated by Dechlorospirillum
sp. DB. The same was not observed with Dechlorosoma sp. PCC. Chlorate accumulation by
Dechlorospirillum sp. DB indicates that perchlorate reduction have different systems within perchlorate
reducing bacteria.
In order to study the growth kinetic with chlorate as the final electron acceptor, it was performed a kinetic
study with 10mM ClO3-. Again CH3COO- was used as electron donor in a concentration of 20mM (Figure 2).
Dechlorosoma sp. PCC
25
25
0.8
ClO3CH3COOClDW (g/l)
20
0.6
0.4
10
0.6
15
mM
mM
15
DW (g/l)
20
0.8
ClO3CH3COOClDW (g/l)
0.4
10
0.2
5
DW (g/l)
Dechlorospirillum sp. DB
0.2
5
0
0
0
1
2
3
4
5
Time (h)
6
7
8
9
10
0
0.0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Time (h)
Figure 2 – Chlorate reduction by Dechlorospirillum sp. DB and Dechlorosoma sp. PCC.
6
Concerning chlorate as the sole final electron acceptor, different behaviours were also observed in both
bacteria. This fact emphasizes once more the presence of different (per)chlorate reduction system within
perchlorate reducing bacteria. Dechlorospirillum sp. DB showed specific growth rate as well as acetate and
chlorate uptake rates higher than Dechlorosoma sp. PCC (Table 2). Comparing each one individually with
the former test with perchlorate, Dechlorospirillum sp. DB showed higher specific growth rate during
chlorate reduction. In this way, it should not be expected chlorate accumulation by Dechlorospirillum sp. DB
in the former test. Chlorate was completely reduced to chloride in both bacteria, which proves the conversion
of chlorate into chloride.
It was also performed a kinetic study with 5mM ClO4- and 5mM ClO3- simultaneously in the media in order
to study the growth kinetic of Dechlorospirillum sp. DB and Dechlorosoma sp. PCC (Figure 3).
Dechlorospirillum sp. DB
Dechlorosoma sp. PCC
20
1.2
ClO4CH3COOClO3DW (g/l)
25
1.0
20
15
mM
DW (g/L)
mM
0.6
10
0.6
10
0.4
0.4
5
0.2
0
0.0
0
1
2
3
4
5
Time (h)
6
7
8
9
10
1.0
0.8
0.8
15
1.2
DW (g/l)
-ClO3
-CH3COO
-Cl
-ClO4
DW
25
5
0.2
0
0.0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
Time (h)
Figure 3 – Acetate and chlorate uptake as function of time during the reduction of 5mM of ClO4- and 5mM of
ClO3- simultaneously, by Dechlorospirillum sp. DB and Dechlorosoma sp. PCC.
In this kinetics it should be stressed the preference for chlorate when both were present in the media.
Perchlorate was not reduced unless chlorate was almost totally reduced. This observation indicates that
chlorate could inhibit perchlorate reduction when both were present in the same concentration of 5mM.
Concerning acetate, it can be observed in each kinetic study two different uptake rates, clearly related with
chlorate and perchlorate reduction respectively (Table 2). The chloride produced was identical to the sum of
perchlorate and chlorate amounts, showing once more a completely conversion of both electron donors into
chloride.
Batch tests with Dechlorospirillum sp. DB and Dechlorosoma sp. PCC were performed in order to determine
kinetic parameters such as specific growth rate (µmax), specific acetate uptake rates (qCH3COO-), specific
perchlorate reduction rates (qClO4-), specific chlorate reduction rates (qClO3-) and biomass yield (g [DW] /
gCH3COO-). They were grown on acetate as the electron donor and perchlorate and/or chlorate as the
electron acceptor.
7
Table 2 – Resume of all kinetic parameters (n, number of data points considered for parameter calculations
Isolate
electron
acceptor
ClO4-
ClO3DB
ClO4+
ClO3-
ClO4ClO3PCC
µmax
(h-1)
-qCH3COO-1
(mmol h
g-1 DW)
-qe-
Y
Y
Y
Y
(mmol h
g-1 DW)
e- acceptor
/CH3COO
e-acceptor /
Cl
g[DW] / g acet
g[DW] / g e- acceptor
acceptor
-1
0.166 ± 0.004
5.3 ± 0.2
2.9 ± 0.1
0.578 ± 0.008
0.85 ± 0.04
1.04 ± 0.04
1.41 ± 0.04
(n = 9)
(n = 5)
(n = 5)
(n = 9)
(n = 7)
(n = 5)
(n= 5)
0.192 ± 0.007
20.5 ± 1.8
11.5 ± 0.9
0.69 ± 0.04
0.84 ± 0.03
0.51 ± 0.07
0.42 ± 0.08
(n = 4)
(n = 4)
(n = 3)
(n = 5)
(n= 5)
(n= 8)
(n= 5)
5.6 ± 0.4
2.5 ± 0.2
0.472 ± 0.004
0.247 ± 0.01
(n = 4)
(n = 4)
(n=4)
0.81 ± 0.04
1.14 ± 0.03
(n= 3)
(n = 5)
17.7 ± 1.8
5.2 ± 0.4
0.43 ± 0.1
(n= 5)
(n= 7)
2.44± 0.01
(n= 3)
(n=3)
(n= 5)
10.9 ± 0.5
7.3 ± 0.4
0.72 ± 0.03
0.167 ± 0.004
0.93 ± 0.05
(n= 7)
0.93 ± 0.04
0.70 ± 0.02
0.56 ± 0.06
(n = 11)
(n= 4)
(n= 4)
(n = 7)
(n= 7)
(n=16)
(n= 11)
0.132 ± 0.005
4.14± 0.07
2.4 ± 0.1
0.64 ± 0.03
0.86 ± 0.03
0.83 ± 0.03
0.72 ± 0.02
(n = 6)
(n= 5)
(n= 5)
(n= 3)
(n= 11)
(n=16)
(n= 16)
3.1± 0.1
1.8 ± 0.1
0.64 ± 0.02
ClO4-
0.149 ± 0.004
(n= 6)
(n= 5)
(n= 6)
0.95 ± 0.02
+
(n = 9)
14.1 ± 0.6
4.8 ± 0.2
0.924 ± 0.03
(n= 15)
(n= 4)
(n= 5)
(n= 3)
ClO3-
0.80 ± 0.04
1.18 ± 0.02
(n= 11)
(n= 5)
0.88 ± 0.03
(n=7)
The specific growth rates determined in this study were within the values found in the literature, ranging
from 0.07 to 0.26 h-1 (Figure 4).
Figure 4 – Specific growth rate determined for both bacteria, Dechlorospirillum sp. DB and Dechlorosoma sp.
PCC with perchlorate and chlorate and values found in the literature: KJ (Logan et al., 2001); GR1 (Rikken et al.,1996);
PDX (Logan et al., 2001) and perclace (Herman and Frankenberger, 1998)
Among the cell yield found in the literature it was observed that Dechlorospirillum sp. DB showed higher
values for perchlorate reduction and that Dechlorosoma sp. PCC showed the highest values for chlorate
reduction compared with the other isolates. In this study, an interesting observation was that for chlorate
reduction the values of the specific growth rate vary indirectly to the cell yield (Figure 5).
8
Figure 5 – Cell yield determined for both bacteria, Dechlorospirillum sp. DB and Dechlorosoma sp. PCC with
perchlorate and chlorate and values found in the literature: KJ (Logan et al., 2001); GR1 (Rikken et al.,1996) and AB1
(Olsen et al., 1997).
CONCLUSIONS AND FURTHER RESEARCH
Each one of the bacteria isolated in this research work had different perchlorate reduction system. The main
evidence was the transient accumulation of chlorate by Dechlorospirillum sp. DB during perchlorate
reduction, which was not observed in Dechlorosoma sp. PCC. Chlorate accumulation can be explained based
on the existence of two enzymes responsible for the conversion of perchlorate into chlorite, in which the
conversion of chlorate into chlorite was the rate-limiting step. If a unique enzyme was present, then chlorate
accumulation could be explained based on the idea that chlorate reduction decreased when perchlorate is
present at 10mM. However, the results found were not sufficient to predict if one enzyme was responsible
for both reduction (perchlorate and chlorate) or if different enzymes were present. Chlorate accumulation
during perchlorate reduction was hardly studied and for further investigation, the enzymes involved in the
perchlorate reduction pathway of these two bacteria, should be purified and studied concerning its
biochemical characterization. Also kinetic test starting with different perchlorate and chlorate concentration
should be done to observe the effect of the initial concentration over the reduction of each electron acceptor.
Regarding the batch test done with chlorate and perchlorate present in the same medium, it was observed the
preference for chlorate over perchlorate in both bacteria. This observation could also indicate that chlorate
inhibit perchlorate reduction, although this finding was not in agreement with chlorate accumulation
observed during perchlorate reduction in the first test. For further elucidation, the effect of chlorate during
perchlorate reduction should be studied. In order to do so a test in which a chlorate spike is added during
perchlorate reduction should be performed.
The genetic characterization of 16S rDNA showed that both isolates have already its sequence deposited,
but no other description was made. For further research concerning characterization it should be done the
construction of the phylogenetic tree. Concerning description of the bacteria it should be done the G + C
content, microbial size and all the tests in order to clarify their characteristics.
9
BIBLIOGRAPHY
Bender, K. S., C. Shang, R. Chakraborty, S. M. Belchik, J. D. Coates, and L. A. Achenbach. 2005. Identification,
characterization, and classification of genes encoding perchlorate reductase. J. Bacteriol., 187:5090-5096.
Bender, K. S., S. M. O’Connor, R. Chakraborty, J. D. Coates, and L. A. Achenbach. 2002. Sequencing and
transcriptional analysis of the chlorite dismutase gene of Dechloromonas agitata and its use as a metabolic
probe. Appl. Environ. Microbiol., 68:4820-4826.
Bruce, R. A., L. A. Achenbach, and J. D. Coates. 1999. Reduction of (per)chlorate by a novel organism isolated from
paper mill waste. Environ. Microbiol., 1:319-329.
Chaudhuri S. K., S. M. O’Connor, R. L. Gustavson, L. A. Achenbach, and J. D. Coates. 2002. Environmental
factors that control microbial perchlorate reduction. Appl. Environ. Microbiol., 68:4425-4430.
Coates, J. D., 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.
Condie, L. 1986. Toxicological problems associated with chlorine dioxide. J. Am. Water Works Assoc., 78:73-78.
Dasgupta, P. K., P. K. Martinnelango, W. A. Jackson, T. A. Anderson, K. Tian, R. W. Tock and S. Rajagopalan.
2005. The origin of naturally occurring perchlorate: the role of atmospheric processes. Environ. Sci. Technol.,
39:1569-1575.
Greer, M. A., G. Goodman, R. C. Pleus and S. E. Greer. 2002. Health effects assessment for environmental
perchlorate contamination: the dose response for inhibition of thyroidal radioiodine uptake in humans. Environ
Health Presp., 110:927-937.
Herman, D. C., and W. T. Frankenberger. 1998. Microbial-mediated reduction of perchlorate in groundwater. J.
Environ. Qual., 27: 750-754
Kengen, S. W. M., G. B. Rikken, W. R. Hagen, C. G. Van Ginkel, and A. J. M. Stams. 1999. Purification and
characterization of (per)chlorate reductase from the chlorate-respiring strain GR-1. J. Bacteriol., 181:6706-6711.
Korenkov, V. N., V. I. Romanenko, S. I. Kuznetsov and J. V. Voronnov. 1976. Process for purification of industrial
wastewaters from perchlorate and chlorates. U. S. patent 3,943,055.
Logan, B. E., Zhang, H., Mulvaney, P., Milner, M. G., Head, I. M. and Unz, R. F. 2001. Kinetics of perchlorateand chlorate-respiring bacteria. App. Environ. Microbiol., 67:2499-2506.
Nerenberg, R., B. E. Rittmann, and I. Najm. 2002. Perchlorate reduction in a hydrogen-based membrane-biofilm
reactor. Journal AWWA., 94:103-111.
Nerenberg, R., Y. Kawagoshi, and B. E. Rittmann. 2006. Kinetics of a hydrogen-oxidizing, perchlorate-reducing
bacterium. Wat. Res., 40:3290-3296.
Olsen, S., 1997. Consortium and pure culture kinetics of chlorate reducing organisms. M. S. thesis. University Arizona.
Tucson.
Olsson, L., J. Nielsen. 1997. On-line and in situ monitoring of biomass in submerged cultivations. TibTech. Vol 15, pp
517-522.
Oltmann, L. F., W. N. Reijnders and A. H. Stoughamer. 1976 Characterization of purified nitrate reductase A and
chlorate reductase C from Proteus mirabilis. Arch. Microbiol., 111:25-35.
Rikken, G. B., A. G. M. Kroon and C. G. van Ginkel. 1996. Transformation of (per)chlorate into chloride by a
newly isolated bacterium: reduction and dismutation. Appl. Microbiol. Biotechnol., 45:420-426.
Rosemarin, A., K. Lehtinen and M. Notini. 1990. Effects of treated and untreated softwood pupl mill effluents on
Baltic sea algae and invertebrates in model ecosystems. Nord Pulp Paper Res. J., 2:83-87.
Sidiqqui, M. 1996. Chlorine-ozone interactions: formation of chlorate. Wat. Res., 30: 2160-2170
Steinberg, L. M., J. J. Trimble and B. E. Logan. 2005. Enzymes responsible for chlorate reduction by Pseudomonas
sp. are different from those used for perchlorate reduction by Azospira sp. FEMS Microbiol. Lett., 247:153-159.
Urbansky, E.T., S.K. Brown, M.L. Magnuson, C.A. Kelty. 2001. Perchlorate levels in samples of sodium nitrate
fertilizer derived from Chilean caliche. Environ. Pollution, 112: 299-302.
van Ginkel, C. G., Rikken, G. B., A. G. M. Kroon and S. W. M. Kengen 1996. Purification and characterization of
chlorite dismutase: a novel oxygen-generating enzyme. Arch. Microbiol. 166:321-326.
Wallace, W., 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.
Wolterink, A. F. W. M., E. Schiltz, P-L. Hagedoorn, W. R. Hagen, S. W. M. Kengen and A. J. M. Stams. 2003.
Characterization of the Chlorate Reductase from Pseudomonas chloritidismutans. J. Bacteriol., 185:3210-3213.
10