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Previous Article | Next Article 
Applied and Environmental Microbiology, December 2000, p. 5110-5115, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Enrichment and Molecular Characterization of a Bacterial Culture
That Degrades Methoxy-Methyl Urea Herbicides and Their
Aniline Derivatives
Said
El-Fantroussi*
Laboratory of Microbial Ecology and Technology,
University of Ghent, B-9000 Ghent, Belgium
Received 10 April 2000/Accepted 6 September 2000
 |
ABSTRACT |
Soil treated with linuron for more than 10 years showed high
biodegradation activity towards methoxy-methyl urea herbicides. Untreated control soil samples taken from the same location did not
express any linuron degradation activity, even after 40 days of
incubation. Hence, the occurrence in the field of a microbiota having
the capacity to degrade a specific herbicide was related to the
long-term treatment of the soil. The enrichment culture isolated from
treated soil showed specific degradation activity towards
methoxy-methyl urea herbicides, such as linuron and metobromuron, while
dimethyl urea herbicides, such as diuron, chlorotoluron, and
isoproturon, were not transformed. The putative metabolic intermediates
of linuron and metobromuron, the aniline derivatives 3,4-dichloroaniline and 4-bromoaniline, were also degraded. The temperature of incubation drastically affected degradation of the
aniline derivatives. Whereas linuron was transformed at 28 and 37°C,
3,4-dichloroaniline was transformed only at 28°C. Monitoring the
enrichment process by reverse transcription-PCR and denaturing gradient
gel electrophoresis (DGGE) showed that a mixture of bacterial species
under adequate physiological conditions was required to completely
transform linuron. This research indicates that for biodegradation of
linuron, several years of adaptation have led to selection of a
bacterial consortium capable of completely transforming linuron.
Moreover, several of the putative species appear to be difficult to
culture since they were detectable by DGGE but were not culturable on
agar plates.
| |
INTRODUCTION |
Contamination of surface water and
groundwater by pesticides is a major environmental concern. General
agricultural application of pesticides and point locations (e.g.,
rinsate wastes from farming operations) are important sources of
contamination. In addition, many pesticide-formulating retailers have
sites sufficiently contaminated to qualify as Superfund sites (11,
18). Phenylurea herbicides are among the most widely used
herbicides in noncrop areas, as well as in tree crops (20).
Given the potential carcinogenic risk of these herbicides (12,
17), there is a serious need to develop remediation processes to
eliminate or minimize contamination of surface water and groundwater.
Biodegradation could be a reliable and cost-effective technique for
pesticide abatement. In this context, I isolated an enrichment culture
capable of degrading the herbicide linuron and 3,4-dichloroaniline
(3,4-DCA). The latter is considered the main intermediate metabolite in
the degradation of several herbicides. The enrichment culture was
derived from an orchard that was regularly treated with linuron for
more than 10 years. Samples of untreated soil collected from the same
location did not express any degradation activity (5). In
the current study denaturing gradient gel electrophoresis (DGGE) was
used to monitor bacterial populations in the enrichment cultures over time and in response to different physiological conditions, such as the
temperature of incubation and the composition of the culture medium.
Application of DGGE in microbial ecology has attracted great interest
in the last few years (10). This technique is suitable for
studying and monitoring enrichment cultures (6, 15, 22).
Teske et al. (19) have used DGGE to identify the bacterial
composition of a coculture capable of sulfate reduction after exposure
to oxic and microoxic conditions. DGGE was used to show changes in
bacterial species composition in enrichment cultures prepared with
various dilutions of inoculum (6). The aim of the present
study was to understand the enrichment process with bacterial strains
capable of degrading the herbicide linuron and its intermediate
metabolite 3,4-DCA.
| |
MATERIALS AND METHODS |
Soil.
Soil samples were taken from the Royal Research
Station of Gorsem (Sint-Truiden, Belgium), an orchard that has been
regularly treated with different urea herbicides since 1987. Details of the soil properties and the soil-sampling procedures have been described previously (5).
Enrichment cultures.
Five grams of soil was added to 95 ml
of minimal medium containing (per liter of MilliQ water) 93.50 mg of
MgSO4 · 6H2O, 5.88 mg of
CaCl2 · 2H2O, 1.15 mg of
ZnSO4 · 7H2O, 1.16 mg of
H3BO3, 1.69 mg of
MnSO4 · H2O, 0.24 mg of
CoCl2 · 6H2O, 0.10 mg of MoO3, 2.78 mg of FeSO4 · 7H2O, and 0.37 mg
of CuSO4 · 5H2O. The pH was adjusted to
about 7.0 by using a phosphate buffer (10 mM
KH2PO4, 10 mM Na2HPO4).
Linuron was added as the sole source of carbon and nitrogen. The
concentration of linuron in the enrichment medium was initially 25 mg/liter and was gradually increased in subsequent flasks to a maximum
of 500 mg/liter. The first enrichment cultures were inoculated with
soil treated with linuron or with the untreated soil. The other
pesticides tested were diuron, chlorotoluron, metobromuron, and
isoproturon, each at a concentration of 25 mg/liter. 3,4-DCA was also
tested at a concentration of 25 mg/liter.
The disappearance of herbicides was monitored by high-performance
liquid chromatography (HPLC) analysis. The HPLC system consisted of a
Kontron liquid chromatograph equipped with a DEGASYS DG-1310 system to
degas the mobile phase, three Kontron 325 high-pressure pumps, a
Kontron MSI 660 injector with a 20-µl loop, a Kontron DAD 495 diode
array detector, and a 450 MT2/DAD software system. The column was a
Hypersil Green Environment 5U column (Alltech, Deerfield, Ill.). The
mobile phase was CH3OH-0.1 M
NH4H2PO4 (pH 3.8) (70:30), the flow
rate was 0.8 ml/min, and the UV detector was set at 210 nm. Products
were identified by comparison with authentic standards.
Nucleic acid extraction and PCR amplification.
DNA
extraction from soil and PCR amplification of 16S rRNA genes with the
primers set (primers P63 and P518) were performed by using the
protocols described previously (4, 5). Colonies isolated on
agar plates were boiled for 10 min in 200 µl of Milli-Q water before
the PCR was performed. For liquid enrichment cultures, RNA extraction
was performed with TRIZOL (Life Technologies, Rockville, Md.) used
according to the manufacturer's instructions. Reverse transcription
(RT)-PCR was performed with rTh DNA polymerase and a buffer kit
obtained from Perkin-Elmer. The RT reaction mixtures (20 µl)
contained 10 mM Tris-HCl (pH 8.3), 90 mM KCl, 1 mM MnCl2, each deoxynucleoside triphosphate at a concentration of 200 µM 0.75 µM primer P518 (7), and 5 U of rTh DNA polymerase. After addition of 1 µl of an RNA sample to each mixture, the mixtures were
incubated for 15 min at 70°C. Following the RT reaction, 80 µl of
each PCR mixture, containing 10 mM Tris-HCl (pH 8.3), 100 mM KCl, 3 mM
MgCl2, 0.75 mM ethylene bis(oxyethylenenitrilo)tetraacetic acid, 5% (vol/vol) glycerol, and 0.15 µM primer P63-GC
(5), was added. The samples were amplified by using 35 cycles consisting of 95°C for 10 s plus 60°C for 15 s.
The reaction was stopped after incubation at 7 min at 60°C.
DGGE.
DGGE analyses were based on the protocol described
previously (5). The polyacrylamide gels were made with a 40 to 55% denaturant gradient (100% denaturant contained 7 M urea and
40% [vol/vol] formamide). Electrophoresis was performed overnight at
60°C and 45 V. DNA fragments were excised from each DGGE gel and
sequenced as previously described (5).
| |
RESULTS AND DISCUSSION |
Enrichment of a microbial culture that degrades linuron.
Five
grams of soil that was either treated with linuron or not treated was
inoculated into liquid medium containing 25 mg of linuron per liter.
Whereas total transformation of linuron was observed in enrichments
inoculated with treated soil, no disappearance of this herbicide was
seen in enrichments inoculated with untreated soil (Fig.
1). The results were highly reproducible
as the samples collected in March 1998 and August 1998 gave the same
results (Fig. 1). Mixing treated and untreated soil samples in
different proportions showed that the degradation ability was found
only in the treated soil. In addition, autoclaved samples of the
treated soil did not express any degradation activity, which clearly
indicated the biological origin of the degradation capacity (Fig. 1B).
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FIG. 1.
Transformation of linuron in enrichment cultures
inoculated with linuron-treated and untreated soil samples obtained in
May 1998 (A) and August 1998 (B). Flasks were inoculated with 5 g
of untreated soil ( ), 5 g of linuron-treated soil ( ), 1 g of linuron-treated soil plus 4 g of untreated soil ( ),
0.1 g of linurom-treated soil plus 4.9 g of untreated soil
( ), and 5 g of autoclaved linuron-treated soil ( ). The error
bars indicate standard deviations based on triplicate measurements.
Where they are not visible, the error bars are smaller than the
symbol.
|
|
DGGE fingerprints of 10-day-old enrichment cultures inoculated with
linuron-treated soil and untreated soil are shown in Fig. 2 (lanes 1 and 2). Differences in band
patterns suggested that some species became more prevalent in the
linuron-degrading culture. After several transfers into minimal medium
containing linuron as the sole source of carbon and nitrogen, a stable
DGGE fingerprint was obtained. The DGGE patterns obtained after RT-PCR,
which targets more active bacteria with high rRNA contents, indicated
that after several transfers there was still substantial diversity in
the enriched bacterial consortium (Fig. 2, lanes 3 and
4). The enrichment culture was plated on
several different solid media, including nutrient agar, (Difco
Laboratories, Detroit, Mich.), R2A medium (Difco), minimal medium
containing linuron as the sole carbon and nitrogen source (MMNL), and
minimal medium without any carbon or nitrogen source (MMN0). The DGGE
fingerprints of the colony mixtures recovered from each solid medium by
resuspension in 2 ml of saline solution are shown in Fig. 2, lanes 5 through 8. There was a difference between the pattern obtained with the
liquid culture (Fig. 2, lanes 3 and 4) and the patterns obtained with the various solid media (Fig. 2, lanes 5 through 8). Indeed, the most
intense DGGE fragments detected in liquid medium (e.g., the fragment
corresponding to the Variovorax paradoxus-like strain [Fig.
2, lanes 3 and 4]) were not detected with the colonies which grew on
solid media (Fig. 2, lanes 5 through 8). On the other hand, species
that were absent in the liquid medium became very abundant after
plating and appeared to be Pseudomonas putida-like (Fig. 2,
lanes 5 through 8). Moreover, the diversity of the enrichment cultures
on solid media, as estimated by the number of DGGE bands, decreased
compared with the diversity of the liquid enrichment cultures.
Interestingly, the resuspended colony mixtures were unable to degrade
linuron, whereas the liquid culture degraded the herbicide completely
within 4 days. It is important to note that MMN0 was prepared with
several pure agar and agarose solidifying agents, including Noble agar
(Difco), Ultrapure agarose (Life Technologies, Paisely, Scotland), and
electrophoresis grade agarose (ICN Biomedicals Inc., Aurora, Ohio).
Despite these efforts there was always a background of bacterial
colonies on all MMN0 plates.
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FIG. 2.
Lanes 1 and 2, DGGE fingerprints of the first enrichment
cultures inoculated with linuron-treated and untreated soil,
respectively; lanes 3 and 4, duplicate DGGE fingerprints of the
bacterial culture that degraded linuron after 15 transfers; lanes 5 through 8, DGGE fingerprints of mixed bacterial colonies recovered from
different solid media (nutrient agar, R2A agar, MMNL, and MMN0,
respectively); lanes 9 through 17, DGGE fragments corresponding to
different strains isolated on R2A agar. Solid and dashed arrows
indicate fragments that correspond to fragments of
Variovorax species (97%) and P. putida (99%),
respectively.
|
|
Separate colonies, which were subjected to DGGE (Fig. 2, lanes 9 through 17), were also picked and tested to determine their capacity to
degrade linuron. None of the isolated colonies, either separately or in
artificial combinations, were capable of degrading linuron. This work
showed that a shift in microbial community structure during transfer
from successful liquid enrichment cultures to solid media could explain
the failure to easily isolate individual strains that degrade
herbicides. To my knowledge, this is the first report that shows a
clear plating bias during enrichment of herbicide-degrading bacteria.
Similarly, using an in situ hybridization technique, Wagner et al.
(21) showed that probing activated sludge with
oligonucleotides specific for the Proteobacteria reveals the
inadequacy of culture-dependent methods for describing microbial communities, since culture-dependent community structure analysis of
activated sludge produced partially to heavily biased results.
Many reports have indicated that degradation of herbicides requires
mixed bacterial cultures (1, 2, 7, 8, 16). This explains the
difficulties encountered by several groups in isolating pure cultures
of herbicide-degrading strains. An illustration comes from the study of
Roberts et al. (14), who tried to isolate pure strains
capable of degrading linuron from an enrichment culture able to do so.
These authors reported that none of the isolates on mineral medium with
linuron or any synthetic mixture of isolates from the mixed culture
degraded the compound. Thus, it was suggested that some carryover of
nutrients occurred during the plating procedures. Nonetheless, after
years of unsuccessful efforts, a pure strain that is capable of
transforming diuron to 3,4-DCA was isolated recently (3),
and several strains that degrade atrazine have also been isolated in
recent years (9, 13). An intriguing point gleaned from
studies of mixed and pure cultures is that most identified bacteria
with a key role in degrading herbicides belong to the genus
Pseudomonas and are species which generally grow easily on
solid media. The ubiquity of pseudomonads in degradation processes has
been attributed to a great diversity of exchangeable genetic elements
in this genus; these elements are both plasmid borne and genomic and
carry catabolic genes. Almost a decade ago, Sayler et al.
(16) stated that one of the reasons why the majority of
catabolic plasmid-bearing strains that had been studied so far were
Pseudomonas-like species could be the enrichment and isolation methods used, which favored the growth of pseudomonads.
In an attempt to isolate a pure strain, serial liquid dilution series
were prepared by using MMNL. Dilutions ranging from 100 to
10
10 were incubated at 28°C for more than 2 months with
25 mg of linuron per liter as the only source of carbon and nitrogen.
The degradation of linuron was monitored regularly, and the dilutions
that showed total transformation of linuron were subjected to DGGE
analysis. The results indicated that no degradation occurred at
dilutions beyond the 105 dilution. Thus, only
100 to 105 dilutions were subjected to DGGE
analysis after complete degradation of linuron. Figure
3 shows that stable, similar mixtures of
DGGE bands were obtained with all dilutions except the
105 dilution, in which one DGGE band seemed to disappear.
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FIG. 3.
Lanes 1 through 6, DGGE fingerprints for serial
dilutions ranging from 100 to 105,
respectively. The arrow indicates the fragment corresponding to
Variovorax-like species.
|
|
Effect of temperature on degradation of linuron.
In addition
to molecular monitoring of the enrichment culture, the effect of
temperature on transformation of linuron was investigated. During
enrichment, which was always carried out at 28°C, no intermediate
metabolites were detected in the enrichments that degraded linuron.
However, when the temperature of incubation was changed, HPLC analyses
showed the appearance of an intermediate metabolite that had the same
retention time as 3,4-DCA. Figure 4 shows
the effect of temperature on degradation of linuron and 3,4-DCA.
3,4-DCA appeared only at 4, 20, and 37°C. The rate of transformation
of linuron by the mixed bacterial culture was affected at 20°C and
decreased at 4°C. On the other hand, whereas no difference was noted
for transformation of linuron at 28 and 37°C, transformation of
3,4-DCA was affected at 37°C. The mass balance for the aromatic ring
showed 100% accumulation of 3,4-DCA when the temperature of incubation
was shifted from 28 to 37°C. To confirm the finding that 3,4-DCA
transformation was influenced by the temperature of incubation, the
enrichment culture was inoculated into minimal medium with 3,4-DCA as
sole carbon and nitrogen source. Total transformation of 25 mg of
3,4-DCA per liter was observed after 4 days of incubation at 28°C,
whereas no significant disappearance was seen when samples were
incubated at 37°C for 3 weeks. A similar effect was also observed
with 4-bromoaniline, the intermediate metabolite of metobromuron (data
not shown). These data suggest that different bacterial species are
involved in the transformation of linuron and 3,4-DCA. This hypothesis
was supported by the DGGE patterns obtained with liquid cultures
incubated at 28 and 37°C with linuron or 3,4-DCA as the sole carbon
and nitrogen source (Fig. 5). Certain
DGGE bands, including the one corresponding to the
Variovorax-like species (Fig. 5), disappeared at 37°C. The
difference in the DGGE patterns observed with linuron and 3,4-DCA at
different temperatures of incubation, together with the results of DGGE
monitoring of dilution series (Fig. 3),
in which similar mixtures of 16S ribosomal DNA genes were always detected after linuron degradation, strongly supports the hypothesis that several strains are involved in complete transformation of linuron. As no pure strain capable of complete mineralization of urea
herbicides has been isolated so far, my findings, together with those
of previous studies, support the concept that consortia are required
for complete transformation of urea herbicides.
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FIG. 4.
Effect of temperature on degradation of linuron and its
intermediate metabolite 3,4-DCA by the enrichment culture. The error
bars indicate standard deviations based on triplicate measurements.
Where they are not visible, the error bars are smaller than the
symbol.
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|
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FIG. 5.
Triplicate DGGE analyses of enrichment cultures
incubated at 28 and 37°C with 25 mg of linuron per liter or of
3,4-DCA (DCA) per liter as the sole carbon and nitrogen source. The
arrow indicates the fragment corresponding to
Variovorax-like species.
|
|
Degradation of urea herbicides by the enrichment culture.
The
ability of the enrichment culture to degrade several urea herbicides
was also investigated. Figure 6 shows the
time course degradation profiles for different urea herbicides.
Herbicides that contain a methoxy-methyl amino group were rapidly
degraded by the enrichment culture (Fig. 6A and C). In contrast,
herbicides that contain a dimethyl-amino urea group were degraded
slowly (Fig. 6B) or not at all (Fig. 6D and E).
The difference in degradation of different urea herbicides by the
enrichment culture could be explained by the fact that different enzymes are involved in biotransformation of urea herbicides
(5). Assuming that the first step consists of breaking the
acylamide bond, it seems that there is a wide spectrum of aryl acyl
amidases. I suggest that the enrichment culture used in this study
contains a specific enzyme capable of transforming
N'-methoxy-methyl urea herbicides, such as linuron and
metobromuron. This enzyme seems not to be involved in transformation of
1,1-dimethyl urea herbicides, such as diuron, isoproturon, and
chlorotoluron. The hypothesis that there are different enzymes involved
in transformation of aryl acyl compounds is supported by the results
obtained by Roberts et al. (14), who enriched a stable mixed
bacterial culture capable of degrading the herbicide linuron. This
culture was also able to degrade related herbicides, such as
monolinuron, but was unable to degrade the 1,1-dimethyl-substituted
urea compounds, such as monuron and diuron. However, it is
important to note that the ring substituents may affect degradation
capacity. The enrichment culture developed by Roberts et al.
(14) was capable of degrading linuron but not metobromuron.
The same result was obtained recently by Cullington and Walker
(3), who showed that a single strain was able to transform
diuron and linuron to 3,4-DCA but was not able to transform
metobromuron. These authors suggested that ring chlorination,
particularly at the 4 position, appears to greatly enhance rates of
degradation. However, my results do not confirm this suggestion, since
linuron and metobromuron were degraded at similar rates. Together, the
data suggest that several pathways are most likely involved in
biodegradation of urea herbicides.
| |
ACKNOWLEDGMENTS |
This work was supported by grant G.O.A. 1997-2002 from the
Ministerie van de Vlaamse Gemeenschap, Bestuur Wetenschappelijk Onderzoek (Belgium).
I thank Siska Maertens for her excellent technical assistance and
E. M. Top for her help in editing the manuscript.
| |
FOOTNOTES |
*
Present address: Civil and Environmental Engineering,
University of Washington, 201 More Hall, Box 352700, Seattle, WA
98195-2700. Phone: (206) 685-3464. Fax: (206) 685-9185. E-mail:
fantrous{at}hotmail.com.
| |
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Applied and Environmental Microbiology, December 2000, p. 5110-5115, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
