Enhanced mineralization of diuron using a cyclodextrin-based bioremediation technology

Article abstract of DOI:10.1021/jf3021909

The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of herbicide formulations and, consequently, it is frequently detected as a major soil and water contaminant in areas where there is extensive use. Diuron has the unfortunate combination of being strongly adsorbed by soil organic matter particles and, hence, slowly degraded in the environment due to its reduced bioavailability. N-Phenylurea herbicides seem to be biodegraded in soil, but it must be kept in mind that this biotic or abiotic degradation could lead to accumulation of very toxic derived compounds, such as 3,4-dichloroaniline. Research was conducted to find procedures that might result in an increase in the bioavailability of diuron in contaminated soils, through solubility enhancement. For this purpose a double system composed of hydroxypropyl-β-cyclodextrin (HPBCD), which is capable of forming inclusion complexes in solution, and a two-member bacterial consortium formed by the diuron-degrading Arthrobacter sulfonivorans (Arthrobacter sp. N2) and the linuron-degrading Variovorax soli (Variovorax sp. SRS16) was used. This consortium can achieve a complete biodegradation of diuron to CO₂ with regard to that observed in the absence of the CD solution, where only a 45% biodegradation was observed. The cyclodextrin-based bioremediation technology here described shows for the first time an almost complete mineralization of diuron in a soil system, in contrast to previous incomplete mineralization based on single or consortium bacterial degradation.

Full text of DOI:10.1021/jf3021909

Article
pubs.acs.org/JAFC
Enhanced Mineralization of Diuron Using a Cyclodextrin-Based
Bioremediation Technology
Jaime Villaverde,* Rosa Posada-Baquero, Marina Rubio-Bellido, Leonila Laiz, Cesareo Saiz-Jimenez,
́
Marıa A. Sanchez-Trujillo, and Esmeralda Morillo
Instituto de Recursos Naturales y Agrobiología (IRNAS-CSIC), Apartado 1052, 41080 Sevilla, Spain
ABSTRACT: The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of
herbicide formulations and, consequently, it is frequently detected as a major soil and water contaminant in areas where there is
extensive use. Diuron has the unfortunate combination of being strongly adsorbed by soil organic matter particles and, hence,
slowly degraded in the environment due to its reduced bioavailability. N-Phenylurea herbicides seem to be biodegraded in soil,
but it must be kept in mind that this biotic or abiotic degradation could lead to accumulation of very toxic derived compounds,
such as 3,4-dichloroaniline. Research was conducted to find procedures that might result in an increase in the bioavailability of
diuron in contaminated soils, through solubility enhancement. For this purpose a double system composed of hydroxypropyl-β-
cyclodextrin (HPBCD), which is capable of forming inclusion complexes in solution, and a two-member bacterial consortium
formed by the diuron-degrading Arthrobacter sulfonivorans (Arthrobacter sp. N2) and the linuron-degrading Variovorax soli
(Variovorax sp. SRS16) was used. This consortium can achieve a complete biodegradation of diuron to CO2 with regard to that
observed in the absence of the CD solution, where only a 45% biodegradation was observed. The cyclodextrin-based
bioremediation technology here described shows for the first time an almost complete mineralization of diuron in a soil system,
in contrast to previous incomplete mineralization based on single or consortium bacterial degradation.
KEYWORDS: cyclodextrin, diuron, 3,4-dichloroaniline, soil contamination, biodegradation, bacterial consortium
variety of guest molecules, which are placed in their
hydrophobic interior cavity.13 A large number of papers
describing the complexation of CDs with pesticides can be
found in the literature. Most pesticide−CD complexes were
aimed to improve their solubility in water.14−18 However, no
research has been reported with the aim of finding correlations
between this increase in solubility, desorption percentage from
soil, and bioavailability by means of mineralizing assays,
confirming the complete dissipation of the pesticides. However,
an increase of bioavailability only will not be enough to reach a
significant soil diuron dissipation, because, although several
diuron-degrading bacteria have been isolated from different
agricultural soils3,8,19,20 and river waters,20 none of them has
been identified as capable of reaching a complete diuron
mineralization in the presence of soil. Sorensen et al.5 used a
two-member diuron-mineralizing consortium, which gave
better results by combining the cooperative degradation
capacities of two bacteria. In this work, we attempt to enhance
mineralization of diuron using a cyclodextrin-based bioreme-
diation technology involving a bacterial consortium, which
resulted in an effective diuron mineralization system thanks to
the increased bioavailability.
INTRODUCTION
■
Diuron is a biologically active pollutant present in soil, water,
and sediments. This substituted urea herbicide inhibits
photosynthesis by preventing oxygen production1 and blocks
the electron transfer at the level of photosystem II of
photosynthetic microorganisms and plants. Diuron has the
unfortunate combination of being strongly adsorbed on soil
organic matter particles and, hence, slowly degraded in the
environment due to its reduced bioavailability.
Diuron is considered a Priority Hazardous Substance by the
European Commission2 because its degradation in soil leads to
3,4-dichloroaniline (3,4-DCA), a very toxic compound, which
has a high tendency to accumulate in the environment with
carcinogenic and mutagenic properties.3−6 Consequently,
diuron has been included in the European Commission’s list
of priority substances for European freshwater resources
(Directive 2000/60/EC) and in the U.S. Contaminant
Candidate List 3.7
Biodegradation has been described as the primary mecha-
nism for diuron dissipation in soils and waters,8 although
dispersion of this compound in agriculture leads to pollution of
the aquatic environment by soil leaching9−11 and runoff.12
In this work, some studies were conducted to find
procedures that might result in an increase in the bioavailability
of diuron in a contaminated soil, through solubility enhance-
ment using biodegradable molecules. These molecules are
cyclodextrins (CDs), which are cyclic oligosaccharides,
containing 6 (α-CD), 7 (β-CD), or 8 (γ-CD) R-(1,4)-linked
glucose units, formed from the enzymatic degradation of starch
by bacteria. It is well-known that they are capable of forming
inclusion complexes both in solution and in solid state with a
The development of an in situ and environmentally friendly
soil decontamination technique, which could give rise to a
complete diuron mineralization by means of increasing the
bioavailability of the pollutant and employing specific chemical
Received: May 21, 2012
Revised: September 13, 2012
Accepted: September 17, 2012
Published: September 17, 2012
© 2012 American Chemical Society
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Figure 1. GC-MS ion chromatograms of diuron and 3,4-dichloroaniline.
showed a 99.567 pairwise similarity with Arthrobacter sulfonivorans
(AF235091), whereas Variovorax sp. SRS16 yielded a 98.780 pairwise
similarity with Variovorax soli (DQ432053). In this study we will refer
to these strains as A. sulfonivorans and V. soli.
bacterium degraders, would involve an improvement from both
economical and environmental points of view.
MATERIALS AND METHODS
■
Methods. Diuron Desorption Studies from Soil. Prior to
desorption studies, triplicate batch adsorption experiments were
performed by mixing 5 g of the soil with 10 mL of 0.01 M Ca(NO3)2
solution, containing various concentrations (5, 10, and 15 mg L−1) of
diuron, in 50 mL polypropylene centrifuge tubes. The samples were
shaken for 24 h at 20 1 °C. This time of reaction was chosen from
preliminary kinetic studies (not shown), which showed that adsorption
had reached pseudoequilibrium. After shaking (on an orbital shaker),
the suspensions were centrifuged, and the concentration of diuron in
the supernatant was determined by using a Shimadzu HPLC equipped
with a UV detector. The difference in herbicide concentration between
the initial and final equilibrium solutions was assumed to be due to
sorption, and the amount of diuron retained by the adsorbent was
calculated.
Desorption experiments were performed after adsorption equili-
brium had been reached by removing half of the supernatant after
centrifugation, replacing it by 5 mL of the extractant solution, allowing
equilibration for an additional 24 h period, and, after that, operating as
in the adsorption experiment. This process was repeated twice.
Desorption experiments were carried out using 0.01 M Ca(NO3)2
solution (named Ca(NO3)2 solution) and the same solution plus
HPBCD with a final concentration of 50 mM (named HPBCD
solution). The percentage of diuron desorbed with respect to that
previously adsorbed during the adsorption process (%D) was
calculated for all of the desorption experiments.
Materials. Technical grade (98%) diuron [N-(3,4-dichlorophenyl)-
N,N-dimethylurea] was provided by Presmar S.L. (Seville, Spain).
Radiolabeled [ring-U-14C]-diuron was purchased from Institute of
Isotopes, Budapest, Hungary (specific activity = 36 mCi mmol−1,
chemical purity = 99.9%, and radiochemical purity = 100%). The
cyclodextrins employed were β-CD (BCD), hydroxypropyl-β-CD
(HPBCD), γ-CD (GCD), and hydroxypropyl-γ-CD (HPGCD) (all
from Cyclolab, Budapest, Hungary, and with a chemical purity of
97%).
The CD hydroxypropyl-β-CD (HPBCD) from Cyclolab was
selected because in previous tests (data not shown) it was
demonstrated that this CD is not used as a carbon source by the
bacteria tested, eliminating its use as a growth substrate by the strains
studied. Likely, this CD showed the best complexation parameters
obtained from the solubility studies.
A southwestern Spain loamy sandy soil with a pH of 8.7, 6.9% of
CaCO3, 1% of organic matter, and a particle size distribution of 82.3%
sand, 4.1% silt, and 13.5% clay was selected for this study. The sample
was taken from the superficial horizon (0−20 cm), air-dried for 24 h,
and sieved through 2 mm, to remove stones, plant materials, etc. The
soil was analyzed for particle size distribution, measured by a
Bouyoucos densimeter, organic matter, measured by K2Cr2O7
oxidation, pH, determined in the 1:2.5 soil/water extract, and total
carbonate content, measured by the manometric method.21
The diuron-degrading organism Arthrobacter sp. N23 was purchased
from the Institut Pasteur Collection. Variovorax sp. SRS16 was kindly
provided by S. R. Sorensen.5 The identification of phylogenetic
neighbors was carried out by applying BLAST22 to the GenBank
sequence database and the EzTaxon database.23 Arthrobacter sp. N2
Inoculum Preparation. After both bacteria had been received, A.
sulfonivorans and V. soli were stored in criovials Microbank, which are 2
mL microtubes containing a specific culture medium and 20 porous
spheres of 3 mm diameter, and kept at −80 °C. Before each
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experiment, the criovials were thawed, and then A. sulfonivorans was
grown in Luria−Bertani (LB) medium and V. soli was grown in an
R2A medium.24 The bacteria were harvested at the beginning of the
stationary phase and, afterward, washed twice in a sterile mineral salts
(MS) solution24 before initiation of the experiments. The initial cell
densities of the bacteria in the degradation experiments were 107 CFU
mL−1. For diuron degradation, the bacteria were grown in a MS
medium supplemented with 40 mg L−1 diuron, as described by
Sorensen et al.24
Solubility in the Aqueous Phase in the Presence of Different CDs.
Phase solubility studies were performed according to the method
reported by Villaverde et al.16 An excess of diuron (5 mg) was added
to aqueous solutions (20 mL) that contained various concentrations of
CDs (0−0.012 M for BCD; 0−0.05 M for GCD; and 0−0.1 M for
HPBCD and HPGCD). The flasks were shaken at 25 °C for 1 week.
The suspensions were subsequently filtered through a 0.22 μm
Millipore glass-fiber membrane, and the concentration of diuron was
determined using HPLC. The apparent stability constants of the
different diuron−CD complexes (Kc) were determined from the
straight line obtained in the phase solubility diagram according to the
equation proposed in Villaverde et al.16
detector was kept at 200 °C. To avoid saturation and to preserve it, the
analyzer was switched off for 4 min during solvent elution and after the
last eluting analyte determination. The limit of quantification for the
metabolite was 1 μg/g of soil. The organic solvent selected to carry out
the extractions from aqueous supernatant was hexane (1:1 ratio MMK
medium/organic solvent, mL) and for extractions from soil pellet
dichloromethane was used (1:1 ratio soil/organic solvent). Recoveries
were 91−102%.
Both mineralization and biodegradation experiments were incu-
bated for 120 days.
Effect of Cyclodextrin Application on Soil Mineralization and
Biodegradation. A HPBCD solution, with a concentration corre-
sponding to 10 times the millimoles of diuron previously added in soil
degradation experiments flasks (50 mg kg−1), was employed to
enhance the herbicide bioavailability and increase its biodegradation
rate. This solution was incorporated into either biodegradation and
mineralization experiments to determine the increase in bioavailability
of the herbicide.
Model of Mineralization Kinetics. Mineralization data [expressed
as the percentage (P) of the initial activity converted to 14CO2 as a
function of time (t)] were fitted to a first-order equation of the
following form:28
Kc = slope/S0(1 − slope)
(1)
P = Pmax(1 − e−kt
)
(2)
where S0 is the diuron equilibrium concentration in aqueous solution
in the absence of the CD and slope refers to the slope of the phase
solubility diagram. Another parameter that can be obtained from the
data of the solubility diagram is the solubilization efficiency (Se), which
is defined as the increment of diuron apparent solubility at the highest
CD concentration with respect to its solubility.
Mineralization and Biodegradation Experiments. Mineralization
of 14C-labeled diuron in the soil was measured (in triplicate) through
the evolution of 14CO2 produced.25 Soil was sterilized using an
autoclave Auster-G, P-Selecta with three cycles at 121 °C, inlet
pressure of 103 kPa, during 20 min. The mineralization assays were
carried out in respirometers: modified 250 mL Erlenmeyers into which
10 g of soil together with 50 mL of mineral salts medium (MMK) was
placed. Acetone stock solution containing 14C-labeled and unlabeled
diuron was added to the soil to obtain a final concentration of 50 mg
kg−1 and a radioactivity of approximately 900 Bq per flask. The flasks
were inoculated with the specific bacterium or the consortium,
prepared as described above, and were closed with Teflon-lined
Nonlinear regression analysis (Sigmaplot v. 8.0) was used to estimate
the parameters Pmax (overall extent of 14C mineralization) and k (first-
order mineralization rate).
The parameters derived from this model accurately describe the
mineralization kinetics of nonsorbed diuron in systems under
equilibrium (instantaneous sorption and/or desorption) and pseudoe-
quilibrium (desorption rates much slower than degradation rates)
conditions.
RESULTS AND DISCUSSION
■
Diuron Solubility in the Aqueous Phase in the
Presence of Different Cyclodextrins. The phase solubility
diagrams of diuron in the presence of the different CDs used in
this work are shown in Figure 2. The initial purpose of this
experiment was to test whether the interaction of diuron with
the different CDs produced the formation of inclusion
complexes in solution. The increase in diuron hydrosolubility
in the presence of CDs indicates that the herbicide forms
inclusion complexes with them. A solubility limit could not be
obtained in the range of CD concentrations used in any of the
stoppers and incubated at 20
1 °C. Noninoculated soil sterile
controls and noninoculated soil sterile with HPBCD addition controls
were also prepared, and no mineralization was detected. Production of
14CO2 was measured as radioactivity appearing in the alkali trap of the
biometer flasks, which contained 1 mL of 0.5 M NaOH. Periodically,
the solution was removed from the trap and replaced with fresh alkali.
The NaOH solution was mixed with 5 mL of liquid scintillation
cocktail (Ready safe from PerkinElmer, Inc., USA) and the mixture
kept in darkness for about 24 h for dissipation of chemiluminescence.
Radioactivity was measured as described by Posada-Baquero et al.26
Biodegradation experiments were performed in parallel in the same
way as mineralization ones, but in this case, only nonradiolabeled
diuron was used, and the main metabolite 3,4-dichloroaniline and the
parent compound were analyzed at different time points by CG-MS. A
gas chromatograph 6890N from Agilent Technologies coupled to an
Agilent Automass quadrupole mass spectrometer 5975A was used.
Injection was in splitless mode with the split valve closed for 48 s.
Helium was employed as gas carrier. A Hewlett-Packard DB 17 ms (30
cm × 0.25 i.d. × 0.17 μm film thickness) capillary column was used.
The temperature program for the chromatographic run was as follows:
injector temperature, 70 °C (hold for 0.5 min), followed by a 100 °C
min−1 ramp to 300 °C (hold for 15 min). For mass spectrometric
detection, a potential of 70 eV was initially imposed in total ion scan or
full scan (m/z 45−300). Then, acquisition was performed under time-
scheduled selected ion monitoring (SIM) using the following product
ion qualifers: 74, 88, 97, 124, 159, and 187, the precursor ion being
m/z 187 for diuron quantification detected as intermediate 1,4-
dichloro-2-isocyanatobenzene.27 In the case of the main metabolite
(3,4-DCA) the precursor ion was m/z 162 (Figure 1). The MS
Figure 2. Phase solubility diagrams of diuron in the presence of the
cyclodextrins studied.
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cases, which is in agreement with an AL classification.16 The
inclusion complexation parameters for all CDs tested are shown
in Table 1. The straight lines of the phase solubility diagrams
pesticide residues adsorbed can increase their removal and pass
to the soil solution, where they become bioavailable.
Diuron Mineralization and Biodegradation Experi-
ments in Soil Inoculated with A. sulfonivorans, V. soli,
and Their Bacterial Consortium. The principal product of
diuron biodegradation, 3,4-DCA, exhibits a high toxicity and is
also persistent in soil, water, and groundwater. Diuron
indirectly possesses a significant amount of toxicity and could
be a potential poisoning herbicide contaminant of groundwater.
Therefore, the ultimate objective of this work was to obtain a
complete diuron mineralization in a soil−water system. The
chosen scenario was a loamy sandy soil from an agricultural site.
A two-member diuron-mineralizing consortium, combining
the cooperative degradation capacities of the diuron-degrading
bacteria A. sulfonivorans and the linuron-mineralizing bacteria V.
soli, was used, in comparison to the individual bacteria.
a
Table 1. Diuron Apparent Stability Constants (Kc) and
b
Solubilization Efficiency (Se) Obtained from the Phase
Solubility Diagrams
CD
Se
Kc (M−1
)
R2
HPBCD
BCD
23.27 1.33
6.76 1.02
6.79 0.88
2.27 0.36
207.70 3.55
175.86 4.21
58.68 1.11
25.74 2.85
0.9886
0.9577
0.9336
0.9985
HPGCD
GCD
a
b
Calculated according to eq 1. Diuron solubility calculated for the
highest CD concentration studied.
exhibited a slope of <1, which is ascribed to the formation of a
1:1 complex stoichiometry in solution. The apparent formation
constants of the inclusion complexes formed (Kc) were
calculated according to eq 1. A comparative study of the Kc
values shows that the highest values were obtained when
HPBCD and BCD were used. The lowest solubilization
efficiency (Se) and lowest Kc values corresponded to GCD
and its derivative, HPGCD. This result reflects the effect of the
size of the CD cavity (BCD and GCD contain 7 and 8 α-(1,4)-
linked glucose units, respectively, which form the toroidal ring)
on the formation of the different inclusion complexes because
the larger size of the internal cavity diameter of GCD would
result in an easy path for the diuron molecule to escape the
GCD cavity. Although the Kc value for BCD was high, which
indicates a strong tendency to form a complex with diuron, the
Se value is low because of the low solubility of this CD (16
mM).
The effects on the mineralization of diuron in soil (50 mg
kg−1) after inoculation of A. sulfonivorans and V. soli,
individually or in a coculture, were determined (Figure 3).
The overall extent of 14C mineralization was estimated using
the first-order production eq 1,28 (Table 3). Inoculation with A.
sulfonivorans resulted in a mineralization of 6.86%. The
metabolite 3,4-DCA was detected in the parallel biodegradation
experiments only in the samples inoculated with this bacterium,
and the amount was equivalent to 8.51% of the initially added
diuron. Inoculation with V. soli alone resulted in a diuron
mineralization in the soil of only 5.22%. Inoculation with the
coculture resulted in rapid diuron mineralization, and an
important mineralization was observed (45.25% of the added
14C diuron was metabolized to 14CO2 during the experiment,
after 120 days) (Figure 3), and no metabolite was determined
after the experiment (Table 3). The highest mineralization rate
values corresponded to the soil slurries inoculated with the
bacterial consortium, k values of 5 and 6 times higher than
those of A. sulfonivorans and V. soli, used separately (Table 3).
The time necessary to reach mineralization values >5% (lag
phase) was only 12.53 days for systems inoculated with both
selected bacteria, in comparison to 120 and 97 days for A.
sulfonivorans and V. soli, respectively, when grown alone.
In Table 3, the residual diuron measured at the end of the
parallel biodegradation experiments is also shown, being
remarkable that in the A. sulfonivorans inoculated system a
60.52% of the diuron initially added was still present,
confirming that about 40% of diuron is biodegraded, but not
mineralized, as can be observed in the mineralization
experiments results (6.86%), remaining in the form of the
toxic metabolite 3,4-DCA (8.51%) or other intermediate
species. A similar result could be observed for the system
inoculated with V. soli, but in this case, the percentage of diuron
remaining at the end of the biodegradation experiment was
lower (38.78%), from which only 5.22% was mineralized.
However, the presence of the toxic metabolite was much lower
(0.61%). Finally, the percentage of diuron measured after soil
biodegradation experiment in the presence of the two-member
bacterial consortium was the lowest, only 21.73%, confirming
again the need to use the two bacteria to reduce drastically the
real risk in a diuron-contaminated soil. Moreover, there is an
extremely high increase in the percentage of diuron
mineralization, from about 5−6% for the individual bacteria
to 45% when the consortium was used. In addition, the toxic
metabolite was not detected. It is also important to highlight
that the lag phase is reduced by 120 or 97 days (only one
degrader) to 12 days in the presence of the consortium, which
Diuron Desorption Experiments Using 0.01 M HPBCD
as Extractant Solution. The desorption percentages (%D)
values obtained for the soil under study when the HPBCD
solution was employed as extractant in comparison to
Ca(NO3)2 solution are shown in Table 2. In this soil for all
Table 2. Percentage of Diuron Desorbed from the Soil
extractant solutions
diuron initial concn (mg L−1
)
0.01 M Ca (NO3)2
0.01 M HPBCD
5
10
15
51.58 0.66
84.01 1.02
87.20 2.55
94.64 2.01
89.79 3.33
100 1.09
diuron initial concentrations about 90% could be desorbed with
HPBCD solution. The results obtained indicate the high
extracting power of HPBCD toward the herbicide previously
adsorbed on the soils in comparison to the percentages
extracted with Ca(NO3)2 solution, due to the formation of
water-soluble inclusion complexes between diuron and
HPBCD. Similar results have been obtained in previous papers
using HPBCD and CDs as extractant solutions for the herbicide
2,4-D and norflurazon from soil.14,15,29−31 In general, low-
polarity pesticides have a high tendency to be adsorbed on soil
surfaces, leading to their inactivation and low bioavailability
and, sometimes, to soil contamination. If these pesticides are
able to form inclusion complexes with CDs and, as a
consequence, to increase their solubility, the application of
CD solutions to soils containing a high concentration of
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Diuron Mineralization and Biodegradation Experi-
ments in the Presence of a HPBCD Solution in Soil
Inoculated with A. sulfonivorans and V. soli and Their
Bacterial Consortium. HPBCD seems to be a good choice
for being applied as decontamination technique in the case of
the herbicide diuron. Similar results were previously reported
for naphthalene and phenanthrene by Badr et al.32 For this
reason diuron mineralization experiments employing soil
slurries in the presence of HPBCD solution were performed
with the aim of enhancing diuron bioavailability and its
subsequent dissipation, ensuring that the potentially bioavail-
able herbicide fraction passes to the soil solution in a faster way,
because, as demonstrated above, higher desorption percentages
of diuron from the soil studied were obtained with the HPBCD
solution employed as diuron extractant. The ability of soils to
release (desorb) pollutants determines their susceptibility to
suffer microbial degradation, thereby influencing the effective-
ness of the bioremediation process. The degradation of
adsorbed contaminants can presumably occur via microbially
mediated desorption of contaminants and the development of a
steep gradient between the solid phase and interfacial
contaminant.33
The use of CD solutions as enhancers in pollutant dissipation
in soil has been postulated as a promising in situ
decontamination tool due to its capacity for pesticide soil
desorption. Previously, our group has reported numerous
results using different types of CD solutions for enhancing soil
desorption of the different herbicides with CDs naturally
originated and their derivates and different aging periods,14,17,31
but no works correlating the CD solution desorption effect with
microbial biodegradation enhancing have been carried out yet.
In Figure 3, diuron mineralization curves obtained from
inoculated soil slurries in the presence of HPBCD in solution at
a concentration equivalent to 10 times the amount of diuron
initially spiked are shown. In the case of inoculation with A.
sulfonivorans (Figure 3a) mineralization was very low (8.55%,
Table 3) despite the fact that this bacterium is a diuron
degrader.3 Then, the addition of the HPBCD solution, which
provokes a slight increase in herbicide bioavailability, provokes
also a slight increase of the main metabolite after degradation,
as confirmed with the data obtained for the 3,4-DCA analysis,
10.26% (Table 3). When the HPBCD solution was applied on
the soil slurries inoculated with V. soli (Figure 3b), a clear
increase in herbicide bioavailability was translated into a higher
percentage of diuron mineralized, reaching an overall extent of
14C mineralization of 57.87%. Finally, in Figure 3c the diuron
mineralization curve, when the HPBCD solution was added,
shows a new significant increase in herbicide mineralization
regarding that observed in the absence of the CD solution,
reaching a maximum percentage of mineralization of 98.67%
and the highest value determined for mineralization rate of 3.08
days−1 (Table 3), demonstrating once again that the increase in
the amount of herbicide that passes to the soil solution is
unequivocally connected with the mineralization rate, especially
when diuron mineralization is managed by an effective bacterial
consortium where cometabolism is necessary. Likewise, in the
presence of the HPBCD solution, diuron biodegradation assays
showed the almost complete disappearance of diuron at the end
of the experiment when the bacterial consortium was applied,
only 6.85% of the initial amount remaining.
Figure 3. Mineralization of 14C-labeled diuron in soil (50 mg kg−1)
and inoculated with (a) Arthrobacter sulfonivorans, (b) Variovorax soli,
and (c) a bacterial consortium, in the presence ( ) and absence ( )
◆
■
of the HPBCD solution.
denotes that the cooperative diuron degradation of the
consortium increases the mineralization rate (0.28 days−1),
clearly turning out to be an effective in situ bioremediation tool.
From these results it can be concluded that this type of
chemical (organochloride persistent compounds) will require a
more complex and conscientious analysis about the best
strategy to reach an optimal bioremediation of a contaminated
soil, especially for those chemical having main metabolites that
are suspected of having unwanted effects on nontarget
microorganism, such as the scenario investigated in this work.
Comparison of the Cyclodextrin-Based Bioremedia-
tion with Other Diuron-Degrading Technologies. A.
sulfonivorans (=Arthrobacter sp. N2) was isolated and
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Table 3. First-Order Diuron Mineralization Kinetic Parameters for Arthrobacter sulfonivorans, Variovorax soli, and the Two-
a
Strain Consortium, in the Absence or Presence of Cyclodextrin
mineralization rate
overall extent of 14C
mineralization (%)
diuron (% at the end 3,4-DCA (% at the end
diuron degrader
A. sulfonivorans
lag phase (days)
(days−1), K × 102
of expt)
of expt)
120.22 0.99
97.41 1.01
12.53 0.88
5.32 0.21
4.81 0.52
28.1 1.1
6.86 0.84
5.22 0.88
45.25 2.27
60.52 1.15
38.78 0.99
21.73 0.91
8.51 0.55
0.61 0.05
0.00 0.03
V. soli
bacterial consortium
A. sulfonivorans + HPBCD
28.33 1.03
6.42 2.52
45.6 6.1
8.55 0.56
22.81 0.02
10.26 0.02
solution
V. soli + HPBCD solution
194.13 2.25
7.62 0.55
57.87 1.21
98.67 1.02
24.34 1.84
6.85 0.44
0.00 0.01
0.00 0.01
bacterial consortium + HPBCD
solution
308
9
a
Percentages of diuron and 3,4-DCA at the end of the parallel biodegradation experiments.
characterized by Widehem et al.3 from a soil by diuron
enrichment procedures. These authors observed that this
bacterium was capable of metabolizing diuron to the
degradation product 3,4-DCA, and this metabolite was
produced in stoichiometric amounts; however, no mineraliza-
tion was observed. On the other hand, Sorensen et al.24 isolated
from a Danish agricultural soil enriched with linuron a
Variovorax sp. strain SRS16 (=V. soli). This was a linuron-
mineralizing bacterium able to use the herbicide as a carbon,
nitrogen, and energy source. Approximately 60−70% of 14C-
linuron was metabolized to 14CO2 within 10 days with only low
concentrations of 3,4-DCA detected. Satsuma34 isolated
Variovorax sp. strain RA8 from Japanese river sediments able
to mineralize 70% of linuron and detected a trace amount of
3,4-DCA.
Funding
This work was supported by the Spanish Ministry of Science
Innovation (cofunded by Fondo Europeo de Desarrollo
Regional, FEDER), Research Projects 2009401184,
CTM2006-04626, and CTM2009-07335.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are indebted to Presmar S.L. for providing the technical
diuron and Dr. S. R. Sorensen for the Variovorax sp. strain
SRS16.
REFERENCES
■
Furthermore, Sorensen et al.5 constructed a linuron- and
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AUTHOR INFORMATION
Corresponding Author
*E-mail: jvillaverde@irnase.csic.es.
■
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