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Effect of immobilization of a bacterial consortium on diuron dissipation
and community dynamics
Stéphane Bazot
a
, Thierry Lebeau
b,*
a
Laboratoire Ecologie, Systématique et Evolution, UMR8079, UPS-CNRS-ENGREF, Département Ecophysiologie Végétale, Université Paris-sud XI, 91405 Orsay Cedex, France
b
Equipe Dépollution Biologique des Sols, Université de Haute Alsace, 29, rue de Herrlisheim, BP 50568, 68008 Colmar Cedex, France
article info
Article history:
Received 10 December 2008
Received in revised form 23 March 2009
Accepted 24 March 2009
Available online 21 April 2009
Keywords:
Biodegradation
Bioremediation
Co-culture
Herbicides
Immobilized cells
abstract
This work intended to study the relationship between diuron herbicide dissipation and the population
dynamics of co-cultivated Delftia acidovorans WDL34 (WDL34) and Arthrobacter sp. N4 (N4) for different
cell formulations: free cells or immobilization in Ca-alginate beads of one or both strains. GFP-tagged
WDL34 and N4 Gram staining allowed analyzing the cell growth and distribution of each strain in both
beads and culture medium in the course of the time. Compared to the free cell co-culture of WDL34 and
N4, immobilization of WDL34 in Ca-alginate beads co-cultivated with free N4 increased the dissipation
rate of diuron by 53% (0.141 mg ml
1
h
1
). In that case, immobilization strongly modified the final equi-
librium among both strains (highest total N4 to WDL34 ratio). Our results demonstrated that the inocu-
lant formulation played a major role in the cell growth of each cultivated strain possibly increasing
diuron dissipation. This optimized cell formulation may allow improving water and soil treatment.
Ó2009 Elsevier Ltd. All rights reserved.
1. Introduction
France is the first consumer of pesticides in Europe. Over
76,200 t of pesticides are applied to agricultural lands each year
in France. This widespread use of pesticides showed a multiple
environmental effects ranging from food safety-related effects to
the deterioration of farmland ecosystems and water resource (Tra-
visi and Nijkamp, 2008). The magnitude of the surface and ground-
water contamination by pesticides depends on both their
susceptibility to sorption by the organic and mineral soil compo-
nents along with bioattenuation, i.e., every natural biological phe-
nomena without any human intervention.
Among pesticides, herbicides such as diuron [3-(3,4-dichloro-
phenyl)-1,1-dimethylurea] were detected at a mean concentration
of 20
l
gL
1
in 34.6% of the samples from superficial water in the
French river basin in 2005 (IFEN, 2007). But concentrations higher
than 1 mg L
1
were recovered in water after diuron applications in
vineyard field (Louchart et al., 2000). Microbial degradation is con-
sidered as the primary mechanism responsible for pesticide miti-
gation in soil (Karpouzas and Singh, 2006; Mulbry and Kearney,
1991; Sørensen et al., 2003). With the aim at optimizing the degra-
dation of pesticides, the development of relevant bioremediation
tools is increasingly encouraged. Bioremediation, i.e., human as-
sisted-microbial treatments, is relevant due to its low cost along
with its low environmental impact compared to chemical treat-
ments. Such a technology has to be optimized for water treatment
in the aim at being used for mitigation of agricultural nonpoint-
source pesticide pollution in artificial wetland ecosystems (Grego-
ire et al., in press).
Some microorganisms were shown to be able to degrade diuron
(Ellis and Camper, 1982; Esposito et al., 1998; Sørensen et al., 2008;
Turnbull et al., 2001; Widehem et al., 2002). Unfortunately all these
studies showed accumulation of 3,4-dichloroaniline (3,4-DCA), the
main metabolite of diuron (Giacomazzi and Cochet, 2004) as a final
degradation product whose toxicity is slightly below 100 times
that of diuron (Tixier et al., 2002). Some other strains were previ-
ously reported to dissipate 3,4-DCA (Dejonghe et al., 2002, 2003;
Travkin et al., 2003; You and Bartha, 1982) without exhibiting
however the capability of degrading diuron to 3,4-DCA.
Recently, with the aim at cleaning water and sediment accumu-
lated in the above mentioned stormwater basin, we achieved the
total dissipation of diuron (Bazot et al., 2007) with a co-culture
of Arthrobacter sp. N4 (N4) and Delftia acidovorans WDL34
(WDL34) strains known to be able to respectively degrade diuron
into 3,4-DCA and to dissipate 3,4-DCA. In this preliminary work,
diuron dissipation was lower with bacteria co-cultivated as free
cells compared to co-cultures where one or both strains were
immobilized. However this work did not study the bacterial co-cul-
ture dynamics, thus preventing the cell growth of N4 and WDL34
from relating to diuron dissipation performances. We hypothe-
sized that immobilization modified the cell growth of each strain
almost as the result of diffusional limitations. In this study, we as-
sumed that the observed accumulation of 3,4-DCA in the culture
media with a co-culture of immobilized WDL34 and free N4 cells
0960-8524/$ - see front matter Ó2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.03.067
*Corresponding author. Tel.: +33 3 89 20 31 35; fax: +33 3 89 20 23 57.
E-mail address: thierry.lebeau@uha.fr (T. Lebeau).
Bioresource Technology 100 (2009) 4257–4261
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
could be the consequence of diffusion constraints in beads (O
2
,
nutrients) which have delayed the contact between 3,4-DCA and
WDL34 and reduced its growth rate.
Artificial immobilization in natural polymers such as Ca-algi-
nate was commonly used for its non microbial toxicity, its role in
the improvement of the microbial survival when microorganisms
were inoculated in environmental matrices such as soil, water,
and its protective effect against contaminant toxicity (mineral
and/or organic) (Cassidy et al., 1996). Immobilization matrices
were also used for inoculation by sustaining a continuous cell re-
lease in environments such as soil, water, sediment (McLoughlin,
1994; Boon et al., 2002; Mertens et al., 2006). Immobilized consor-
tia were already successfully used for the degradation of contami-
nants such as chlorinated phenol, pentachlorophenol and
dichloroacetic acid (Bettmann and Rehm, 1984; Cassidy et al.,
1997; Feodorov et al., 1993; Heinze and Rehm, 1993; Keweloh
et al., 1989; Lee et al., 1994). In these studies, the results showed
the degradation of contaminants without considering growth
dynamics within beads of the strains making consortia.
This work intended to study the relationship between diuron
herbicide dissipation and the population dynamics of WDL34 and
N4 making consortium for different cell formulations.
2. Methods
2.1. Microbial strains and preculture conditions
Arthrobacter sp. N4 (N4) and Delftia acidovorans WDL34
(WDL34) were used respectively for their ability to degrade diuron
into 3,4-dichloroaniline (3,4-DCA) (Tixier et al., 2002) and to dissi-
pate 3,4-DCA (Dejonghe et al., 2003). Bacterial strains were stored
at 4 °C on agar LB (tryptone, 10 g l
1
; yeast extract, 5 g l
1
; NaCl,
10 g l
1
; agar, 15 g l
1
). Each strain was pre-cultivated on LB med-
ium during 24 h at 28 °C and 200 rpm. For both strains, bacterial
cell concentrations were determined by measuring optical density
(OD) of the samples at 600 nm (Beckman Coulter DU 530) and
relating the value to a calibration curve previously obtained
(OD
600
vs. cell number).
2.2. GFP-WDL34 construction
Escherichia coli S17–1 kpir (Herrero et al., 1990) was transformed
with plasmid pUTGFP (Tombolini et al., 1997) as described by
Chung et al. (1989). This plasmid contains a mini-Tn5transposon
with the nptII (Kanamycin resistant (Km)) and GFP genes, and was
used to insert the latter two genes into the chromosome of Genta-
mycin (Gm)-resistant WDL34. This procedure was done by means
of biparental mating between E. coli S17–1 kpir(pUTGFP) and
WDL34 (Boon et al., 2000). Pre-culture of tagged E. coli S17–1
kpir(pUTGFP) strain supplemented with 25 mg l
1
of Km and pre-
culture of WDL34 strain supplemented with 25 mg l
1
of Gm were
obtained in 10 ml LB liquid medium after incubation overnight at
28 °C. For each culture, 5 ml were centrifuged (8200g, 10 min). Cells
were then washed with 1% NaCl and resuspended in 1 ml 1% NaCl.
Both cell cultures were mixed (1 ml) and were centrifuged at
3800gfor 15 min and resuspended in 100
l
l 1% NaCl before plating
on minimal acetate medium (MM9-acetate) supplemented with
25 mg l
1
of both Gm and Km. Only the transformant strain, i.e.,
the GFP-tagged WDL34, developed on the culture medium with
both Km and Gm and showed green fluorescence under UV light.
2.3. Cell immobilization
According to previous experiments (Jezequel et al., 2005), a
sterile solution of sodium alginate (30 g l
1
) was mixed with the
bacterial pre-culture to obtain a final concentration of 1.5 10
7
cells ml
1
for each strain. Ca-alginate beads of about 3 mm diam-
eter were obtained by dropping the alginate cell mixture into a
solution of CaCl
2
(30 g l
1
).
2.4. Dissipation of diuron and 3.4-DCA
Culture medium: experiments were carried out in Erlenmeyer
flasks containing 75 ml of sediment extract medium (Lebeau
et al., 2002) incubated at 28 °C and 200 rpm. Sediment was col-
lected in the Waldweg stormwater basin (Rouffach, Alsace, France).
Briefly the sediment was mixed with the same weight of water (tap
water) and autoclaved at 130 °C during 1 h. Filtrate was autoclaved
at 115 °C during 0.5 h. A stock solution of diuron (99.5%, Sigma–Al-
drich, Germany) sterilized by filtration (0.2
l
m, Millipore Millex
PTFE, Japan) was obtained by dissolving in dimethylsulfoxyde
(30 g l
1
).
Culture conditions: the concentration of diuron in the culture
medium was 20 mg l
1
. The sediment culture medium was inocu-
lated with different formulations of the bacterial co-culture, i.e.,
free cells (FC) of N4 and FC of WDL34 (FCN4 FCWDL34), immobi-
lized cells (IC) of N4 and IC of WDL34 (ICN4 ICWDL34), FC of N4
and IC of WDL34 (FCN4 ICWDL34), and IC of N4 and FC of
WDL34 (ICN4 FCWDL34).All formulations were tested with and
without diuron. 10
6
cells ml
1
of each strain were inoculated.
For IC, 5 g of Ca-alginate beads at 1.5 10
7
cells ml
1
of Ca-algi-
nate beads were introduced in each flask. Concentrations of IC
brought back to the volume of the culture medium were thus the
same as FC, i.e., 10
6
cells ml
1
.
Compared to the control (diuron-free medium), growth of N4
and WDL34 was not significantly affected by diuron (data not
shown). We checked also that alginate beads did not sorb diuron.
2.5. Analysis
Growth of total free and immobilized bacteria was determined
by measuring OD
600
as above mentioned. For IC, 10 beads per flask
were previously dissolved in 3 ml of sodium tricitrate (50 mM)
(Bréant et al., 2002). Growth of WDL34 was determined by using
a Neubauer counting chamber under an epifluorescent microscope
(Eclipse E600, Nikon). N4 cells number was calculated by subtract-
ing WDL34 cells from the total bacteria concentration. GFP-tagged
cells immobilized in Ca-alginate beads were visualized in situ from
samples consisting in thin bead layers sections (some
l
m) by using
a microtome.
WDL34 cells were visualized as above mentioned. For N4, a
Gram staining was used (Madigan et al., 2004). Indeed, WDL34 is
a Gram
bacterium while N4 is Gram
+
.
Concentrations of diuron and 3.4-DCA were measured after fil-
tration (0.20
l
m Millipore Millex PTFE, Japan). About 20
l
l of fil-
trate were injected in a reverse phase (RP) HPLC system (Waters
2487 detector/1525 dual pump, UK) equipped with a RP column
(Lichrosorb RP18, 250 4 mm, Merck) at room temperature. The
mobile phase was a mixture of acetonitrile and water (45/55,
vol/vol), at a flow rate of 1 ml min
1
. Diuron and 3.4-DCA were de-
tected at 254 nm.
All the experiments were performed in triplicate. Data were sta-
tistically analyzed by ANOVA (JMP Statistics 1989–97 v. 3.2.1, SAS
Institute Inc., Cary NC, USA). All tests were carried out at the 95%
confidence level.
3. Results
Fig. 1 showed both cell growth of N4 and WDL34 and diuron
dissipation according to inoculant formulations. Growth of N4
4258 S. Bazot, T. Lebeau / Bioresource Technology 100 (2009) 4257–4261
started directly after the culture medium was inoculated while a
48 h lag time was observed with WDL34. When co-cultivated as
free cells (FCN4 FCWDL34), maximum N4 concentration was
slightly higher (P< 0.001) than WDL34 (5.5 10
7
vs.
1.9 10
7
cells ml
1
,Fig. 1A) and diuron was totally dissipated in
216 h. When N4 and WDL34 were co-cultivated respectively as
free and immobilized cells (FCN4 ICWDL34), diuron was dissipated
in 144 h (Fig. 1B). N4 and WDL34 maximal cell concentrations
were respectively higher (8.4 10
7
cells ml
1
) and lower
(0.39 10
7
cells ml
1
)(P< 0.001) than those reported with the
free cells co-culture. ICN4 FCWDL34 and ICN4 ICWDL34 formula-
tions both allowed total diuron dissipation in 192 h (Fig. 1C, 1D).
In beads where N4 and WDL34 were co-immobilized (Fig. 1D),
the cell concentration of both strains slightly decreased in the
course of the time but N4 concentration related to the WDL34
one remained close to 1, i.e., the chosen one ratio for inoculation.
Conversely, free cells resulting from both the release of immobi-
lized cells from Ca-alginate beads and their subsequent growth
in the culture medium showed N4 concentration largely exceeding
that of WDL34.
Maximum concentrations of immobilized N4 (1.4 10
7
and
3.0 10
7
cells ml
1
) were slightly but significantly lower
(P< 0.05) than those recorded with free cells resulting from a free
cell culture (5.5 10
7
cells ml
1
) or from the cell release out of
beads with subsequent growth in the liquid culture medium
(3.7 10
7
and 7 10
7
cells ml
1
)(Fig. 1). Immobilized WDL34
biomass (P< 0.05) was also slightly lower compared to free cells,
but only by considering free-cultivated cells (1.9 10
7
vs.
1.0 10
7
and 1.1 10
7
cells ml
1
).
Microscopic observations of GFP-tagged WDL34 cells within
beads (ICN4 ICWDL34 formulation) just after bead inoculation
(Fig. 2A) and after 144 h of incubation (Fig. 2B) showed that
WDL34 grew mainly in the outer part of beads in the form of clus-
ters. Contrary to what was observed with WDL34, N4 visualization
after Gram staining (data not shown) revealed colonization of the
whole beads as mainly single cells or some small clusters.
4. Discussion
Diuron dissipation as the cells grow revealed a primary metab-
olism. Contrary to what was hypothesized with N4 and WDL34 co-
cultivated as free cells (Bazot et al., 2007), i.e., a larger develop-
ment of WDL34, growth analysis here revealed the contrary
(Fig. 1A and Table 1) as significant growth of WDL34 was observed
after only 48 h of incubation. Diuron was completely dissipated in
216 h without any accumulation of 3,4-DCA in the culture medium
meaning a same or higher 3,4-DCA dissipation rate than diuron.
Compared to free cells co-cultures, inoculants formulation with
one or both strains being immobilized, showed quite different
growth kinetics of N4 and WDL34 (Fig. 1B–D) – although total cell
concentrations of both strains were the same at the beginning of
incubation – revealing the ability of immobilization techniques
to act upon the population dynamic and hence to the final equilib-
rium among co-cultivated strains. This technique was successfully
used for several applications such as nitrate removal from ammo-
nium (Santos et al., 1996) and simultaneous fermentation of a xy-
lose and glucose mixture to ethanol (Lebeau et al., 1997). In this
study immobilization of one or both strains increased diuron dissi-
Fig. 1. Diuron dissipation (d) (20 mg l
1
) by co-cultures of Arthrobacter sp. N4 () and Delftia acidovorans WDL34 (N). A: FCN4 FCWDL34 formulation, B: FCN4 ICWDL34
formulation, C: ICN4 FCWDL34 formulation, D: ICN4 ICWDL34 (dashed line, immobilized cells; continuous line, free cells including those resulting from both the release of
immobilized cells from Ca-alginate beads and their subsequent growth in the culture medium). Vertical bars indicate confidence interval (n= 3).
S. Bazot, T. Lebeau / Bioresource Technology 100 (2009) 4257–4261 4259
pation rate (144 h for ICN4 FCWDL34 formulation and 192 h for
ICN4 FCWDL34 and ICN4 ICWDL34 formulations).
Total N4 biomass to total WDL34 one ratio was 1 at the begin-
ning of incubation and reached up to 26 (FCN4 ICWDL34, Table 1).
This ratio was correlated (R
2
= 0.95) with diuron dissipation rate
(Table 1) which increased in 53% at the maximum (FCN4
FCWDL34: 0.092 mg ml
1
h
1
vs. FCN4 ICWDL34: 0.141 mg
ml
1
h
1
). Free cells, including cells released from beads in the cul-
ture medium, mainly explain this correlation (see line ‘FCN4/
FCWDL34’, Table 1) with a quite same R
2
value. Indeed, considering
the low volume of beads (5 g) compared to that of the culture med-
ium (75 ml), most of the cells were free at the end of the incubation
even though all cells were first co-immobilized (ICN4 WDL34)
(Table 1). At the end of incubation, immobilized N4 and WDL34
cells were indeed respectively about 2% and 20% of whole cells,
i.e., free and immobilized cells, which is consistent with data of
Boon et al. (2002). Conversely, neither total N4 nor WDL34 were
correlated to diuron dissipation rate demonstrating that diuron
dissipation was controlled more by the equilibrium among the
two bacterial strains rather than by their biomass. Compared to
free co-cultivated cells, total N4 to total WDL34 ratio of other inoc-
ulant formulations were mainly the consequence of WDL34 whose
concentration varied up to 0.8 log unit (0.17 10
7
and
1.16 10
7
cells ml
1
for FCN4 ICWDL34 and ICN4 FCWDL34,
respectively) whereas differences in N4 concentration did not ex-
ceed 0.4 log unit. We concluded that WDL34 was more sensitive
than N4 to shifts in environmental conditions due to immobiliza-
tion, e.g., mass transfer limitations. The average biomass ratio of
free co-cultivated cells was higher than that of co-immobilized
cells – without considering released cells – (5.6 vs. 0.75) indicating
that immobilization modified the population dynamic of each
strain. Now by taking into account both immobilized and released
cells, N4 to WDL34 ratio also increased (from 5.6 with FCN4
FCWDL34 up to 26 with FCN4 ICWDL34) indicating that immobili-
zation modified both the equilibrium among strains into the beads
and indirectly that of free cells.
The specific diuron dissipation rate gives information of the
physiological status of N4 (Table 1). The values varied depending
mainly of the total WDL34 biomass, not N4. We concluded that
WDL34 was able to act upon the physiological state of N4 altering
specific diuron dissipation rate.
GFP-tagged WDL34 formed cell clusters mainly in the outer part
of beads. Such cluster revealed active cell growth. Optimal condi-
tions for the growth of this strain most probably require minimal
oxygen and/or nutrient level which only diffused in the periphery
of the beads. Due to the peripheral development of WDL34 while
N4 colonized the whole beads, it could have been expected a larger
WDL34 release out of the beads, compared to N4, and then a low
FCN4 to FCWDL34 ratio. Indeed active cell growth almost observed
in the periphery of the beads revealed by cluster formation is usu-
ally associated with bead degradation and massive cell release, as
already observed by several authors (Bréant et al., 2002; Jobin
et al., 2005; Lebeau et al., 1998). Surprisingly, opposite result was
observed here, i.e., a higher N4 cell release. However, when free
cells were counted in the culture medium, it was not possible to
make a distinction between the cells resulting from the beads
and those resulting from the cell growth of the released cells. We
can thus hypothesize that the physiological state of the released
cells was different to that of free-cultivated cells, thus modifying
the competitiveness of each strain to the benefit of Arthrobacter
sp. N4.
Fig. 2. Ca-alginate beads distribution of GFP-tagged Delftia acidovorans WDL34. A: t
0. B: 144 h of incubation. 1 cm represents 0.25 mm.
Table 1
Biomass of immobilized (IC) and free cells (FC), diuron dissipation rate and specific diuron dissipation rate, for each formulation of the Arthrobacter sp. N4 (N4) and Delftia
acidovorans WDL34 (WDL34) co-culture.
FCN4 FCWDL34 FCN4 ICWDL34 ICN4 FCWDL34 ICN4 ICWDL34
N4 WDL34 N4 WDL34 N4 WDL34 N4 WDL34
Biomass
a
IC 0 0 0 2.7 (7.4) 8.8 (7.9) 0 4.2 (7.6) 5.6 (7.7)
FC 368 (9.6) 66 (8.8) 356 (9.6) 11 (8)
b
394 (9.6)
b
87.4 (8.9) 153 (9.2)
b
18.6 (8.3)
b
Total N4 368 (9.6) – 356 (9.6) – 403 (9.6) – 157 (9.2) –
Total WDL34 – 66 (8.8) – 13.7 (8.1) – 87.4 (8.9) – 24 (8.4)
IC N4/IC WDL34 – – – 0.75
FCN4/FCWDL34 5.6 32.3 4.5 8.2
Total N4/Total WDL34 5.6 26 4.6 6.5
Diuron dissipation rate (mg ml
1
h
1
) 0.092 0.141 0.102 0.102
Specific diuron dissipation rate (mg 10
9
N4 cells
1
h
1
). 1.9 3.0 1.9 4.9
a
Biomass is expressed as cell numbers (10
7
) (in brackets, log cell number).
b
Free cells resulting from both the release of immobilized cells from Ca-alginate beads and their subsequent growth in the culture medium.
4260 S. Bazot, T. Lebeau / Bioresource Technology 100 (2009) 4257–4261
5. Conclusions
This study demonstrated that immobilization had a strong
influence on diuron dissipation by modifying the global equilib-
rium among N4 and WDL34 in the course of the incubation. Our re-
sults showed that the best diuron dissipation rate required the
largest N4 to WDL34 biomass obtained with free N4 cells co-culti-
vated with immobilized WDL34 cells. In practice however, it could
be more relevant to co-immobilize the two strains although diuron
dissipation rate is lower. Indeed, some carriers can retain biomass
avoiding liquid medium to be contaminated. This is a real advan-
tage in water treatment, thus avoiding subsequent expensive puri-
fication steps. Also, when growth rate is lower than the reactor
dilution rate in continuous process, immobilisation avoids reactor
cell leakage. Last advantage is about bacterial cells protection
against possible other toxic compounds such as metals often ob-
served in mixed pollutions.
Acknowledgements
This study was financially supported by the European Commis-
sion under the ARTWET Project (LIFE ENVIRONMENT, LIFE 06 ENV/
F/000133) and by the Alsace Région (no. 930/05). We are very
grateful to M. Sancelme, W. Dejonghe and N. Boon for providing
us strains of Arthrobacter sp. N4, Delftia acidovorans WDL34 and
Escherichia coli S17–1 kpir(pUTGFP), respectively.
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