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Applied and Environmental Microbiology, August 1998, p. 2937-2942, Vol. 64, No. 8
Centre de Pédologie Biologique du CNRS,
UPR 6831 Associée à l'Université Henri
Poincaré (Nancy I) B.P. 5, 54501 Vandoeuvre-les-Nancy Cedex,
France
Received 12 January 1998/Accepted 3 June 1998
An enzyme-linked immunofiltration assay (ELIFA) has been developed
in order to estimate directly and specifically Thiobacillus ferrooxidans attachment on sulfide minerals. This method derives from the enzyme-linked immunosorbent assay but is performed on filtration membranes which allow the retention of mineral particles for
a subsequent immunoenzymatic reaction in microtiter plates. The
polyclonal antiserum used in this study was raised against T. ferrooxidans DSM 583 and recognized cell surface
antigens present on bacteria belonging to the genus
Thiobacillus. This antiserum and the ELIFA allowed the
direct quantification of attached bacteria with high sensitivity
(104 bacteria were detected per well of the microtiter
plate). The mean value of bacterial attachment has been estimated to be
about 105 bacteria mg The chemolithotrophic
acidophilic bacterium Thiobacillus ferrooxidans, which uses
sulfide mineral oxidation (S2 The observation and measurement of bacterial attachment to mineral
surfaces and the role of this process are of major interest to
understand and control the mechanisms of sulfide dissolution and
weathering. T. ferrooxidans attachment has been
estimated by microscopic counting of nonadhering bacteria after contact between bacterial and mineral suspensions (17, 24, 29). This
method has a low sensitivity and needs at least 5 × 105 to 106 bacteria ml The adhesion of T. ferrooxidans was also evaluated by
total protein determination with sulfide minerals collected after
inoculation (32) and by epifluorescence microscopy
(31). Accurate and reproducible results by these methods
require large amounts of samples or high cell densities and a large
number of assays (16, 32). To improve epifluorescence
measurements, complementary analyses such as image analysis were
recently developed. But image analysis requires regular surfaces
(33) and would not be suitable for the determination of cell
attachment onto mineral particles. To determine cell attachment, a
spectrofluorimetric assay was also used to measure the intensity of
fluorescently labeled adhering bacteria in the upper layer of
sedimented inoculated mineral particles (8). The sensitivity
of this measurement of adhering cells is also very low compared to that
of fluorescence measurement of nonadhering cells and requires at least
106 cells mg The most-probable-number (MPN) technique was also used to indirectly
count ferrous-iron-oxidizing adhering bacteria, after their desorption
from the mineral by vortexing (30). This method was also
tedious and in addition nonspecific. In fact, all these previous
methods do not distinguish specifically T. ferrooxidans from other similar bacteria.
Jerez and Arredondo (13, 14) and Amaro et al. (1)
have developed immunological methods for the enumeration of acidophilic bacteria. These methods are very sensitive and can also be serotype specific (11, 18). Consequently, they can allow the
enumeration of bacteria belonging to one species by the selection of an
antiserum which recognizes all the serotypes of that species. However,
those methods have only been developed for counting nonadhering
acidophiles. Muyzer et al. (21) have attempted the use of
polyclonal antibodies for a combined
immunofluorescence-DNA-fluorescence staining procedure to observe the
abundance of T. ferrooxidans on coal particles. But
unfortunately, detection of T. ferrooxidans was not
observed at the end of the experiments; detection was not possible
probably because this bacterium was outcompeted by other acidophilic
microorganisms.
So, in order to observe and estimate directly, more
specifically, and accurately T. ferrooxidans
attachment on sulfide minerals during leaching processes, experiments
have been performed to develop and evaluate an enzyme-linked
immunofiltration assay (ELIFA). The assessment of this method, derived
from the enzyme-linked immunosorbent assay (ELISA), has been performed
by comparison of the method with indirect and more classical bacterial
cell counting methods. To directly perform ELIFA on mineral particles, microtiter plates with wells having a filter membrane at the bottom have been used.
Bacterial strains and preparation of cultures.
The bacteria
used in this study are listed in Table 1.
T. ferrooxidans strains and isolates were grown in 72 mM ferrous sulfate or 2% (wt/vol) pyrite-M2 basal salts
medium (with compounds at the following concentrations [in grams per
liter]: (NH4)2SO4, 1.0;
KH2PO4, 0.4;
MgSO4 · 7H2O, 0.4), pH 1.8 (19).
Various other bacteria were also used to determine the specificity of
the antiserum raised against T. ferrooxidans DSM 583. Leptospirillum ferrooxidans CF12, Thiobacillus
thiooxidans DSM 504, Acidiphilium strains, and the
acidophilic heterotroph isolate T23 were grown in the basal salts
medium described by Postgate (25). L. ferrooxidans CF12 was grown at pH 1.8 on a 100 mM ferrous
sulfate-0.05% (vol/vol) trace element solution (15).
T. thiooxidans DSM 504 and Acidiphilium strains were grown at pH 2.5 on 1% (wt/vol) elemental sulfur-0.05% (vol/vol) trace element solution and on 10 mM glycerol-0.02% (wt/vol) yeast extract, respectively. Isolate T23 was grown at pH 2.0 on 10 mM
ferrous sulfate-0.02% (wt/vol) yeast extract.
Leptospirillum-like strain L8 was grown in the liquid medium
of Silverman and Lundgren (28) containing 144 mM ferrous
sulfate and 100 mM ferric sulfate, pH 1.6 (3). Culturing was
performed on a rotary shaker at 130 rpm at 28°C. Bacteria were
harvested by centrifugation and then washed three times with 0.01 M
H2SO4 and resuspended in Tris-buffered saline
(TBS; pH 7.6). The strains of nonacidophilic gram-negative bacteria
Pseudomonas corrugata ATCC 29736, Burkholderia
cepacia ATCC 25416, and Azospirillum brasilense SP7
were also used to determine the specificity of the antiserum. They were
cultivated in nutrient broth (Difco) at 28°C, harvested by
centrifugation, and then washed twice and resuspended in TBS.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Enzyme-Linked Immunofiltration Assay To Estimate
Attachment of Thiobacilli to Pyrite
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 of pyrite at a
particle size of 56 to 65 µm. The geometric coverage ratio of pyrite
by T. ferrooxidans ranged from 0.25 to 2.25%. This
suggests an attachment of T. ferrooxidans on the
pyrite surface to well-defined limited sites with specific
electrochemical or surface properties. ELIFA was shown to be compatible
with the measurement of variable levels of adhesion. Therefore, this
method may be used to establish adhesion isotherms of T. ferrooxidans on various sulfide minerals exhibiting
different physicochemical properties in order to understand the
mechanisms of bacterial interaction with mineral surfaces.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, S0,
Fe2+) for growth, is of environmental and industrial
interest due to its involvement in the biocycling of sulfur and iron,
in the bioleaching and biovalorization of minerals, and in the
acidification of waters and soils (5). Such bacterial
oxidation and dissolution processes for nonsoluble minerals involve
interfacial phenomena between bacteria and minerals, suggesting that
microbial contact and attachment play a major role in sulfide oxidation
(6, 22, 27).
1 to get a
good measurement. Furthermore, cell multiplication does not allow the
use of such a method to determine the dynamics of bacterial adhesion
over time.
1 of particles. In addition, this
method also may be prone to error due to the interference of cell
treatment during the labeling reaction with the attachment ability of
the treated bacteria.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Bacterial strains used in these experiments
Sulfide minerals.
The pyrites used in these experiments
originated from a hydrothermal Peruvian deposit (pyrite K4)
and a sedimentary deposit in Spain (pyrite ES). The mineral particles
were collected by wet sieving after dry grinding in a tungsten carbide
mill. The particle size distribution was determined by laser scattering particle size analysis (Malvern Master Sizer). The mean particle diameter was 65 µm for pyrite K4 and 56 µm for pyrite
ES. From these data, and considering the particles as spheres, particle surface areas were estimated to be 190 and 220 cm2
g1, respectively. Infrared spectrometry indicated more
pronounced mineral-oxidized species [FeSO4,
Fe2(SO4)3] at the surface of pyrite K4 than at the surface of pyrite ES. Chemical
analyses of the pyrites are presented in Table
2.
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Preparation of antiserum and specificity determination.
A
polyclonal antiserum was raised against the pure strain of
T. ferrooxidans DSM 583. The bacteria were grown
in the presence of pyrite, as the sole source of energy, and harvested
at exponential and early stationary phases. Bacteria were prepared as
described by Muyzer et al. (21), and a mixture of equal
amounts of cells from these culture stages (109 cells
ml1) was used for the immunization of a rabbit (A. Dorier, Institut de Biologie Appliquée, Villeurbanne, France).
Determination of attachment of T. ferrooxidans
to pyrite.
T. ferrooxidans DSM 583 was used for
attachment experiments. Cells adapted either to pyrite K4
(Tf/K4 cells) or to ferrous sulfate (Tf/Fe2+
cells) by three successive cultures were inoculated onto pyrite ES and
pyrite K4. A total of 2 × 109 bacteria
ml1 were resuspended in 200 ml of M2 basal
salts medium containing 4 g of pyrite and incubated for 24 h
at 30°C with stirring (750 rpm) in batch reactors described
previously (20).
ELIFA. ELIFA is a modified ELISA method using a microtiter plate with wells having a 0.2-µm-pore-size Durapore filter membrane at the bottom (Multiscreen MAGVN; Millipore). Inoculated pyrite particles were retained on the filter. Prior to deposition in the wells of the microtiter plate, pyrite samples were washed in a tube two times with 10 volumes of M2 basal salts medium to remove nonattached bacteria, i.e., bacteria present in the interstitial solution between pyrite particles. For comparison and estimation of standard curves, the reaction was also performed at the same time with bacteria remaining in the supernatant after harvesting the pyrite. The ELIFA was derived from the method described by Gouzou et al. (10).
Suspended bacteria (100 µl of suspension) or inoculated and washed pyrite particles were added to each well of the microtiter plate. The medium was removed by filtration using an accessory vacuum filter holder (Multiscreen filtration system; Millipore) which allows the simultaneous processing of 96 individual samples. The wells were washed three times with 200 µl of TBS. To saturate the remaining binding sites on the filter and on pyrite particles, the wells were incubated at 37°C for 1 h with 100 µl of 1% (wt/vol) bovine serum albumin (BSA; Sigma) in TBS. They were then rinsed three times with 200 µl of TBS-0.05% (vol/vol) Tween 20 (Prolabo). Then, 100 µl of diluted antiserum (1:10,000) in TBS containing 1% BSA was added to each well. After 1 h of incubation at 37°C, the wells were again rinsed three times with TBS-Tween 20 and then incubated with 100 µl of goat anti-rabbit globulin (Sigma; A 75-39) alkaline phosphatase conjugate, diluted (1:2,000) in TBS-Tween-BSA. The wells were rinsed two times with TBS-Tween and one time with 10% (vol/vol) diethanolamine (Prolabo) buffer (pH 9.8). Thereafter, the samples were incubated with 100 µl of p-nitrophenylphosphate (Boehringer GmbH, Mannheim, Germany; 1 mg/ml, in diethanolamine buffer) as a substrate. Absorbance at 405 nm was measured with a MR 700 RS Dynatech spectrophotometer. Controls in each experiment consisted of a 0.2-µm filtrate of the bacterial suspension and of noninoculated pyrite particles. To determine the number of bacteria fixed per milligram of pyrite, the weight of pyrite particles used in the ELIFA reaction was determined as follows. Pyrite particles present in each well were dissolved in 10 ml of nitric acid (69%; analytical reagent; Prolabo). Total solubilized iron, determined by inductive coupled plasma analysis (Jobin Yvon JY 38) using an internal yttrium standard (40 ppm), was used to calculate the amount of pyrite according to the chemical composition of the pyrite. The statistical analysis of the results obtained by the ELIFA has been performed by using the Student t test. The mean and its confidence limits have been calculated considering that the probability of the results to be excluded from these limits was 5%.Enumeration of nonadhering thiobacilli by the MPN technique. The MPN technique was performed with microtiter plates as described by Rouas (26). Each bacterial dilution was tested in 40 wells. Twenty-five microliters of culture dilution was added to each well, which contained 200 µl of M2 basal salts medium supplemented with 72 mM ferrous sulfate. After 14 days of incubation at 30°C, the wells presenting an orange color due to bacterial iron oxidation were counted, and the MPN and its confidence limits were determined by using a statistical program (12).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
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Specificity of the antiserum. The antiserum raised against T. ferrooxidans DSM 583 showed a strong positive reaction with cells of T. ferrooxidans DSM 583 and Tf 2 and T. ferrooxidans-like isolate OP14 but not with cells of T. ferrooxidans ATCC 33020 (Fig. 1). It also exhibited a strong reaction with cells of T. thiooxidans DSM 504. Only weak and nonsignificant reactions were obtained with L. ferrooxidans CF12 and Leptospirillum-like strain L8 and with the acidophilic heterotrophs including Acidiphilium isolates Ao and SJH. The iron-oxidizing heterotroph T23 and the gram-negative neutrophiles P. corrugata, B. cepacia, and A. brasilense SP7 also exhibited no significant reaction (Fig. 1). These results indicate that the antiserum raised against T. ferrooxidans DSM 583 can recognize some cell surface antigens present on bacteria belonging to the genus Thiobacillus.
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Sensitivity of ELIFA reaction for T. ferrooxidans detection. Bacterial suspensions of T. ferrooxidans DSM 583, previously grown in the presence of either ferrous sulfate (Tf/Fe2+ cells) or pyrite K4 (Tf/K4 cells), were incubated for 23 h in the presence of pyrite ES or K4. Standard curves for ELIFA have been determined from bacteria remaining in the supernatant after the harvesting of the pyrite. The optical density resulting from the ELIFA reaction for nonadhering bacteria was compared with the number of bacteria estimated by the MPN method (Fig. 2). The MPN method was chosen after controlling the viability of nonadhering bacteria at about 100%.
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) and pyrite
K4-adapted bacteria (Tf/K4; 
) incubated in
the presence of pyrite ES. (B) Ferrous sulfate-adapted bacteria after
incubation with either pyrite K4 (1 (Fig.
2). Therefore, this method is more sensitive than the
microscopic counts, for which a minimum of 5 × 105 to 106 bacteria ml1 were
necessary. The optical densities obtained at 405 nm were significantly
different for bacterial concentrations differing by 0.5 log units and
showed the accuracy of this reaction.
Better sensitivity of detection was obtained for pyrite-adapted
bacteria (Tf/K4) than for ferrous sulfate-adapted bacteria (Tf/Fe2+) (Fig. 2A). This difference could be explained by
the preparation of antibodies with T. ferrooxidans
grown on pyrite.
After 1 day of incubation of the bacteria with two different pyrites
(K4 and ES), similar ELIFA reactions were observed (Fig. 2B).
Indirect measurement of attached bacteria by enumeration of
nonadhering bacteria.
The concentrations of nonadhering bacteria
in the medium before and after contact with pyrite were significantly
different (data not shown). The numbers of attached bacteria, estimated from this difference by either MPN or direct counting, were
similar (Fig. 3). Furthermore, similar
levels of attachment were observed with Tf/Fe2+ and
Tf/K4 inocula in the presence of both pyrites ES and K4
(3 × 105 to 9 × 105 bacteria
mg1 of pyrite).
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1 or lower, because above this value the difference
between the concentrations of nonadhering bacteria before and after
contact was not significant. These results indicated the need for a
direct method to measure bacterial attachment.
By estimating the number of attached bacteria per milligram of pyrite,
it was possible to determine the coverage ratio of pyrite by bacteria.
It was assumed that the average bacterial cell surface was about 0.5 µm2. On the other hand, the specific surface area of
pyrite particles calculated after laser scattering particle size
analysis was 200 cm2 g1. Thus, the area
covered by attached bacteria represented about 0.75 to 2.25% of the
total geometric pyrite surface. As a consequence, the number of
bacteria attached to each mineral particle was estimated to be 170 to
500 cells, assuming that the mean particle diameter was 60 µm. This
low coverage ratio value suggests that an immunological method can
allow an easier direct estimation of the adhesion level of these
bacteria.
ELIFA reaction on T. ferrooxidans attached to pyrite particles. Controls consisted of autoclaved mineral particles. The optical density obtained with sterile mineral particles was lower than 0.2 units after a 30-min reaction. In the presence of inoculated and washed pyrite, a fast and positive reaction was noted (data not shown). This result demonstrated the presence of attached bacteria on inoculated mineral particles.
In measurements performed on samples of pyrite ES and K4 inoculated with T. ferrooxidans and having a weight less than 1.5 mg, a linear relationship between the amount of pyrite and the optical density was obtained (Fig. 4). These results confirmed that attached bacteria can be measured by ELIFA. As for the observed reaction with nonadhering bacteria, a better sensitivity was obtained for bacteria previously cultivated in the presence of pyrite (Tf/K4) than in the presence of ferrous sulfate (Tf/Fe2+).
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1 of pyrite) were observed for the different inocula
(Tf/Fe2+ and Tf/K4) on both pyrites, showing
that this ELIFA method gave dependable and homogeneous results. From
this measurement of attachment level, the coverage ratio of pyrite by
bacteria could be estimated to be about 0.25%, corresponding to a mean
number of 57 attached bacteria per mineral particle.
The measurement of the attachment of T. ferrooxidans on
pyrite was determined in another set of experiments. A comparison of
the different methods was made with two different amounts of inoculum
(Table 3). With an inoculum of about
107 bacteria ml1, the adhesion level
determined was similar to the previous one. For a larger inoculum
(6.9 × 107 bacteria ml1) high
attachment levels were observed by indirect methods (1.6 × 106 to 3.6 × 106 bacteria
mg1). This high number of attached bacteria was confirmed
by ELIFA (up to 106 bacteria mg1), showing
that this method allows the determination of variable levels of
adhesion.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The knowledge of reactions at the interface between sulfide minerals (e.g., pyrite) and bacteria such as T. ferrooxidans and of other phenomena such as chemotaxis and attachment is of major interest to understand and control the mechanisms involved in the bacterial dissolution and weathering of minerals and in bioleaching processes of metals.
Compared to previous quantitative indirect methods using counting of cells remaining in suspension (4, 19) and to qualitative methods using a combined immunofluorescence-DNA-fluorescence staining procedure (21), the immunoenzymatic ELIFA method performed on filtration membranes and presented in this paper allowed the direct quantification of T. ferrooxidans cells attached to pyrite particles. This method, performed with microtiter plates, is sensitive (104 bacteria were detected per well) and accurate. A 95% confidence interval lower than 0.3 log units in the optical density at 405 nm was observed regularly.
The mean values of attached bacteria estimated by ELIFA and by the
indirect determinations were 105 and 5 × 105
bacteria mg1, respectively. Compared with indirect
measurements, ELIFA was shown to slightly underestimate bacterial
adhesion. Such a result suggests that the accessibility of antiserum to
bacterial surfaces was limited by the contact with the mineral. It can
be considered that some antigenic determinants recognized by the
antiserum may also be cell surface components that play a role in
bacterial adhesion. Using immunoblotting of T. ferrooxidans lysates, Koppe and Harms (18) identified
LPS and membrane proteins as antigenic determinants.
Independently, bacterial adhesion studies performed by Arredondo
et al. (2) suggested that both components are involved in
the attachment of T. ferrooxidans to solid
surfaces.
The ELIFA reaction obtained with T. ferrooxidans grown on pyrite was more sensitive than that observed with ferrous-iron-grown cells. This result suggests that the mineral can induce a modification in the expression of bacterial surface components. The presence of a solid substrate such as pyrite enhances the excretion of exopolymeric substances (EPS), and bound ferric ions in these EPS are responsible for the attachment of T. ferrooxidans to mineral particles (9).
The antiserum produced against the strain of T. ferrooxidans DSM 583 has been shown to recognize cell surface antigens present on bacteria belonging to the genus Thiobacillus. As ELIFA enables direct measurement of attached bacteria, the preparation and selection of an antiserum which recognizes the serotypes of one bacterial species may allow the determination of the attachment dynamics of this bacterial species during a complete bioleaching process. ELIFA can also be very useful to examine how various microorganisms interact with each other and with the surfaces of mineral particles. Attached T. ferrooxidans cells were recently evaluated by measuring their ability to oxidize ferrous iron (7). But this method is based on the assumption that the specific rate of ferrous iron oxidation does not vary significantly during the bioleaching process. In fact, ferrous iron oxidation is both a biological and chemical process which varies significantly between exponential and stationary phases, and according to growth conditions (4, 20).
The measurements of coverage ratio for T. ferrooxidans
attached to pyrite ranged from 0.25 to 2.25% for inoculum
concentrations promoting optimal bioleaching (107
bacteria ml1). In other experiments not
reported in this paper (4), attachment measurements
indicated that for inocula ranging from 107 to
108 bacteria ml1, the surfaces of mineral
particles were not saturated by bacteria. Under saturation conditions
and for similar-size mineral particles, the coverage ratio
was estimated to range from 20 to 45% (23, 24, 29). From
these observations, it can be proposed that T. ferrooxidans could adhere only to specific and limited sites on
the pyrite surface. It can also be suggested that bacteria need a
well-defined area with proper electrochemical properties to attach to
and oxidize a sulfide mineral surface. Finally, it may also be possible
that only a reduced proportion of the bacterial population is able to
attach at the surface due to the presence of specific cell surface
components which allow efficient bacterial adhesion.
The ELIFA method presented here was shown to be compatible with the measurement of different levels of adhesion. It would be possible to use ELIFA to determine and study adhesion isotherms of T. ferrooxidans on various sulfide minerals exhibiting different physicochemical properties to better understand the mechanisms of bacterial interaction with mineral surfaces. In the present experiments performed with two pyrites exhibiting different surface properties, equivalent attachment levels were observed despite different bio-oxidation rates for both sulfides. Measurements of bacterial attachment and of its dynamics, complemented by the determination of bacterial activity and mineral solubilization, may contribute to a better understanding of the role of direct bacterial contact with mineral particles in biological oxidation processes.
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ACKNOWLEDGMENTS |
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We are extremely grateful to G. Belgy (Centre de Pédologie Biologique, Nancy, France). We thank also D. B. Johnson (School of Biological Sciences, Bangor, United Kingdom) for providing acidophilic strains and K. B. Hallberg (School of Biological Sciences) for reviewing this paper.
This research was supported by a grant from ADEME, BRGM, COGEMA, and ECODEV CNRS, France.
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FOOTNOTES |
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* Corresponding author. Present address: School of Biological Sciences, University of North Wales, Bangor, Gwynedd LL57 2UW, United Kingdom. Phone: 44-(0)1248 351151. Fax: 44-(0)1248 370731. E-mail: bsr005{at}bangor.ac.uk.
Present address: DSV-DEVM, Laboratoire d'Ecologie Microbienne de
la Rhizosphère, UMR 163, CNRS-CEA, CEA Cadarache, 13108 Saint
Paul Lez Durance, France.
Present address: TREDI, Technopôle de Nancy-Brabois, 54505 Vandoeuvre-les-Nancy Cedex, France.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Applied and Environmental Microbiology, August 1998, p. 2937-2942, Vol. 64, No. 8
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