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Appl Environ Microbiol, July 1998, p. 2566-2571, Vol. 64, No. 7
Environmental Technology,
Received 29 December 1997/Accepted 17 April 1998
A new aerobic bacterium was isolated from the sediment of a
freshwater pond close to a contaminated site at Amponville (France). It
was enriched in a fixed-bed reactor fed with 2,6-dichlorophenol (2,6-DCP) as the sole carbon and energy source at pH 7.5 and room temperature. The degradation of 2,6-DCP followed Monod kinetics at low
initial concentrations. At concentrations above 300 µM (50 mg
· liter Chlorinated phenols are widely used
by the chemical industry as intermediate products in synthesis and
previously were frequently applied as wood preservatives and fungicides
(4, 13). Due to their high toxicity, strong odor emission,
and persistence in soil these compounds pose serious ecological
problems as environmental pollutants (26, 29).
Aerobic degradation of polychlorinated phenols has been studied
extensively during the last few years (12). Several strains of bacteria that are able to completely mineralize
polychlorinated phenols have been described (5).
Degradation of 2,4-dichlorophenol (2,4-DCP) by pure cultures,
especially cultures of Ralstonia eutropha (formerly
Alcaligenes eutrophus [34]) and
Burkholderia cepacia, has been reported repeatedly (8,
11, 33), as has degradation of 2- and 4-monochlorophenols by
Ralstonia eutropha, Pseudomonas putida, and
Alcaligenes sp. strain A 7-2 (4, 16, 17). The
aerobic 2,4-DCP degradation pathway has been elucidated (6, 9,
25). Degradation of the 2,6 isomer has also been observed in
laboratory and field experiments, and transformation of this compound
by resting cells of Mycobacterium chlorophenolicum,
Azotobacter sp., and Pseudomonas cepacia has been
reported (3, 21, 32). Complete degradation of 2,6-DCP in
sediment samples has been observed (22). However, no pure
culture capable of growing on 2,6-DCP aerobically has been described
previously.
In this study we describe the isolation and characterization of a pure
culture that grows on 2,6-DCP as a sole source of carbon and energy.
The potential of this organism to purify contaminated groundwater or
wastewater from soil washing and to remove a series of chlorophenols
under nonsterile conditions was studied with fixed-bed reactors. We
tested removal rates for short hydraulic residence times (HRTs) for
water containing low chlorophenol concentrations, and we also
investigated the importance of pH homeostasis and oxygen control for
optimal degradation of high concentrations of 2,6-DCP for practical
applications.
Bacterial strain and culture conditions.
Ralstonia sp.
strain RK1 was isolated from a fixed-bed reactor with 2,6-DCP as the
sole carbon and energy source. A sample of the reactor effluent was
diluted 1:1,000 in a shake flask containing 100 ml of mineral medium
supplemented with 120 µM 2,6-DCP. When the 2,6-DCP had been consumed,
fresh medium was inoculated with 100 µl of this preculture. This
enrichment procedure was repeated four times. A sample from the fifth
shake flask was streaked onto nutrient agar plates. Four
morphologically distinct types of colonies appeared. One colony of each
colony type was picked and streaked onto a separate nutrient agar
plate, and a single colony from this plate was removed and incubated in
liquid mineral medium supplemented with 2,6-DCP.
(i) Mineral medium.
The chloride-free mineral medium used
for growth of the pure culture and the experiments with the fixed-bed
reactors was designed to correspond to groundwater conditions
(28). It consisted of 152 µM
(NH4)2HPO4, 368 µM
KH2PO4, 575 µM
K2HPO4, 0.5 ml of each of the trace element
solutions per liter, and 0.5 ml of a vitamin solution per liter. Trace
element stock solution 1 contained (per liter) 6.5 mg of
Na2SeO3 · 5H2O and 12.1 mg
of Na2WO4 · 2H2O in 0.1 N NaOH, and trace element stock solution 2 contained (per liter)
2,250 mg of FeSO4 · 7H2O, 90 mg of
H3BO3, 150 mg of MnSO4 · H2O, 210 mg of CoSO4 · 7H2O,
55 mg of NiSO4 · 6H2O, 150 mg of ZnSO4 · 7H2O, 10 mg of
CuSO4 · 5 H2O, and 80 mg of
(NH4)6Mo7O24 · 4H2O in 0.001 N H2SO4. The vitamin
solution contained (per liter) 2 mg of biotin, 20 mg of nicotinic acid,
10 mg of thiamine, 10 mg of 4-aminobenzoate, 5 mg of pantothenate, 20 mg of cyanocobalamin, and 50 mg of pyridoxamine; it was filter
sterilized and added after autoclaving.
(ii) Complex medium.
Complex medium contained 10% nutrient
broth and was prepared by using the specifications of the supplier
(Difco).
Microscopy.
Morphological examinations were carried out by
phase-contrast microscopy (model M-20 microscope; Wild, Heerbrugg,
Switzerland) and scanning electron microscopy (model S-4000 microscope;
Hitachi, Tokyo, Japan). For the latter, cells were harvested by
centrifugation at 3,000 × g, washed twice with mineral
medium, and fixed with 3% glutaraldehyde. Then the medium was
gradually changed to 100% acetone, cells were transferred to a
polycarbonate filter, and critical point drying was carried out with
CO2 and sputtering with argon.
Taxonomic determination.
Physiological and biochemical tests
were carried out with an API 20NE kit (BioMerieux, Marcy l'Etoile,
France). Cells grown on complex medium were used to sequence the 16S
ribosomal DNA. The total DNA was obtained by phenolic extraction
(27). The RNA was removed by incubating an aqueous solution
for 15 min at 37°C with 5 U of DNase-free RNase. A PCR with the
extracted DNA was performed with two eubacterial primers,
S-D-Bact-0008-a-S-20 (15) and S-D-Bact-1492-a-A-19
(20). The designations of the primers were standardized as
described by Alm et al. (1). Amplification was carried out
with a thermocycler (Techne, Duxford, United Kingdom) by using a
25-µl sample. Each reaction tube contained Pfu reaction buffer, 1.5 mM MgCl2, 100 µM deoxynucleotide
triphosphate, 1 U of Pfu DNA polymerase (Stratagene, La
Jolla, Calif.), 5 pmol of each primer, and 100 ng of template DNA. The
PCR steps included initial denaturation at 95°C for 3 min, 25 cycles
consisting of 95°C for 1 min, 50°C for 1 min, and 72°C for 2 min,
and a final extension step consisting of 72°C for 10 min. The PCR
products were separated on a 1.5% agarose gel. The bands expected for
the 16S ribosomal DNA were cut out, and the DNA was purified by
phenolic gel extraction as described previously (27).
Sequencing of the 16S rRNA gene was carried out with a model ABI 310 Prism Collector (Perkin-Elmer, Foster City, Calif.) according to the
supplier's instructions. The following primers were used for the
partially double-stranded sequencing analysis: S-D-Bact-0008-a-S-20
(15), S-D-Bact-0338-a-A-18 (2),
*-Univ-0519-a-S-18, *-Univ-0519-a-A-18, *-Univ-1392-a-A-15
(24), and S-D-Bact-1492-a-A-19 (20).
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Aerobic Mineralization of 2,6-Dichlorophenol by
Ralstonia sp. Strain RK1
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1), 2,6-DCP increasingly inhibited its own
degradation. The base sequence of the 16S ribosomal DNA allowed us to
assign the bacterium to the genus Ralstonia (formerly
Alcaligenes). The substrate spectrum of the bacterium
includes toluene, benzene, chlorobenzene, phenol, and all four
ortho- and para-substituted mono- and
dichlorophenol isomers. Substituents other than chlorine prevented
degradation. The capacity to degrade 2,6-DCP was examined in two
fixed-bed reactors. The microbial population grew on and completely
mineralized 2,6-DCP at 2,6-DCP concentrations up to 740 µM in
continuous reactor culture supplied with H2O2
as an oxygen source. Lack of peroxide completely stopped
further degradation of 2,6-DCP. Lowering the acid-neutralizing capacity
of the medium to 1/10th the original capacity led to a decrease in the
pH of the effluent from 7 to 6 and to a significant reduction in the
degradation activity. A second fixed-bed reactor successfully removed
low chlorophenol concentrations (20 to 26 µM) with
hydraulic residence times of 8 to 30 min.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1, and the
acid-neutralizing capacity was 182 mM equivalents of H+ at
pH 7.5. This medium corresponded well to the groundwater conditions at
the Amponville site (pH 7.6; conductivity, 320 µS · cm1).
Growth experiments.
Growth experiments were performed in
duplicate in 500-ml shake flasks containing 100 ml of mineral medium
and, unless stated otherwise, 20 mg of the corresponding test substance
per liter. The precultures used for inoculation were grown for 3 days
on 120 µM 2,6-DCP (20 mg of 2,6-DCP per liter). The concentration of
the remaining 2,6-DCP was below the quantification limit, 1.8 µM. A
0.1-ml aliquot (1
) of the preculture was transferred into new
medium, and growth was allowed to proceed at 20°C on a rotary shaker
at 144 rpm. Both substrate disappearance and cell growth to at least 10 times the inoculum concentration were used as criteria for growth.
Reactor setup. The fixed-bed reactors consisted of glass columns with an inner diameter of 3 cm and a total volume of 210 ml that were filled with 120 ml of sintered glass beads (diameter, 2 to 4 mm; Siran; Schott, Mainz, Germany). The reactors were run with mineral medium (pH 7.5) in upstream mode.
The flow rate of reactor 1 was 750 ml · day1,
which yielded an HRT of 3.84 h as related to the interstitial
volume of the fixed bed. The 2,6-DCP feed concentration was gradually
increased from 250 to 740 µM. Simultaneously, the acid neutralization
capacity and the trace element concentration of the mineral medium were increased up to threefold. Hydrogen peroxide (30%, vol/vol) was diluted to a molar ratio of about 12:1 in relation to the actual 2,6-DCP concentration to provide enough oxygen for complete
mineralization of the substrate. Sufficient oxygen was indicated by
oxygen effluent concentrations of 1 to 8 mg · liter1.
Reactor 2 was run with constant feed concentrations of 2,4-DCP and
2,6-DCP (9 µM each) and 2,4,6-trichlorophenol (2,4,6-TCP) (7.6 µM).
The flow rate was gradually increased from 6 to 78 liters · day1, which yielded HRTs between 0.48 and 0.04 h.
Analytical methods.
Aromatic compounds were analyzed by
reversed-phase high-performance liquid chromatography performed with a
model 300 system (Kontron Instruments, Everett, Mass.) equipped with a
Nucleosil C18 column (Bischoff, Leonenberg, Germany).
Chlorophenols were separated by using a 30 to 90% acetonitrile
gradient (pH 2 adjusted with H3PO4). The flow
rate was 1 ml · min1, and data were
quantified by absorbance at 290 nm. The typical retention time for
2,6-DCP was 8.7 min. The quantification limits were 1.8 µM for
2,6-DCP and 0.6 µM for 2,4-DCP and 2,4,6-TCP. The chloride ion
concentration was determined with the Spectroquant system (catalog no.
1.14755; Merck, Darmstadt, Germany), and hydrogen peroxide was measured
with the Reflectoquant peroxide test system (catalog no. 16974; Merck).
The glucose concentration was determined with Trinder reagent (Sigma
Chemical Co., St. Louis, Mo.) by using the supplier's instructions.
Proton activity was measured with a portable pH meter (Portamess 751;
Knick, Darmstadt, Germany). CFU were determined by direct plating
following 3 days of incubation on 1.5% agar (Bacto Agar and nutrient
agar; Difco Laboratories, Detroit, Mich.) amended with 0.3 beef
extract and 0.5 peptone. The optical densities of liquid cultures
were measured at 546 nm.
Chemicals. The chemicals used in this study were analytical grade. 3,5-DCP and 2,3,5,6-tetrachlorophenol were purchased from Aldrich-Europe, Beerse, Belgium; acetonitrile, ortho-phosphoric acid, 2,4,6-TCP, 3,4-DCP, 2,6-DCP, toluene, and tetrachloroethylene were obtained from Merck. All other chemicals were purchased from Fluka, Buchs, Switzerland.
Nucleotide sequence accession number. The 16S rRNA sequence of strain RK1 has been deposited in the EMBL nucleotide sequence database under accession no. AJ002302.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Taxonomic characteristics.
Strain RK1 was the only isolate
from fixed-bed reactor 1 that was able to grow in pure culture on
2,6-DCP as the sole carbon and energy source. This organism is a
white-pigmented, gram-negative, oxidase- and catalase-positive rod that
is 1.5 to 4 µm long and 0.6 to 0.8 µm wide. No extracellular
structures and no spores are visible when preparations are examined by
light microscopy or scanning electron microscopy. Small, white,
transparent, circular colonies with smooth surfaces can be observed
after incubation for 2 days at room temperature on nutrient agar
plates. The colonies change to a flowerlike shape when plates are
incubated for longer periods of time (up to 5 days). The bacterium is
urease positive and arginine dihydrolase and 
-galactosidase
negative; it lyses neither esculin nor gelatin, cannot reduce nitrate,
is not able to grow anaerobically, and does not form acids from sugars.
It is able to grow on the following substrates: gluconate, caprate, adipate, malate, citrate, succinate, pyrocatechol, benzene, toluene, chlorobenzene, phenol, 2-chlorophenol (2-CP), 4-CP, 2,4-DCP, and 2,6-DCP. It does not grow on glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, 3-CP, 2,3-DCP, 3,4-DCP,
3,5-DCP, any isomer of trichlorophenol, 2,3,5,6-tetrachlorophenol,
pentachlorophenol, 2,4-dichlorophenoxyacetic acid, 1,3-dichlorobenzene,
1,2,3-trichlorobenzene, 2,6-dichlorotoluene, 2,6-dinitrophenol,
2,6-dimethylphenol, or 1,2,3-trichloropropane. Important taxonomic
features of strain RK1 and its closest phylogenetic neighbors are
listed in Table 1.
|
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Growth characteristics.
Ralstonia sp. strain RK1
completely degrades 2,6-DCP under oxic conditions, and chloride ions
are produced stoichiometrically (Fig.
1A). No UV-detectable intermediate
products were detected in the fixed-bed reactor experiments and in
batch assays with substrate concentrations below 1,200 µM. The
bacterial cell yield was 119 ± 34 CFU · pmol of
2,6-DCP1 and was constant for initial 2,6-DCP
concentrations greater than 120 µM; the bacterial cell yield was
slightly higher for lower concentrations (data not shown). The yield
obtained corresponds to about 7.5 g of protein · mol of
C1, assuming a weight of 1 µg/microorganism and a
protein content of 50% (wt/wt) (31). The high protein
yield, the consumption of approximately 12 mol of hydrogen peroxide
(yielding 6 mol of O2) per mol of 2,6-DCP in fixed-bed
reactor 1, and the fact that no aromatic or other conjugated
intermediates were detected justify the assumption that 2,6-DCP was
completely mineralized.
|
, 2,6-DCP concentration; 
,
chloride ion concentration; 
, CFU per milliliter. (B) Lag phase of
Ralstonia sp. strain RK1 with increasing initial
concentrations of 2,6-DCP. (C) Growth rate of Ralstonia sp.
strain RK1 at different initial concentrations of 2,6-DCP. Solid line,
curve obtained with the Monod equation (1) was reached when the 2,6-DCP concentration was
approximately 300 µM, and the growth rate decreased rapidly with
higher substrate concentrations (Fig. 1C). The maximal growth rate,
KS, and Ki were
determined by Lineweaver-Burk linearization (23) of the data
shown in Fig. 1C. At initial 2,6-DCP concentrations less than 300 µM,
the growth rate followed Monod kinetics, with a maximal growth rate of
0.082 h1 and a KS of 24.1 µM.
The decrease in the growth rate at higher initial concentrations was
attributed to inhibitory effects of 2,6-DCP. The decreasing part of the
curve could be described by the Haldane equation (14).
Neither the Monod model nor the Haldane model could be used to describe
the growth rate over the whole concentration range, however.
Since nonsterile conditions and mixed bacterial cultures are prevalent
in field applications, we compared the performance of the pure culture
of strain RK1 with the performance of a mixed culture obtained from an
aliquot from a fixed-bed reactor (reactor 1) (see below). The mixed
culture was transferred five times in fresh mineral medium containing
120 µM 2,6-DCP as described above for the isolation of
Ralstonia sp. strain RK1. The resulting mixed culture had a
bacterial cell yield of 172 ± 7 CFU · pmol of
2,6-DCP1 and a growth rate of 0.15 h1 at an
initial 2,6-DCP concentration of 120 µM (data not shown). Ralstonia sp. strain RK1 was the only isolate obtained from
reactor 1 that grew on 2,6-DCP as a sole carbon and energy source. More than 90% of all colonies from reactor 1 that grew on complex medium were morphologically similar to Ralstonia sp. strain RK1
colonies. We assumed, therefore, that Ralstonia sp. strain
RK1 accounted for a major portion of the reactor biomass and that it
grows well and maintains a metabolically active population under
nonaseptic conditions.
Reactor experiments. Reactor experiments with two different reactors were conducted to determine whether mixed cultures could be used to remove chlorophenols from water and soil. Both reactors were inoculated with sediment samples from a freshwater pond at the Amponville site, which is contaminated with 2,4-DCP, 2,6-DCP, and 2,4,6-TCP.
Fixed-bed reactor 1 was run with high concentrations of 2,6-DCP (220 to 740 µM) to investigate the feasibility of treating wastewater from soil washing. The reactor was allowed to adapt for 55 days to 220 µM 2,6-DCP before the feed concentration was increased stepwise to 740 µM over a period of 22 days (Fig. 2, arrow 1). This led to an almost stoichiometric increase in chloride ions in the outlet. At day 92 (arrow 2), the addition of hydrogen peroxide was interrupted, and mineralization ceased almost instantaneously. Then 11 days later, hydrogen peroxide was added again, and simultaneously the concentration of 2,6-DCP was lowered to 400 µM (arrow 3). 2,6-DCP degradation and chloride release resumed quickly, and the feed concentration was readjusted to its former value. Lowering the acid-neutralizing capacity of the medium to 1/10th the initial capacity (arrow 4) led to a decrease in the pH from 7 to 6 in the effluent (data not shown). Concomitantly, the performance of the reactor decreased significantly. It took 10 days before the system began to adapt to the lower pH and reactor performance increased again.
|
,
2,6-DCP effluent concentration; 
, chloride ion effluent
concentration. The arrows indicate experimental changes described in
the text. d, days.
1 · day1
with a conversion coefficient as high as 0.85. The maximal volumetric degradation yield achieved in this experiment was as high as 1.6 g
· literR1 · day1 (Fig.
3C).
|
, HRT. (B) Effluent concentrations
of the three chlorophenol congeners. Symbols: 
,
2,6-DCP; 
outflow concentration)/feed concentration].
The same x axis applies to panels A through C. d, days;
lR, liter reactor volume.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Degradation of chlorophenols has been reported very often. Ralstonia sp. strain RK1 is the first pure culture capable of growing on and mineralizing 2,6-DCP.
The 16S rRNA gene of the newly isolated bacterium described in this paper exhibits the highest levels of homology with the 16S rRNA genes of members of the genera Alcaligenes and Burkholderia which have been transferred to the genus Ralstonia (34). According to Stackebrandt and Goebel (30), the generally accepted limit for a relationship at the species level is 70% DNA similarity, and DNA similarities of more than 60% are very unlikely to occur with 16S rRNA homologies of 97% or less. Since (i) the most closely related Alcaligenes sp. has not been identified to the species level (Table 2), (ii) the levels of 16S rRNA homology between strain RK1 and the three previously described Ralstonia species (Ralstonia eutropha, Ralstonia solanacearum, and Ralstonia pickettii) are less than 96% (Table 2), and (iii) the new Ralstonia sp. strain has a different phenotype than the previously described species of the genus Ralstonia (Table 1), we suggest that the new strain should be placed in a new species in the genus Ralstonia and that strain RK1 should be regarded as the type strain. We propose the name Ralstonia basilensis (from basilea, Latin for Basel, Switzerland, where the strain was isolated) for this organism.
The substrate spectrum of strain RK1 includes nonhalogenated aromatic compounds, like benzene, toluene, and phenol, as well as chlorobenzene and all mono- and dichlorophenols with a chloride substituent(s) in the ortho and/or para position.
Hydroxylation in the ortho position, which produces chlorocatechol, has been reported to be the first step in the aerobic degradation of mono- and dichlorophenols (9, 12). The initial steps used by R. basilensis RK1 for ring cleavage must be different from the pathway described by Don et al. (9). 2,4-DCP hydroxylase cannot act on the ortho positions of 2,6-DCP, because both of these positions are occupied by chlorine atoms. In addition, R. basilensis RK1 also lacks the ability to degrade 2,4-dichlorophenoxyacetic acid. Further investigations are in process to evaluate the 2,6-DCP degradation pathway.
Lengthening of lag phases with increasing initial substrate concentrations may be interpreted as a consequence of metabolic inhibition by 2,6-DCP at concentrations above 300 µM, since 2,6-DCP and other chlorophenols can act as uncoupling agents in biological membranes (7, 21, 29). Escher et al. (10) reported that the concentration required to inhibit the NAD+ reduction rate by 50% was 8.9 mM for 2,6-DCP. In our experiments, which were performed with concentrations that were 10 times lower, the bacterial cell yield remained unchanged, while the lag phase increased from up to 3 days at 2,6-DCP concentrations below 300 µM to 16 days at 1,200 µM 2,6-DCP. The prolonged lag phase at high initial 2,6-DCP concentrations may reflect slow growth of the bacteria due to large energy dissipation from simultaneous chlorophenol degradation and uncoupling. The lower growth rates at high initial 2,6-DCP concentrations also reflect substrate inhibition.
Under comparable conditions, a mixed culture from reactor 1 degraded
2,6-DCP faster and with a higher cell yield than a pure culture of
R. basilensis RK1. Our observation that the maximum growth
rate of an enriched, specialized pure culture is only one-half the
maximum growth rate of a mixed culture (0.076 versus 0.15 h1) is in contrast to observations made with
phenol-degrading strains of Acinetobacter calcoaceticus and
Pseudomonas fluorescens (19). There are several
possibilities which might account for our experience. Community
synergism based on 2,6-DCP products might prevent the buildup of
metabolic intermediates and thus minimize feedback regulation of the
consumption of 2,6-DCP. Alternatively, the community might contain
other bacterial populations that are able to degrade 2,6-DCP, which we
were not able to isolate on nutrient agar plates.
With the fixed-bed reactors used in this study we were able to demonstrate the practicability of eliminating chlorophenols in water through biological degradation. The ability to degrade high concentrations of 2,6-DCP demonstrated with reactor 1 opens up new possibilities for treating wastewater from chemical soil washing without further dilution. Elimination of 46 mmol of 2,6-DCP per liter of reactor volume per day (at a 2,6-DCP concentration of 720 µM) consumed 750 ml of mineral medium, 62 ml of hydrogen peroxide (30%), and very little energy for pumping. With fixed-bed reactor 2, we treated a simulated groundwater containing low concentrations of 2,4-DCP, 2,6-DCP, and 2,4,6-TCP. The concentrations of all three isomers were reduced to below the detection limits with an HRT of 20 min and were reduced 95% with an HRT of 8.2 min. Longer adaptation times might improve reactor performance.
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ACKNOWLEDGMENTS |
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This work was supported by grant 245067 from the Safety, Health, and Environment Departments of Ciba Specialty Chemicals Inc. and Novartis Inc. to P.S.
We thank Urs Jauch for his help with the electron microscope.
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FOOTNOTES |
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* Corresponding author. Mailing address: Ciba Specialty Chemicals Inc., WS-2090.K1.26, CH-4133 Pratteln, Switzerland. Phone: 41 61 468 23 46. Fax: 41 61 468 21 65. E-mail: patrick.steinle{at}cibasc.com.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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