Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2 ~
MICROBIAL DE_RADATION OF POLYH_LOGENATED AROMATIC
HYDROCARBONS
Research into the microbial degradation of halogenated
dibenzo-p-dioxins has been limited and is confined to two
studies by KLECKA and GIBSON using Beijerinckia spec. (1)
and BUMPUS et al using the white rot fungus Ph. chryso-
sporium (2)~ XLECKA and GIBSON observed the reaction of
dibenzodioxin, 1- and 2-chlorodibenzodioxin to the corre-
sponding dihydrodiol derivatives (without dechlorination)
by a Beijerinckia spec. after culture with succinate. Fur-
ther degradation with release of chloride was not observed.
BUMPUS et al (2) described the formation of 27.9 p mol l4co2
(-2% of the theoretically releasable quantity) from 1.25
nmol 2,3,7,8 TCDD after incubation for 30 days with the
white rot fungus Phanerochaete chrysosporium during growth
on glucose (56 ~mol). Whereas nothing is known of the use
of the degradation reaction by Beijerinckia spec., HUTTER-
MANN and TROJANOWSKI (3) for example experimented with the
use o~ the white rot fungus for soil decontamination by
using straw as the C source.
On the other hand, applicants' own investigations into
the degradation of halogenated benzenes revealed strains
which were capable of oxidizing dibenzodioxin, dibenzofuran
and 2-bromodibenzofuran to a high degree, but were not able
to degrade these compounds completely. Accordingly, the
problem addressed by the present invention was to enrich
and isolate microorganisms, which are able to use diben-
zofuran, diben~odioxin and 2-bromodibenzofuran as the carbon
source, and to obtain strains which are capable of effec-
tively degrading even halogenated diben20dioxins.
According to the invention, this problem was solved by
the enrichment and isolation from Rhine water samples of
new microorganism strains of the genus Brevibacterium
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designated RST 69-211, RST 69-233 and RST 69-1361. These
strains were deposited on the 20.07.1988 under the numbers
DSM 4714, 4715 and 4716 in the Deutschen Sammlung von
Mikroorganismen (DSM), GrisebachstraBe8, D-3400 ~ottingen,
~ederal Republic of Germany, under the provisions of the
Budapest Treaty on the international recognition of the
deposition of microorganisms for patent purposes.
The present invention also relates to the mutants and
variants of these strains which have the features and
properties essential for carrying out the invention.
According to the invention, these new strains are used
for the microbial degradation of polyhalogenated polycyclic
aromatic hydrocarbons, more especially halodibenzo-p-
dioxins and dibenzofurans. ~onocyclic aromatic hydrocar-
bons, preferably phenol or toluene, may be added as induc-
tors.
Principal applications for the new strains include
microbiological wastewater treatment and soil decontamina-
tion. The second of these two applic:ations is of consid-
erable importance for the decontamin~tion and reclamation
of old dumps contaminated with halogenated polycyclic
aromatic hydrocarbons.
The invention is illustrated by the following Ex-
amples.
Growth conditions and characterization of the pure cultures
Bacteria capable of using dibenzo-p-dioxin and di-
benzofuran as sole carbon source were isolated from Rhine
water. To this end, mineral salts were added to the Rhine
water, dibenzo-p-dioxin or dibenzofuran (0.5 g/l~ was added
as sole carbon source and the whole was introduced in 250
ml portions into a 1 liter Erlenmeyer flask and incubated
for several weeks at 28C while shaking at 250 r.p.m. Af--
ter the appearance of a yellow or brown coloration, the
flasks were inoculated onto fresh mineral salt medium with
Le A 26 751 2
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dibenzofuran and, finally, the bacteria were isolated as
pure cultures via dilution series on sterile agar media.
To this end, the mineral salt medium was supplemented with
yeast extract (0.5 g/l), dibenzo-p-dioxin or dibenzofuran
~0.5 g/l) was added in the form of a 10% DMSO solution and
Tween 80 (50 mg/l) was additionally introduced for uni-
formly distributing the insoluble dibenzofuran. Dibenzo-
furan- and dibenzo-p-dioxin-degrading bacteria cGuld be
recognized from the discoloration of the medium (yellow or
brown).
Isolated pure cultures were kept on agar plates with
complex medium ~Merck Standard I). Long-term cultures were
stored in liquid nitrogen.
10 Different pure cultures capable of using dibenzo-
furan and dibenzo-p-dioxin aS the ~arbon source were enriched
and isolated from Rhine water (Table 1~. A test under the
same conditions showed that only the gram-positive isolates
were capable of degrading 2-bromodibenzofuran. According-
ly, these gram-positive isolates were used for the further
investigations because degradation of the halodibenzo-p-
dioxins was expected to occur soonest.
Closer characterization of the isolates RST 69-211,
RST 69-233 and RST 69-1361 produced the following results:
gram-positive, immobile coccoidal rodlets with a distinct
rodlet coccus cycle in the early growth phase. Catalase
was positive, oxidase and the KOH test were negative. Cell
wall preparations contained directly attached p-DAP;
mycolic acids and N-glycolyl ester were not present. All
strains used the following as carbon sources (Table 2):
glucose, sucrose, glycerol, pyruvic acid, acetic acid,
benzoic acid and salicylic acid, but not mannitol. These
results allow clear assignment to the genus Brevibacterium.
By contrast, there were distinct differences with each of
the four species described in Bergey's Manual of Systematic
Bacteriology. Accordingly, further determination of the
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species was not possible. The strains were deposited as
patent strains on the 20.07.88 under the numbers DSM 4714,
4715 and 4716 (for RST 69-211, RST 69-233 and RST 69-1361).
Di~ferences between the three strains were observed
where phenol, toluene and lactic acid were used as th~ carbon
source. Aniline and chlorobenzene could not be used as the
carbon source in the concentration used~ However, the
appearance of brown or orange coloration indicated in-
complete degradation of these compounds. In addition,
strain 233 was capable of growing with benzene or o-xylene
as the carbon source and of degrading 4-chlorophenol and 5-
chlorosalicylic acid. By virtue of the broad degradation
spectrum, including in particular the monocyclic halogenat-
ed aromatic hydrocarbons, in strain 233, further degrada-
tion of the dioxins was tested with this strain.
Deqradation tests
Degradation tests with polycyclic aromatic hydrocar-
bons were carried out in 1 liter Erlenmeyer flasks with
Teflon-coated screw closures which contained 20 ml mineral
medium with the corresponding additives. For the residue
analyses, the entire contents of the flask were worked up.
It was only in this way that reproducible measurements
could be carried out, even in low concentrations.
Relatively large quantities of cells were cultured in
a 10 liter BE fermenter (Braun, Melsungen). The ~ulture
medium used was Standard I with phenol (0.8 g/l) or mineral
medium with yeast extract (0.5 g/l). In the latter case,
further carbon sources were introduced in the form of DMSO
solutions (2 to 10~, sterile-filtered) through a separate
inflow air stream saturated with solvent (toluene, benzene,
o-xylene) or in the form of concentrated aqueous solutions
(phenol, salicylic acid; 20 g/l). The fermentation con-
ditions were: 800 r.p.m., 28C, 1 1 air/min.
The residue analysis of the aromatic compounds was
Le A 26 751 4
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carried out by HPLC. To this end, the entire contents of
a 1 liter Erlenmeyer flask (20 ml) were shaken for 30
minutes at 28C with 2 ml of a 30% Brij 58 solution (in
DMSO), 20 ml dimethyl formamide was then added and, after
shaking for 30 minutes, the mixture was introduced into 50
ml Falcon centrifuge tubes. After centrifugation for 15
minutes at 4,000 g, the concentration of the compounds to
be tested was measured in the supernatant phase. Mixtures
adjusted to pH 2 with HCl were used as controls. The
supernatant phases were separated in an RP8 column using as
eluent water/acetonitrile mixtures differing in composition
(from 8:2 to 2:8) according to the polarity of the aromatic
hydrocarbons. Detection was carried out with a W detector
(Shimadzu) of variable wavelength at the W maximum of the
compounds to be tested. This method of residue analysis
produced better recovery rates (> 90~) than the considerab-
ly more expensive axtraction methods. In order further to
improve the results (less scattering), 1,4-diaminoanthra-
~uinone could be added as an internal standard with the
dimethyl formamide. In this case, the supernatant phases
had to be alkalized with 6 N NaOH for the HPLC analysis.
The detection limit of the HPLC analysis was between 10 and
100 ~g/l.
A chloride-free, purely mineral medium containing
relatively little phosphate (10 mM) was used for determin-
ing the chloride and bromide balances. To determine the
concentration of halide ions, the samples were adjusted to
pH 2 with H2SO4, centrifuged, sterile-filtered and stored at
4C pending analysis. Concentration was determined using
a Dionex 2000 i/SP ion chromatograph with an HPIC AS4A
separation column and an HPIC AG4A preliminary column. 1.7
mM Na2CO3/1.8 mM NaHCO3 was used as eluent for the bromide
determination. For the chloride determination, chloride
first had to be eluted with 2 mM NaOH before the other ions
were ~luted with Na2CO3/NaHCO3. In this way, a base line
Le A 26 75I 5
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separation could readily be obtained from chloride. The
detection limit of the method was at 0.1 ~M, i.e. the
samples could be diluted in a ratio of 1:10 for ion chroma-
tography.
For the enrichment and isolation of degradation
products of the various dibenzo-p-dioxins, the substances
were dissolved in high concentrations (2 to 10%) in DMS0/
Tween 80 (9:1), introduced with stirring into 450 ml 0.1 M
phosphate buffer, pH 7.~, in a l liter Erlenmeyer flask up
to a final concentration of 40 mg/l and 50 ml of a cell
suspension (40 g centrifuged cell mass in 120 ml 0.1 M
phosphate buffer, pH 7.2) of active cells was added. The
Erlenmeyer flask was shaken at 250 r.p.m. at 28C, an ali-
quot was removed at hourly intervals and the concentration
of the starting compound was determined. After substan-
tially complete conversion of the starting compounds, the
entire mixture was repeatedly extracted with n-hexane or
dichloromethane, the extract was dried, concentrated and
chromatographically purified on preparative TLC plates
(Merck) in toluene/dioxaneJacetic acid (90:25:5). Bands
which showed a blue coloration on spraying with Folin
reagent was scraped off, eluted and t:he structure further
investigated by GC-MS and NMR.
Conditions for ~ M~ analysis:
The analysis was carried out using a Bruker AM 360
360-MHZ-Supercon-FT-NMR spectrometer. For the analysis,
the chromatographically isolated, dried sample was dis-
solved in C6D6. 32 Scans ~ere accumulated. The chemical
shift was based on the internal standard tetramethyl silane
(0 ppm). The results were evaluated on the stretched
spectrum tlO Hz/cm) on the basis of the aromatic proton
signals in the 6 to 7 ppm range.
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Mass spectrometry
Analysis by mass spectrometry was carried out by GC-
MS coupling using a Finnigan MAT 8230 mass spectrometer.
The preceding gas chromatograph was of the Varian 3700
type. The gas chromatography was carried out with Split
1:130, injection block 250~C, and the following temperature
program: 3 minutes isothermal 70C, gradient from 70 to
320C at 15C/minute using an SE 30 capillary (20 m) for an
injection volume of 1 ~1. Entry into the ~ass spectrometer
is by direct coupling. The mass-spectrometric analysis
conditions were: 70 eV ionic collision activation and 3 kV
acceleration voltage. Accuracy was +/- 0.2 amu up to l,Ooo
amu.
Measurement of the breathina rates
To measure the breathing rates, freshly cultured or
frozen cells were suspended in 0.1 M phosphate buffer p~
7.2, in a concentration of 0.2 g DM per 10 ml. 1 ml of
this cell suspension was pipetted inlo a 100 ml heatable,
sealable and stirrable incubation vessel with an Orion 2
electrode, the basic breathing was rec:orded over 5 minutes,
100 ~1 of the test substrate solution (10 mg/ml in DMSO)
were added and the 2 uptake measured l`or another 5 minutes.
From the difference between the two breathing rates, it was
possible to calculate the specific 2 upta~e rate with the
aromatic test substrates.
Dearadation of 2-bromodibenzofuran and haloqenated dibenzo-
~-dioxins by strain ~ST 69-233
Strain ~33 was able to degrade 2-bromodibenzofuran (80
~M) to less than 10 ~g/l (= 40 mN) in 24 hours for a cell
density of 2 g DM/l. The delay in the release of inorganic
bromide indicated a temporary enrichment of intermediate
products. After 72 hours, approximately 75~ of the organi-
cally bound bromine used was recovered as free bromide.
Le A 26 751 7
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The cell culture method had only a slight influence on the
degradation of 2-bromodibenzofuran.
A totally different result was observed in the degrad-
ation of 2,3-dichlorodibenzo-p-dioxin. Whereas non-induced
cells showed only partial degradation of 2,3-dichlorodi-
benzo-p-dioxin after 4~ hours, complete disappearance of
the starting substance was observed after only 6 hours in
the case of phenol-induced cells. In this case, however,
the delay in the release of chloride was observed even more
clearly. Chloride could only be detected in the incubation
mixture during the measurement carried out after 24 hours,
its concentration reaching more than 130 ~M after 120
hours. This corresponded to a substantially quantitative
release of the organically bound chlorine to chloride.
Strain 233 evidently required an additional inductor, for
example pheno~, to induce the enzyn~es degrading halodi-
benzo-p-dioxin. Accordingly, the degradation of various
halodibenzo-p-dioxins and of 2-bromodibenzofuran by phenol-
induced cells was comparatively measured in another test.
Whereas the ring-halogenated substrates had been degraded
to the detection limit after 6 hours, complete disappear-
ance of the starting compound was only observed after 30
hours in the case of 2,7-dichlorodibenzo-p-dioxin. With
all halogenated derivatives, the release of halide was
substantially quantitative (Table 3).
Induction of the deqradation of halodibenzo-p-dioxin in
strain RST 69-233
To clarify the induction of the degradation of di-
benzo-p-dioxin, strain 233 was cultured with various
aromatic compounds and the breathing of the various mono-
cyclic and polycyclic aromatic hydrocarbons was measured.
As expected from the degradation results, the dibenzodioxin
oxygenase activity was induced only relatively weakly, if
at all, by 2-bromodibenzofuran or 2,3-dichlorodibenzo-p-
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dioxin on its own (Table 4). Various monocyclic aromatic
hydrocarbons (phenol, toluene) acted as good inductors of
the dibenzodioxin oxygenase. Benzene and salicylic acid as
growth substrates showed no induction effect.
Where phenol was used as an additional carbon source,
a distinct difference was observed in relation to culture
with phenol as sole carbon source. In every case, phenol
hydroxylase and salicylic acid oxygenase activities were
additionally measured. The result with benzene-grown cells
pointed to various enzymes for the oxidation of mono~yclic
and polycyclic aromatic hydrocarbons because, despite very
high conversion rates with toluene, no oxidation of di-
benzo-p-dioxin was measured. In all cultures with aromatic
hydrocarbons, high ring cleavage activity with pyrocatechol
was observed.
Another test was carried out to investigate the sub-
strate specificity of the oxidation system after growth
with a good inductor, namely toluene (Table 5). Of the
polycyclic aromatic hydrocarbons, phenoxazine was oxidized
at the highest rate while dibenzofuran, dibenzodioxin,
phenothiazine, naphthalene and indene were all oxidized at
a distinctly lower rate. Additional halogen substituents
also reduced the conversion rates. No 2 consumption could
be measured with xanthone, carbazole, phenazine, thioxan-
thone, anthraquinone or 2,7-dichloroclibenzo-p-dioxin. In
this case, the oxidation rates were partly below the
measurement limit because, with prolonged incubation times,
slow degradation, for example of 2,7-dichlorodibenzo-p-
dioxin, was measured. Another indication of slow degrada-
tion was the appearance of discoloration (Em~X ~ 510 nm) in
the event of prolonged incubation with thioxanthone.
~eavily discolored degradation products were also formed in
the oxidation o~ phenoxazine (Ema~ = 571 nm) and phenothi-
azine (EmAX = 591 nm).
The high conversion rat~s of 4-chloropyrocatechol and
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the oxidation of 3,5-dichloropyrocatechol, 5-chlorosalicyl-
ic acid, 4-chlorophenol and chlorobenzene were in accord-
ance with the release of halide from 2-bromodibenzofuran
and dichlorodibenzo-p-dioxins observed in degradation
tests. strain 233 clearly has the enzymes for the complete
degradation both of monocyclic and of polycyclic aromatic
halogenated hydrocarbons.
Characterization of degradation products
In the degradation of 2-bromodibenzo-p-dioxin, 2,3-
dichloro- and 2,7-dichlorodibenzo-p-dioxin, intermediate
products appeared in the first ~ew hours after addition,
disappearing again after a prolonged incubation time. If
mixtures were extracted at an earlier stage (3 to 6 hours),
degradation products could be chromatographically purified
from these extracts, showing the typical blue coloration of
aromatic hydroxy compounds when sprayed with Folin reagent.
GC-MS mass spectra of all the degradation products showed
an increase in molecular weight of 16, i.e. monohydroxy
derivatives of the starting compounds (Table 6). However,
it was not possible from the decomposition pattern to
identify the hydroxylation site. To ~etermine the substi-
tion site of the hydroxy group, h.lgh-field proton NMR
spectra of 2-bromodibenzodioxin, 2,7-dibromodibenzodioxin
and the unsubstituted dibenzodioxin were measured for com-
parison. As ~xpected, the unsubstituted dibenzodioxin
gives an A2-B2 spectrum with a narrow chemical shift range
while the ~,7-dichloro derivative, for reasons of symmatry,
gives an aromatic 1,2,4-proton system. The 2-bromo deriva-
tive shows the A2B2 part and the 1,2,4-substitution pattern
of the brominated ring. The A2B2 part of the spectrum is
missing in the case of the the hydroxylated 2-bromodi-
benzodioxin. This suggests that hydroxylation has taken
place in the unsubstituted benzene ring. The spectrum may
be interpreted as a mixture of 2-bromo-7-hydroxydibenzo-
Le A 26 751 lO
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dioxin and 2-bromo-8-hydroxydibenzodioxin (four different
aromatic 1,2,4-proton groups). In the case of the hydrox-
ylated product, the use of hexadeuterobenzene as NMR
solvent was crucial to the separation of the proton sig-
nals.
Table lDegradation properties and gram behavior of various micro-
organisms degrading dibenzofuran and dibenzo-p-dioxin
(Rhine water isolates)
Strain Gram Degradation Degradation Degradation
staining of dibenzo- of dibenzo- of 2-bromo-
furan p-dioxin dibenzofuran
69-7 - - +
69-8 - - + ~:
69-41 - - +
69-42 - + +
69-112 - - +
69-234 - - +
69-2351 - - +
6~-211 + + + +
69-233 + + - +
69-~361 + ~ + +
Le A 26 ?51 11
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Table 2
Biochemical properties of the 2-bromodibenzofuran-degrading
strains RST 69-211, -233 and -1361. For the growth test
with volatile aromatic hydrocarbons, the substrates were
added via the vapor phase.
Property - 69-211 69-233 69-1361
Gram behavior + + +
Oxidase - - -
Catalase + + +
Growth with C source (g/1):
.~ .
Glucose (10) + + +
Sucrose (10) + + +
Mannitol (1) - - -
Glycerol (2) + + +
Pyruvic acid (2) + + +
Lactic acid (2) + - +
Acetic acid (2~ + + +
Benzoic acid ~2) + + +
Salicylic acid (2) +
Phenol (0.8) - +
Aniline ~0.8) - - -
Benzene nm + nm
Toluene - + +
o-Xylene nm + nm
Chlorobenzene _ _ -
Degradation of (g/l~:
5-Chlorosalicylic acid (0.1) nm + nm
4-Chlorophenol (0.1) nm + nm
Le A 26 751 12
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Table 3
Halide balance in the degradation of 2-bromodibenzofuran
and various halodibenzo-p-dioxins by phenol-induced 69-233
cells after 5 and 10 days. The halide concentration in a
control with no addition of halogenated aromatics was < 2
~m.
Test substrate Substrate Final Final
concen- substrate halide
tration concen- concen-
usedtrationtration
(~M)(~M) (~M)
2-Bromodibenzofuran 73< 0.05 60
2-Bromodibenzo-p-dioxin 68 < 0.05 63
2,3-dichlorod:ibenzo-
p-dioxin 61 < 0.1 129
2,7-dichlorodibenzo-
p-dioxin 30 ~ 0.1 56
Le A 26 751 13
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Q ~ S: ~ ~ ~ O a) ~ O
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Table 5
Specific 2 uptake rates with various mono- and polycyclic
aromatic hydrocarbons by strain 69-233 after growth with
toluene. 0 - < 0.5 nMol/min X mg DM.
Substrate Specific 2 uptake
(nMol/min x mg DM)
Dibenzo-p-dioxin 3.8
2-Bromodibenzo-p-dio~in 3.3
2,3-Dichlorodibenzo-p-dioxin 0.6
2,7-Dichlorodibenzo~p-dioxin 0
Dibenzofuran 5.2
2-Bromodibenzofuran 3.3
Phenoxazine 22.2
Phenazine o
Carbazole 0
Phenothiazine 3.3
Thioxanthone 0
Xanthene 6.6
Xanthone o
Anthraquinone 0
Naphthalene 4~5
Indene 7.8
Benzene 16.9
Toluene 52.1
Chlorobenzene 1.6
Phenol 62.5
Chlorophenol 16.3
Salicylic acid 22.2
5-Chlorosalicylic acid 1.2
Benzoic acid 0
Pyrocatechol 312.5
4-Chloropyrocatechol 160.2
3,5-Dichloropyrocatechol 16.9
Le A 26 751 15
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Table 6
GC-MS data o~ 2-bromodibenzo-p-dioxin, 2,3-dichloro- and
2,7-dichlorodibenzo-p-dioxin and their degradation products
by strain 69-233
Compound m/L ~rel. Decomposi~ion
intensity) products
2-Bromodibenzo-p-dioxin 262 (100) Molecule ion
183 ( 15) -Br
155 ( 40) -C0
125 ~ 30) -C0
Degradation product of 278 (100? Molecule ion
2-bromodibenzo-p-dioxin 199 ( 12) -Br
171 ( 20) -co
143 ~ 5)-C0
115 ( 20) -C0
2,3-Dichlorodibenzo-p-dioxin 252 (100) Molecule ion
217 ( 5)-Cl
189 ( 50) -C0
161 ( 90) . -C0
126 ( 90) -Cl
Degradation product of 268 (100) Molecule ion
2,3-dichlorodibenzo-p-dioxin 23:3 ( 5) -Cl
205 ( 15) -C0
177 ( 3)-C0
14!3 ( 12) -C0
114 ( 3)-Cl
, .. . _
2,7-Dichlorodibenzo-p-dioxin 252 (100) Molecule ion
217 -Cl
189 -C0
161 -C0
Degradation product of 268 (100) Molecule ion
2,7-dichlorodibenzo-p-dioxin 233 ( 15) -Cl
205 ( 30) -C0
177 ( 2) -C0
149 ( 12) -C0
Le A 26 751 16
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Literature
1) G.M. Klecka, D.T. Gibson: Me~abolism of dibenzo-p-
dioxin and chlorinated dibenzo-p-dioxins by a Beijer-
inckia species. Appl. Environ Microb. 39 (1980), 288
2) J.A. Bumpus, M.T.D. Wright, S.D. Aust: Oxidation of
persistent environmental pollutants by a white rot
fungus, Science, 228 (1985), 1434
3~ A. Huttermann, J. Trojanowski: Ein Konzept fur eine
in-situ Sanierung von mit schwer abbaubaren Aromaten
belasteten Boden durch Inkubation mit dafur geeigneten
WeiBfaulepilzen und Stroh.
V. Franzius (Ed.): Sanierung kontaminierter Standorte
1986 "Neue Verfahren zur Bodenreinigung", Abfallwirt-
schaft in Forschung und Praxis, Vol. 18, 205-218,
Berlin, E. Schmidt Verlag (1987)
4) W. Springer, H.G. Rast: Biologischer Abbau mehrfach
halogenierter mono- und polyzyklischer Aromaten. GWF
Wasser/Abwasser 129 (1988), 70.
Le A 26 751 17
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