Transformation of difluorinated phenols by Penicillium frequentans Bi 7/2

Article abstract of DOI:10.1023/A:1008230926973

The Penicillium frequentans strain Bi 7/2, using phenol as a sole source of carbon and energy, transformed the fluorinated phenols 2,3-, 2,4-, 2,5- and 3,4-difluorophenol rapidly. After growth on phenol, resting mycelia of the fungus converted the difluorophenols completely at an initial concentration of 0.5mM within 6 hours. The corresponding difluorinated catechols were found to be intermediates of all difluorophenols investigated. A relatively unspecific phenol hydroxylase catalyzed this hydroxylation step and showed activities towards all difluorophenols tested. One difluorocatechol was formed from each difluorophenol substituted with fluorine in the ortho-position, whereas two catechols were formed from 3,4-difluorophenol, due to its two vacant ortho-positions. A partial defluorination (50-77%) was observed in all cases.

Full text of DOI:10.1023/A:1008230926973

Biodegradation 8: 379–385, 1998.
379
c
1998 Kluwer Academic Publishers. Printed in the Netherlands.
Transformation of difluorinated phenols by Penicillium frequentans Bi 7/2
Ulrike Wunderwald1, Martin Hofrichter2, Gu¨nter Kreisel1 & Wolfgang Fritsche2
1 Universita¨t Jena, Institut fu¨r Technische Chemie, Lessingstrasse 12, 07743 Jena, Germany; 2 Universita¨t Jena,
Institut fu¨r Mikrobiologie, Bereich Technische Mikrobiologie, Philosophenweg 12, 07743 Jena, Germany
( author for correspondence)
Accepted 15 January 1998
Key words: defluorination, difluorocatechols, difluorophenols, metabolism, Penicillium, phenol hydroxylase
Abstract
The Penicillium frequentans strain Bi 7/2, using phenol as a sole source of carbon and energy, transformed the
fluorinated phenols 2,3-, 2,4-, 2,5- and 3,4-difluorophenol rapidly. After growth on phenol, resting mycelia of
the fungus converted the difluorophenols completely at an initial concentration of 0.5 mM within 6 hours. The
corresponding difluorinated catechols were found tobe intermediates ofall difluorophenols investigated.A relatively
unspecific phenol hydroxylase catalyzed this hydroxylation step and showed activities towards all difluorophenols
tested. One difluorocatechol was formed from each difluorophenol substituted with fluorine in the ortho-position,
whereas two catechols were formed from 3,4-difluorophenol, due to its two vacant ortho-positions. A partial
defluorination (50-77%) was observed in all cases.
Abbreviations: DFC – difluorocatechol; DFP – difluorophenol; DFV – difluoroveratrol; IU – integration units;
MFP – monofluorophenol
Introduction
duced and stored by certain plants in spite of its high
toxicity (Goldman 1965; Wong et al. 1992; Viss-
cher et al. 1994). The bacterial metabolism of p-
fluorophenylacetic acid, 2-, 3- and 4-fluorobenzoic
acid has been reported in detail by Harper and Blak-
ley (1970; 1971), Oltmanns et al. (1989), Engesser
et al. (1990) and Schlo¨mann et al. (1990). A partial
defluorination was observed, and in some cases the
fluorobenzoic acids were used as sole sources of car-
bon and energy.
There are only few reports on the degradation of
fluorinated compounds by fungi. Neujahr and Varga
(1970) have described the utilization of phenol as car-
bon source by Trichosporon cutaneum. After growth
on phenol, this yeast was also able to oxidize the MFPs.
The fungal growth on MFPs has only been observed
for the deuteromycetous mold Penicillium simplicis-
simum SK9117 (Marr et al. 1996). Hofrichter et al.
(1993) have reported the utilization of phenol as sole
source of carbon and energy by Penicillium frequen-
tans Bi 7/2. Phenol pregrown resting mycelia of this
Halogenated compounds are important environmental
pollutants of soil, water and air. Detailed investiga-
tions were carried out on the degradation of chlori-
nated compounds, because they are the most wide-
spread halogenated substances (Neilson 1990; Rei-
necke 1988; Rochkind-Dubinsky 1987). In addition,
the production and the use of fluorinated substances
(pesticides, pharmaceutics, polyfluorinated polymers
and tensides) have been increased enormously in the
recent years (Gerstenberger & Haas 1981; Naumann
1994; Cociglio et al. 1996; Key et al. 1997). There-
fore, our interest has been focused on the microbial
degradation of fluorinated aromatics, especially on the
metabolism of fluorinated phenols by fungi.
Several studies have described the oxidative degra-
dation of monofluorinated aliphatics and aromatics by
bacteria, but polyfluorinated compounds were scarce-
ly considered. Monofluoroacetate is the mostly inves-
tigated fluoroaliphatic compound, since it is pro-

380
fungus metabolized the MFPs via the corresponding
catechols and muconic acids. The initial hydroxyla-
tion in the ortho-position is effected by phenol hydrox-
ylase and subsequently catechol dioxygenase cleaves
the aromaticring. The defluorinationof MFPs occurres
(Hofrichter et al. 1994). Until now, the transformation
of polyfluorinated phenols has not been investigated in
detail, neither for bacteria nor for fungi. Thus, we have
focused our attempts on the degradation of DFPs by
the fungus P. frequentans. The degradability, the extent
of degradation, the release of fluoride and the primary
enzymatic attack were studied.
(E.C. 1.13.11.1.) activity was determined by measur-
ing the increase in absorbance at 260 nm, due to the
formation of cis,cis-muconic acid (Neujahr & Varga
1970). Molar absorption coefficients were published
by Dorn and Knackmuss (1978).
Chemical and physical analysis
A Gynkotek high performance liquid chromatograph
(HPLC) with a Gynkotek diode-array detectoroperated
from 200 to 400 nm and fitted with a Sepserv Ultrasep
ES RP-18 column (125 4.0 mm ID, 5 m particle
size) was used to determine the concentration of the
phenols and to detect the metabolites using isocratic
conditions (solvent: 0.05% phosphoric acid/methanol;
45: 55, v/v for 2,3-, 2,4-DFP and 55: 45, v/v for 2,5-,
3,4-DFP). As reference standards of 3,4-, 3,6- and 4,5-
DFC were not available, concentrations are given as
integration units.
To analyse the metabolites by gas chromatogra-
phy/mass spectrometry (GC/MS), the organic sub-
stances were extracted with diethylether at pH 2. If it
was necessary, the extracted metabolites were methy-
lated with diazomethane. The extracts obtained were
dried with Na2SO4 and used for GC/MS analysis car-
ried out with a Hewlett/Packard GC/MS HP 5890 series
II fitted with a HP 5 column.
Materials and methods
Chemicals
2,3-, 2,4-, 3,4-DFP, and 4,5-difluoroveratrol were
obtained from Aldrich-Chemie, Steinheim/Germany.
2,5-DFP and 3,5-difluoro-2-hydroxyacetophenone
were purchased from ABCR GmbH & Co., Karls-
ruhe/Germany. The purity of all chemicals was
between 97 and 99%.
3,5-DFC was synthesized by Dakin oxidation of
3,5-difluoro-2-hydroxyacetophenone according to the
prescription for 4-fluorocatechol of Corse and Ingra-
ham (1951). Diazomethane was synthesized from N-
nitroso-N-methylurea (Becker et al. 1996). HPLC
grade methanol was obtained from Merck, Darm-
stadt/Germany.
A UV/VIS spectrometer Shimadzu UV-2102PC
was used to determine the enzyme activities. The con-
centration of fluoride in the culture fluids was measured
with a fluoride sensitive electrode, Orion Model 94-09.
Organism and culture conditions
Results
The soil fungus Penicillium frequentans [Westling]
strain Bi 7/2 (ATCC-number: 96048) was isolated
from a soil contaminated with aromatic hydrocarbons
as described previously (Hofrichter et al. 1993).
All investigations were carried out with resting
mycelia precultivated on phenol in 0.05 M potassium-
phosphate buffer (pH 7.8) (Hofrichter et al. 1993). The
DFPs were added to a final concentration of 0.5 mM,
the average dry weight of mycelia was about 1 g/l.
Cell free extracts were prepared from phenol
grown washed mycelia by disruption with dry ice
and a subsequent ultracentrifugation as reported ear-
lier (Hofrichter et al. 1994).
Transformation of difluorophenols
2,4-difluorophenol was completely converted within 6
hours. In contrast, the releasing of fluoride stopped
after 23 hours and amounted about 50%. During the
time course of degradation a metabolite with a typical
catechol UV-spectrum was detected by HPLC/DAD
analysis (Figure 1A). At the maximum of its concen-
tration, the cultivation was stopped and the metabo-
lite was extracted with diethylether. The neutralized
extract was dried and divided in two fractions. The first
fraction was directly analyzed by GC/MS. The mass
spectrum of the metabolite showed a molecule peak
of m/z 146, which corresponds to a compound with
the composition C6H4O2F2. The followingdefragmen-
tation peaks were determined: m/z 128 (M - H2O),
The activity of phenol hydroxylase (E.C.
1.14.13.7.) was measured by following the disappear-
ance of its specific cosubstrate NADPH at 340 nm
(Neujahr & Varga 1970). Catechol-1,2-dioxygenase

381
A
B
C
D
Figure 1. Elimination of DFPs ( ) by resting mycelia of P. frequentans pregrown on phenol as sole source of carbon and energy (final dry
weight: 1 g/l). The initial concentration amounted 0.5 mm DFP. During the degradation, the transient formation of the corresponding DFCs (
) and a partial defluorination ( ) were observed.
,
A). Conversion of 2,4-DFP [mM] ( ) under formation of 3,5-DFC [mM] ( ) and fluoride [mM] ( ).
B). Conversion of 2,3-DFP [mM]( ) under formation of 3,4-DFC [IU] ( ) and fluoride [mM] ( ).
C). Conversion of 2,5-DFP [mM]( ) under formation of 3,6-DFC [IU] ( ) and fluoride [mM] ( ).
D). Conversion of 3,4-DFP [mM]( ) under formation of 3,4-DFC [IU] ( ), 4,5-DFC [IU] ( ) and fluoride [mM] ( ).
m/z 117 (M - CHO). The other fraction was treated
with diazomethane and also analyzed by GC/MS. The
chromatogram showed a peak with a molecule peak
of m/z 174, which corresponds to a compound with
the elemental composition C8H8O2F2. The follow-
ing defragmentation peaks were unequivocal: m/z 159
(M - CH3), m/z 131 (M - CH3 - CO), m/z 111 (M -
CH3- CO - HF). Therefore, the methylated product
should be 3,5-DFV and the found metabolite was sup-
posed to be 3,5-DFC. To substantiate this assumption,
3,5-DFC was synthesized. The comparison of both
UV-spectra (HPLC/DAD), mass spectra (GC/MS), and
retention times confirmed the identity of the metabo-
lite formed. Thus, the extraction and methylation pro-
cedure used and the subsequent analysis by GC/MS
have been shown to be a suitable method to detect the
catecholic metabolites of the fungal degradation of the
DFPs.
To get further information on the degradation path-
way, the activity of catechol-1,2-dioxygenasewas esti-
mated in cell free extracts towards 3,5-DFC. The
enzyme activity was about 2% of that detected for
unsubstituted catechol. An exact determination of the
enzyme activity, however, was difficult, as it was
estimated by following the increase in absorbance at
260 nm and using the extinction coefficient of unsub-

382
Table 1. Release of fluoride and metabolites identified during the degradation of difluorinated phenols
compound
release of
metabolites identified
phenolhydroxylase activity
in the crude extract
fluoride [%]
[mU/mg] [%] compared
with phenol
2,3-DFP
2,4-DFP
2,5-DFP
3,4-DFP
50
50
50
77
3,4-DFC
107
131
86
23
28
18
50
3,5-DFC; 2,4-difluoro-cis,cis-muconic acid
3,6-DFC
3,4-DFC; 4,5-DFC
232
stituted cis,cis-muconic acid. This extinction coeffi-
cient was used as approximate value, since that of
2,4-difluoro-cis,cis-muconicacidwas not available and
no extinction coefficients were found in the literature.
Controls omitting cell-free extract showed no decrease
of 3,5-DFC concentration within this space of time. A
direct qualitative determination of the metabolite was
possible by HPLC/DAD analysis after treatment of 3,5-
DFC with cell-free extract and a following acidifica-
tion with HCl to pH 2. The UV-absorption maximum
is 273 nm. 2,4-Difluoro-cis,cis-muconic acid can be
assumed as an intermediate.
The degradation of 2,3-DFP was accompanied by
a removal of 50% fluoride (Figure 1B) and the tempo-
rary accumulation of a metabolite having the typical
UV-spectrum of catechols. As in the case of 2,4-DFP,
2,3-DFP has also one unsubstituted ortho-position and
therefore only one difluorinated catechol is expect-
ed. To clarify this assumption by GC/MS analysis,
methylation of the etheric extract of the culture filtrate
was performed. The mass spectrum showed a mole-
cule peak of m/z 174 corresponding to a composition
of C8H8O2F2. The defragmentation is similiar to that
described for 3,5-DFV. Therefore, the metabolite was
proposed to be 3,4-DFC.
2,5-DFP was a degraded in a similar way to 2,3-
and 2,4-DFP, due to its vacant ortho-position (Fig-
ure 1C). A release of 50% fluoride and a transient
accumulation of a metabolite with a UV-spectrum typ-
ical for catechols were observed. The GC/MS analysis
of the methylated intermediate showed also a mole-
cule peak of m/z 174 corresponding to the compo-
sition C8H8O2F2 and the defragmentation mentioned
above. These mass data are also in agreement with that
found for 3,6-DFV by Ladd & Weinstock (1981). The
metabolite was supposed to be 3,6-DFC.
unsubstituted ortho-positions, the phenol hydroxylase
of P. frequentans has two possible positions for attack-
ing the molecule. Thus, the formation of two interme-
diary metabolites having UV-spectra typical for cate-
chols was observed during the metabolism of 3,4-DFP.
One of these intermediates has a UV-spectrum and
retention time (HPLC/DAD) identical to that of 3,4-
DFC found in the metabolism of 2,3-DFP. To confirm
this finding, the metabolites were extracted and methy-
lated as described above. GC/MS analysis demonstrat-
ed that the mass spectrum and the retention time of
the methylated 3,4-DFC (3,4-DFV) detected during
the degradation of 2,3-DFP also correspond to the data
of one of both metabolites. Thus, 3,4-DFC is one of
both metabolites in the degradation pathway. The oth-
er metabolite was identified by comparing its mass
spectrum and retention time (GC/MS) with those of
authentic 4,5-DFV. It was found to be 4,5-DFC.
Formation and disappearance of 4,5-DFC proceed-
ed faster than of 3,4-DFC. Possibly the attack of phe-
nolhydroxylase at the 6-position is preferential com-
paredwith the attack at the 2-position. The faster disap-
pearance indicates that the ortho-cleavage of 4,5-DFC
is easier than that of 3,4-DFC.
Table 1 shows the data of fluoride released, the
metabolites identified during the degradation of the
fluorophenols by the fungus P. frequentans as well as
the activities of phenol hydroxylase towards the fluo-
rinated phenols.
In Table 2, the most important data of the mass
spectra of the different DFV and the relative maxima
of UV-spectra of the DFC are summarized. The defrag-
mentation patterns of the DFVs show a high similarity.
A brownish coloring was observed for all fluo-
rophenol cultures tested due to autoxidation. This
process causing oxidative polymerization of catechols
(Ziechmann1994)and leading to humic-acidlike com-
pounds (Bilton & Cain 1968, Hofrichter et al. 1994)
probably gives also a small contribution to the release
3,4-DFP was transformed with the highest rate
(within 2 hours) and the release of the highest amount
of fluoride (77%) (Figure 1D). Since 3,4-DFP has two

383
Table 2. Relative maxima of the UV-spectra of the DFC and the mass spectra of the methylated metabolites (difluoroveratrols, DFV)
metabolite
UV-Spectra:
relative
mass spectra: m/z (% relative intensity) of the corresponding DFV
maximum
[nm]
3,4- DFC
3,5-DFC
4,5- DFC
3,6- DFC
273
272
285
260
175 (9), 174 (100), 160 (4), 159 (46), 131 (14), 116 (16), 111 (15), 101 (16), 88 (25), 83 (48)
175 (9), 174 (100), 160 (6), 159 (74), 131 (30), 116 (13), 111 (30), 88 (23), 83 (39)
175 (9), 174 (100), 160 (4), 159 (52), 131 (33), 116 (14), 113 (33), 111 (30), 101 (20), 88 (46), 83 (58)
175 (9), 174 (100), 160 (3), 159 (38), 131 (6), 116 (17), 111 (23), 101 (17), 88 (18), 83 (36)
OH
OH
OH
OH
F
F
F
F
F
F
F
F
1
1
1
1
1
OH
OH
OH
OH
HO
F
F
HO
HO
2
F
HO
F
F
F
F
F
F
2
2
2
2
CO2H
CO2H
3
3
3
3
a
b
F
50 % F-; u.p.
50 % F-; u.p.
50 % F-; u.p.
77 % F-; u.p.
fluorinated humic acid-like substances
Figure 2. Proposed pathway for the metabolism of difluorophenols in P. frequentans. The first step is the formation of the corresponding DFCs by
phenolhydroxylase. The release of fluoride proceeds via unknown metabolites probably initialized by the attack of catechol-1,2-dioxygenase (2).
A part of the DFCs is transformed to fluorinated humic acid-like substances, which becomes visible by dark-brown coloring of the culture fluid
and is probably accompanied by releasing of a small amount of fluorid. 1 - phenolhydroxylase, 2 - catechol-1,2-dioxygenase, 3 - spontaneous
oxidative polymerization; u.p. - unknown products; a - is concerned to 3,4-DFP, b - is concerned to 2,3-DFP.

384
of fluoride (Figure 2). Investigations using monofluo-
rocatechols demonstrated a spontaneous partial deflu-
orination under degradation conditions in the absence
of fungal mycelium. The rate depends on the posi-
tion of the fluorine. 3-fluorocatechol is significantly
more stable than 4-fluorocatechol. Information about
the spontaneous defluorination of DFCs have not been
found in the literature, but it can be assumed according
to the behaviour of the monofluorocatechols.
responding catechols from dichlorophenols has been
reported for the yeast Candida maltosa (Polnisch et
al. 1993) and P. frequentans (Hofrichter et al. 1994),
but no release of chloride was observed. Thus, it can
be concluded that the same initial hydroxylation step
(caused by phenol hydroxylase) took place both dur-
ing the metabolism of difluoro- and dichlorophenols.
In contrast to dichlorophenols, a subsequent dehalo-
genation was observed for DFPs. Information about
the course of defluorinationand the further degradation
process were not obtained, since other metabolites than
the catechols and 2,4-difluoro-cis,cis-muconic acid in
the case of 3,5-DFC degraded by crude extract were
not detectable (Figure 2). The activity of catechol-1,2-
dioxygenase towards 3,5-DFC in the crude extract of
P. frequentans indicates an intradiol ring cleavage to
form the corresponding muconic acid, probably fol-
lowed by the release of fluoride. Similiar findings have
been made for various 4-monohalocatechols (metabo-
lites of 4-monohalophenols), which are converted to
the instable 3-halo-cis,cis-muconic acids and subse-
quently to dienlacton and halogenide (Schlo¨mann et
al. 1990; Polnisch et al. 1992; Hofrichter et al. 1994;
Marr et al. 1996). Further studies will have to clarify
the degradation process of other polyfluorinated phe-
nols e.g. 2,6-DFP, tri- and tetrafluorophenols.
Discussion
After precultivation on phenol, resting mycelia of
the deuteromycetous soil fungus Penicillium frequen-
tans metabolized, in addition to monofluorophenols
(Hofrichter et al. 1994), also difluorophenols at an ini-
tial concentration of 0.5 mM rapidly. The degradation
of all DFPs proceededvia formationof the correspond-
ing difluorinated catechols. The subsequent degrada-
tion led, in all cases, to a partial defluorination. The
identical data of defluorination (50%) of the ortho-
substituted DFPs are conspicuous, but this value was
reached only after about 1 day (not shown in Figure 1).
In contrast, a removal of fluoride of about 77% took
place in the case of 3,4-DFP.
On the other hand, this mold is unable to uti-
lize fluorinated phenols as sole source of carbon
and energy, since e.g. the defluorination product 4-
carboxymethylenebut-2-ene-4-olideformedduringthe
degradationof 3- and 4-MFP can not be further degrad-
ed (Hofrichter et al. 1994).
References
Becker HGO & Berger W et al. (1996) In: Organikum: Organisch-
chemisches Grundpraktikum (p 599). Johann Ambrosius Barth
Verlag, Heidelberg; Leipzig
The potential for attacking fluorophenols is prob-
ably due to the low specificity of the phenol degrad-
ing enzyme system. Marr et al. (1996) have recently
reported the utilization of monofluorinated phenols as
carbon source by the soil fungus Penicillium simplicis-
simum, but the amounts of fluorophenols degraded and
the biomass formed were comparatively low.
Bilton RF & Cain RB (1968) The metabolism of aromatic acids by
microorganisms. A reassessment of the role of o-benzochinone
as a product of protocatechuate metabolism by fungi. Biochem.
J. 108: 1113–1117
Cociglio M, Brandissou S, Alric R & Bressolle F (1996) High-
performance liquid chromatographic determination of flucona-
zole in plasma. J. Chromatogr. B. 686: 11–17
Corse J & Ingraham LL (1951) The monofluorocatechols. J. Org.
Chem. 16: 1345–1348
As far as we know, there is only one report in
the literature on the microbial metabolism of DFP
and TFP. Hofrichter et al. (1994) have described the
cometabolic transformation and partial defluorination
of 2,4- and 3,4-DFP and 2,3,4-TFP (initial concen-
tration: 1 mM) by P. frequentans, but no intermedi-
ates were observed. Furthermore, activities of phe-
nol hydroxylase were detectable towards these sub-
strates. In our present experiments, the corresponding
DFCs of all DFPs (2,3-, 2,4-, 2,5- and 3,4-DFP) test-
ed were identified as major metabolites, their appear-
ance, however, was transient. The formation of the cor-
Dorn E & Knackmuss H-J (1978) Chemical structure and biodegrad-
ability of halogenated aromatic compounds. Biochem. J. 174:
85–94
Engesser KH, Auling G, Busse J & Knackmuss H-J (1990) 3-
fluorobenzoate enriched bacterial strain FLB 300 degrades ben-
zoate and all three isomeric monofluorobenzoates. Arch. Micro-
biol. 153: 193–199
Gerstenberger WRC & Haas A (1981) Fluorierungsmethoden in
der Organischen Chemie. Angew. Chem. 93: 659–680, Angew.
Chem. Int. Ed. Engl. 20: 647–668
Goldman P (1965) The enzymatic cleavage of the carbon-fluorine
bond in fluoroacetate. J. Biol. Chem. 240: 3434–3438
Harper DB & Blakley ER (1970) The metabolism of p-fluoro-
phenylacetic acid by a Pseudomonas sp. I. Isolation and iden-

385
tification of intermediates in degradation. Can. J. Microbiol. 17:
635–644
Oltmanns RH, Mu¨ller R, Otto MK & Lingens F (1989) Evidence for
a new pathway in the bacterial degradation of 4-fluorobenzoate.
Appl. Environ. Microbiol. 55: 2499–2504
Polnisch E, Kneifel H, Franzke H & Hofmann HH (1992) Degra-
dation and dehalogenation of monochlorophenols by the phenol
assimilating yeast Candida maltosa. Biodegradation 2: 193–199
Reinecke W (1988) Microbial degradation of haloaromatics. Ann.
Rev. Microbiol. 42: 263–287
Rochkind-Dubinsky ML, Sayler GS & Blackburn JW (1987) Micro-
bial decomposition of chlorinated aromatic compounds. Marcel
Dekker, New York and Basel
Schlo¨mann M, Fischer P, Schmidt E, Knackmuss H-J (1990) Enzy-
matic formation, stability, and spontaneous reactions of 4-
fluoromuconolactone, a metabolite of bacterial degradation of
4-fluorobenzoate. J. Bacteriol. 172: 5119–5129
Harper DB & Blakley ER (1971) The metabolism of p-fluorobenzoic
acid by a Pseudomonas sp. Can. J. Microbiol. 17: 635–644
Hofrichter M, Gu¨nther Th & Fritsche W (1993) Metabolism of phe-
nol, chloro- and nitrophenols by the Penicillium strain Bi 7/2
isolated from a contaminated soil. Biodegradation 3: 415–421
Hofrichter M, Bublitz F & Fritsche W (1994) Unspecific degradation
of halogenated phenols by the soil fungus Penicillium frequentans
Bi 7/2. J. Basic Microbiol. 34: 163–172
Hofrichter M, Bublitz F & Fritsche W (1995) Cometabolic degrada-
tion of o-cresol and 2,6-dimethylphenol by Penicillium frequen-
tans Bi 7/2. J. Basic Microbiol. 35: 303–313
Key BD, Howell RD, Criddle CS (1997) Fluorinated organics in the
biosphere. Environ. Sci. Technol. 31: 2445–2454
Ladd DL & Weinstock J (1981) Improved synthesis of fluorovera-
troles and fluorophenethylamines via organolithium reagents. J.
Org. Chem. 46: 203–206
Visscher PT, Culbertson CW & Oremland RS (1994) Degradation of
trifluoroacetate in oxic and anoxic sediments. Nature 369: 729–
731
Marr J, Kremer S, Sterner O & Anke H (1996) Transformation
and mineralization of halophenols by Penicillium simplicissimum
SK9117. Biodegradation 7: 165–171
Naumann K (1994) Neue Insektizide. Nachr. Chem. Tech. Lab. 42:
255–260
Wong HD, Kirkpatrick WE, King DR & Kinnear JE (1992) Defluo-
rination of sodium monofluoracetate (1080) by microorganisms
isolated from western australian soils. Soil Biol. Biochem. 24:
833–838
Ziechmann W (1994) Humic Substances. BI Wissenschaftsverlag,
Mannheim; Leipzig; Wien; Zu¨rich
Neilson AH (1990) The biodegradation of halogenated organic com-
pounds. J. Appl. Bacteriol. 69: 445–470
Neujahr HY & Varga JM (1970) Degradation of phenols by intact
cells and cell-free preparations of Trichosporon cutaneum. Eur.
J. Biochem. 13: 37–44