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International Biodeterioration & Biodegradation 59 (2007) 220225

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Degradation of phenol by a halotolerant strain

of Penicillium chrysogenum
A.L. Leitao, M.P. Duarte, J.Santos Oliveira
Grupo de Ecologia da Hidrosfera, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, and Unidade de Biotecnologia Ambiental,
Quinta da Torre 2829-516 Caparica, Portugal
Received 18 March 2005; received in revised form 13 November 2005; accepted 6 September 2006
Available online 1 November 2006

Abstract

The lowest 50% lethal (effective) concentration, L(E)C50, of phenol in a battery of seven microbiotests with species representing
different trophic levels was 110 mg l 1, classifying it as toxic. A phenol-degrading microorganism was isolated from soil samples of
the salt mine of Clona in Portugal, after enrichment in the presence of phenol and high salt concentration. Based on cultural and
morphological characteristics, the strain CLONA2 was identied as belonging to Penicillium chrysogenum. It was found to be a
halotolerant fungus able to grow in a nutrient-rich medium with 5.8% NaCl. It degraded at least 300 mg l 1 phenol as sole source of
carbon and energy, without accumulation of intermediates. The samples were also tested for toxicity using the Microtoxs assay. Data
showed that P. chrysogenum CLONA2 could be effectively utilized to reduce phenol toxicity. The results suggest also that phenol under
saline conditions can be successfully mineralized by P. chrysogenum CLONA2.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Phenol; Halotolerant; Biodegradation; Metabolites; Toxicity

1. Introduction Phenol is aerobically biodegraded to catechol and the

Phenol is a major pollutant present in several types of
industrial wastewater, such as that from coal reneries,
phenol manufacturing pharmaceuticals, industries of re-
sins, paints, dyes, petrochemicals, and textiles, and pulp
and paper mills (Kumaran and Paruchuri, 1997). It is
currently removed by costly and inefcient chemical or
physical methods. Biological degradation has been utilized
as an alternative, since it has low associated costs and leads
to complete mineralization of the xenobiotic (Singleton,
1994). The ability to degrade phenol and other phenolic
compounds is widespread in mesophilic microorganisms.
Pseudomonas spp. have been the most common organisms
used for phenol degradation studies (Allsop et al., 1993;
Hill and Robinson, 1975; Hinteregger et al., 1992;
Margesin and Schinner, 2001; Margesin et al., 2004).
Corresponding author. Grupo de Ecologia da Hidrosfera, Faculdade
de Ciencias e Tecnologia, Universidade Nova de Lisboa, Quinta da torre
2829-516 Caparica, Portugal. Tel./fax: +351 21 2948543.
E-mail address: aldl@fct.unl.pt (A.L. Leitao).
metabolic pathway is initiated by either ortho or meta
cleavage (Leonard and Lindley, 1998; Muller and Babel,
1994). Both pathways have a phenol hydroxylase in the
rst step of degradation, but in the second degradation
step, eukaryotes generally utilize catecol 1,2-(ortho) dioxy-
genase and prokaryotes use 2,3-(meta) dioxygenase (Muller
and Babel, 1994; Neujahr and Gaal, 1973). On the basis of
enzymatic studies, Jones et al. (1995) proposed two
pathways for the metabolism of phenol in Aspergillus
fumigatus. In one route, phenol undergoes ortho-hydro-
xylation to give catechol; in the other, phenol is hydro-
xylated in the para-position to produce hydroquinone,
which is converted to 1,2,4-trihydroxybenzene and then, by
the action of 1,2,4-trihydroxybenzene dioxygenase, to
maleylacetate.
As some industrial wastewaters have high concentrations
of salts (43.5%), degradation of phenolics by halophilic or
halotolerant microorganisms seems to be an attractive
option for the overall treatment of phenolic wastewater
(Woolard and Irvine, 1995). Halophilic and halotolerant
microorganisms that degrade phenol have been investigated

0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2006.09.009
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A.L. Leitao et al. / International Biodeterioration & Biodegradation 59 (2007) 220225
previously (Bastos et al., 2000; Hinteregger and Streichsbier,
1997; Maskow and Kleinsteuber, 2004). These microorgan-
isms have the potential to mineralize or transform the
phenol in the presence of osmotic stress that may be
uctuating in amplitude and frequency (Oren et al., 1993).
In spite of the studies already done in this eld, we found
only one study that reported halotolerance of Penicillium
spp. (Razak et al., 1999), and another on the ability of
Penicillium strain Bi 7/2 to metabolize phenol (Hofrichter
et al., 1993). But we found no other information about the
ability of Penicillium spp. strains to degrade phenol under
saline conditions.
Due to its toxicity, as shown by ecotoxicological studies
(Kahru et al., 2000; Kaiser and Palabrica, 1991), several
microbiotests have analyzed phenol compounds of oil-
shale industry wastewaters. The most sensitive test utilizes
the inhibition of bioluminescence as an indication of
toxicity (Kahru et al., 2000; Vismara et al., 1996).
We report here the phenol-degradative capacity of a new
halotolerant fungus isolated from hypersaline soil in the
Clona Salt Mine (Algarve, Portugal). The Microtoxs assay
was used to determine the toxicity of compound(s)
produced from biodegradation of phenol.
2. Materials and methods
2.1. Chemicals
Phenol, hydroquinone, catechol, resorcinol, pyrogallol, phloroglucinol,
and benzoic acid were of chromatographic grade (purity X99%), with the
exception of trans, trans-muconic acid (purity 98%) and were obtained
from Sigma-Aldrich (St. Louis, USA). HPLC acetonitrile was from Lab-
Scan (Dublin, Ireland). All other reagents were of analytical reagent grade.
Water puried by the Mili-Q system was used in all experiments. Nutrient
agar (NA) was purchased from Difco (Detroit, USA).
2.2. Screening and isolation of phenol-degrading microorganisms
Soil samples were collected from Clona, a salt mine in the Algarve
region of Portugal. The soil samples (3 g weight) were mixed in 10 ml
sterile nutrient broth (3.0 g peptone l 1, 5.0 g beef extract l 1) and
incubated at 2571 1C. Strains were puried by subculturing in a mineral
medium (330 mg KH2PO4 l 1, 1200 mg Na2HPO4 l 1, 100 mg NH4Cl l 1,
100 mg MgSO4
7H2O l 1, 440 mg CaCl2
2H2O l 1, 58500 mg NaCl l 1,
pH 6.8) supplemented with 10 mg l 1 phenol as sole carbon source. After
four transfers, serial dilutions of the culture were prepared and spread on
mineral medium agar plates supplemented with phenol. The plates were
incubated at 2571 1C for 3 days. Colonies were isolated from this
medium.
Isolates were individually inoculated into 100 ml mineral medium
(330 mg KH2PO4 l 1, 1200 mg Na2HPO4 l 1, 100 mg NH4Cl l 1, 100 mg
MgSO4
7H2O l 1, 440 mg CaCl2
2H2O l 1, 58500 mg NaCl l 1, pH 6.8) or
mineral medium with iron (1000 mg K2HPO4 l 1, 1000 mg (NH4)2SO4 l 1,
200 mg MgSO4
7H2O l 1, 33 mg FeCl3
6H2O l 1, 100 mg CaCl2 l 1,
58500 mg NaCl l 1, pH 6.8). Both media were initially supplemented with
0.55 mM glucose and 50 mg l 1 phenol. When microbial growth was
observed, 10-ml aliquots were aseptically inoculated into 100 ml of mineral
medium with iron, supplemented with increasing phenol concentrations
(18.8150 mg l 1 phenol). All cultures were incubated at 2571 1C in an
INNOVA 4000 Incubator Shaker (New Brunswick Scientic, NJ, USA)
operating at 160 rpm in the dark in order to avoid photodestruction of
phenol.
221
To identify strain CLONA2, several tests were done. The strains were
cultivated on NA plates. After 72 h of incubation at 2571 1C, individual
colonies appeared.
2.3. Identification of strain CLONA2
Identication was done mainly on the basis of cultural and
morphological characteristics (micromorphological and macromorpholo-
gical features). The strain was identied as belonging to the genus
Penicillium according to Barnett and Hunter (1999) and the species
chrysogenum as described in Pitt (1979).
2.4. Culture conditions
A preculture of the microorganism was produced in 100 ml of sterile
complex medium (MC) (30.0 g glucose l 1, 3.0 NaNO3 l 1, 0.5 g
MgSO4
7H2O l 1, 10 mg NH4Fe(SO4)2
12H2O l 1, 1.0 g K2HPO4 l 1,
5.0 g yeast extract l 1, 58.5 g NaCl l 1, pH 5.6) and incubated at 2571 1C
for 3 days with shaking at 160 rpm.
To test the ability of the strain to utilize phenol, several concentrations of
this aromatic were used. The nal concentrations added to sterile MMFe
(1000 mg K2HPO4 l 1, 1000 mg (NH4)2SO4 l 1, 200 mg MgSO4
7H2O l 1,
33 mg FeCl3
6H2O l 1, 100 mg CaCl2 l 1, 58500 mg NaCl l 1, pH 5.6) were
50, 100, 200, 250, and 300 mg l 1 ; CLONA2 was then inoculated and
incubated at 2571 1C in the dark, at 160 rpm. Uninoculated Erlenmeyer
asks served as controls. Phenol content and biomass of the halotolerant
strain were measured at various intervals over 4 days. All experiments
were carried out in triplicate, except the control that was carried out in
duplicate.
2.5. Biomass estimation
Microbial dry biomass was estimated gravimetrically by centrifugation
of 10 ml of each suspension at 10000g for 10 min. The supernatant was
discarded and the pellet was dried at 90 1C for 24 h and weighed (Gunther
et al., 1995).
2.6. Analytical analysis
The concentrations of phenol and metabolites were estimated by HPLC
(LaChrom HPLC Systeme, Merck), apparatus L-7100, equipped with a
quaternary pump system, and an L-7400 UV detector. A personal
computer equipped with HPLC System Manager software for windows
NTTM was used to acquire and process chromatographic data. The
separation was achieved with a LiChroCart 250-4 RP-18 endcapped
(5 mm) column (Merck, Darmstadt, Germany). A deionized water/
acetonitrile (70:30, vol/vol) isocratic system was used as solvent and the
ow rate was maintained at 1.0 ml/min. Ultraviolet analysis was carried
out at 254 nm. Under these conditions, baseline separation for phenol and
its intermediates could be obtained within 10 min. These compounds were
identied by comparing their retention time with those of similarly treated
external standards. Under the above conditions, the retention times of
phenol, hydroquinone, catechol, resorcinol, pyrogallol, phloroglucinol,
trans, trans-muconic acid, and benzoic acid were, 8.29, 4.39, 4.04, 3.21,
2.59, 2.51, 1.76, and 1.45, respectively.
2.7. Phenol degradation
The organism was cultivated in 100 ml of MC, for 3 days at 160 rpm
and 2571 1C. Ten milliliters of this culture were incubated in 500-ml
Erlenmeyer asks containing 100 ml of phenol-MMFe medium supple-
mented with (i) 3% glucose, (ii) 3% glucose and 250 mg l 1 phenol, (iii)
250 mg l 1 phenol. Cells were harvested by centrifugation (10000g, 10 min)
before inoculation and washed three times with sterile 0.85% NaCl.
Cultures were incubated at 160 rpm and 2571 1C for 4 days. The time
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course of phenol metabolism was determined by collecting and analyzing

samples (1 ml) at intervals of 24 h. They were harvested and puried by
HPLC, using the above system. Another HPLC method was developed
using the same system but with isocratic conditions (solvent: H3PO4
(0.01%)/acetronitrile (90:10, vol/vol)) and a variable wavelength absor-
bance detector operating at 254 and 280 nm.

2.8. Batch cultures

For degradation experiments at various saline concentrations, cells

were harvested by centrifugation (10000g, 10 min) from 10 ml of preculture
in MC containing NaCl (2%), and washed three times with sterile 0.85%
NaCl. The resulting inoculum was incubated in 500-ml Erlenmeyer asks
containing 100 ml of a mineral medium (1000 mg K2HPO4 l 1, 1000 mg
(NH4)2SO4 l 1, 200 mg MgSO4
7H2O l 1, 33 mg FeCl3
6H2O l 1, 100 mg
CaCl2 l 1 with 58500 mg NaCl l 1 or 20000 mg NaCl l 1, and pH 6.8).
Phenol (200 mg l 1) was added to 100 ml of both media with cell
suspension, and cultures were incubated in the dark for 4 days at
2571 1C, at 160 rpm on a rotary shaker. The assays were carried out in
duplicate.
Uninoculated Erlenmeyer asks in 100 ml of both media with Fig. 1. Micromorphological structure of P. chrysogenum CLONA2
200 mg l 1 of phenol were used as controls. (400  ).
Periodically, 1 ml from each Erlenmeyer ask was centrifuged (10000g,
10 min) and 20 ml of the supernatant analyzed. Phenol concentrations were
determined by HPLC as described in Section 2.6.

2.9. Toxicity testing

The toxicity of samples collected at different times was evaluated by the

Microtoxs assay using Microtoxs Analyser 500. Microtoxs is the
registered trademark of Azur Environmental (formerly Microbics
Corporation, CA). In this assay, the light emitted by the bacteria
(Photobacterium phosphoreum) is reduced upon exposure of the test
organisms to a toxic sample. This luminescence reduction is directly
related to the relative toxicity of the sample. The experimental procedures
were carried out according to the Microtoxs instructions. The effective
concentration for 50% inhibition of luminescence (EC-50) after 15 min
was calculated with the Microtoxs software. All the samples were
centrifuged (10000g, 10 min), to prevent interference from suspended
particles during bioassay.

3. Results and discussion

3.1. Isolation and identification of Penicillium spp. strain
CLONA2
One fungal species (CLONA2 strain) able to grow
utilizing phenol as the sole carbon and energy source was
isolated from the salt mine. As the microorganism was able
to grow in the absence, as well as in the presence of salt, it
must be designated as halotolerant (Margesin and Schin-
ner, 2001). Optical microscopy showed fruit-bodies char-
acteristic of the genus Penicillium, and conidiophores with
three branching points, characteristic of subgenus Penicil-
lium (Fig. 1). Conidia showed at spherical or ellipsoidal
shapes with an estimated diameter of 2.8 m. The fungus
demonstrated thermal dimorphism.
3.2. Degradation of phenol by Penicillium chrysogenum
To determine the ability of P. chrysogenum to degrade
phenol under saline conditions (5.9%, weight/vol NaCl),
Fig. 2. Degradation of phenol in 5.9% NaCl by P. chrysogenum. Initial
concentration of phenol: () 50 mg l 1; (K) 100 mg l 1; (m) 200 mg l 1;
(&) 250 mg l 1; (J) 300 mg l 1. Each point represents the mean of
triplicates.
the culture was exposed to different initial concentrations
of this aromatic compound. Phenol degradation at
initial concentrations from 50 to 300 mg l 1 is shown in
Fig. 2. Fifty and 100 mg l 1 of phenol were completely
degraded within 48 h. Higher concentrations, 200, 250,
and 300 mg l 1, were degraded after 72, 96, and 100 h,
respectively.
The degradation of phenol by fungi has previously been
observed in Fusarium flocciferum (Anselmo et al., 1985),
A. fumigatus (Jones et al., 1994), Graphium spp. FIB4
(Santos et al., 2003), Trichosporon cutaneum (Santos and
Linardi, 2001; Shiverova et al., 1999; Shoda and Udaka,
1980) and Candida tropicalis (Bastos et al., 2000; Chang
et al., 1998). Only the C. tropicalis, isolated in Amazonia,
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was able to degrade and tolerate higher concentrations of
phenol and salt (16 mM and 15%, respectively; Bastos et
al., 2000).
However, the degradation ability did not correspond to
the visible growth of P. chrysogenum CLONA2 mycelia.
None of the culture conditions tested, where fungi used
phenol as the sole source of carbon and energy, showed a
decrease in biomass at the end of the experiment. These
results were consistent with published data (Kang and
Park, 1997; Wang and Loh, 2000). Phenol degradation by
Pseudomonas species began before growth commenced
(Kang and Park, 1997).
3.3. Metabolism of phenol by P. chrysogenum
When P. chrysogenum was grown in the presence of
phenol (200 mg l 1) or in combined solutions of phenol
with glucose (200 mg l 1 phenol and 3% glucose), the
phenol concentration decreased to 125 and 150 mg l 1,
respectively, within 48 h. Analytical reverse-phase HPLC
(Fig. 3) revealed the presence of one phenol metabolite
formed by the fungus in the combined solution. Never-
theless, this did not occur in phenol alone. Phenol eluted
with a retention time of 8.29 min. The metabolite that
eluted at 4.39 min could be hydroquinone by comparison
with a standard solution. This result could be supported by
those published by Jones et al. (1995), who proposed two
pathways for the metabolism of phenol, one giving
catechol and the other hydroquinone. The degradation
rate of phenol in the presence of glucose was lower than in
simple phenol solution (Fig. 3), indicating that phenol
degradation by P. chrysogenum was delayed in the cultures
with 3% glucose. Wang et al. (1996) found that Pseudo-
monas putida (ATCC 17514) used the individual compo-
nents of mixed glucose and phenol substrates
simultaneously but with lower specic rates than for the
separate substrates. Later Wang and Loh (2000) reported
the effect of cross-inhibition between phenol and sodium
glutamate in Pseudomonas putida (ATCC 49451). Cata-
bolic repression of phenol degradation by glucose and
acetate was also observed in other fungi (Bastos et al.,
2000; Gaal and Neujahr, 1981).
After 80 h of culture in combined solutions of phenol
and glucose, chromatograms revealed the absence of
phenol and hydroquinone. This result suggests that either
phenol or hydroquinone were completely utilized by P.
chrysogenum.
With the aim of separating the compounds that appear
at the beginning of the chromatograms, a second method
was used (see materials and methods section). Chromato-
grams showed the presence of ve peaks, apparently
related to the metabolic products, which are constantly
present in the other culture conditions. In the presence of
phenol (combined with glucose or not), an additional

Fig. 3. HPLC chromatograms of the metabolite formed by P. chrysogenum after 48 h (panels A and B) and 72 h (panels C and D) of incubation with
200 mg l 1 phenol (panels B and D) or in combined solution (200 mg l 1 phenol and 3% glucose; panels A and C). The compound was identied as
hydroquinone. Note the accumulation of hydroquinone (Hq) (retention time 4.39) at 48 h only in cultures in the presence of glucose and the disappearance
of Hq and phenol (Phe) (retention time 8.29) at 72 h in phenol or combined solution cultures.
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chromatographic peak was observed (retention time not accumulate any metabolite in the presence of different
11 min). Chromatograms were obtained at different wave- saline concentrations at twice that phenol concentration.
lengths (254 and 280 nm), and the relationship A280/A254 All the toxicity tests were performed using samples from
showed that the phenol-specic peak does not have an cultures grown at 2% NaCl, since samples containing 5.9%
aromatic structure (data not shown). This chromato- NaCl showed direct toxicity effects, probably due to the
graphic behavior suggests the absence of an aromatic concentration of salt that conditioned the osmotic stability
compound from fungal phenol metabolism. of the luminescent bacteria.
Results of the toxicity test are shown in Table 1 and
3.4. Toxicity bio-assays Fig. 5. Toxicity levels, expressed as EC50, were approxi-
mately constant until 53 h of assay. At this point they
In order to test the presence of toxic compounds before began to decrease and became undetectable at 81 h. These
and after the treatment of phenol solutions with P. data suggest that toxicity was reduced by decreasing phenol
chrysogenum, a Microtoxs assay was performed. This is concentrations.
one of the most widely employed bioassays for biological In order to evaluate the contribution of phenol to
monitoring and toxicity assessment because it is simple, toxicity, solutions of different standard phenol concentra-
rapid, and sensitive. One important point in its application tions in MMFe (5200 mg l 1) were used. Fig. 5 shows that
is to ensure the osmotic stability of the marine bacterium. both curves are coincident, indicating that phenol is the
The manufacturer recommends the use of NaCl concentra- main agent responsible for sample toxicity. The absence of
tions of 20 g l 1 (Vismara et al., 1996). In order to have a toxicity at the end of the assay suggests that the end
nal concentration of 2% NaCl for toxicity testing, products of phenol degradation are not toxic according to
cultures were made with 5.9% and 2% NaCl for phenol
degradation studies. When P. chrysogenum was incubated
with 2% NaCl, complete degradation of phenol was Table 1
observed in 72 h, although the degradation rate was faster Microtox acute toxicity testing of phenol biodegradation by P.
chrysogenum
than at 5.9% NaCl (Fig. 4). HPLC analysis of samples
from 2% and 5.9% NaCl cultures in the presence of Time (h) EC50 (at 15 min, %) 95% condence
200 mg l 1 phenol revealed the same prole in all the cases range
(data not shown), demonstrating that the phenol degrada-
0 9.271 [9.898.69]
tion mechanisms are the same at both saline concentra- 23 11.45 [12.1510.80]
tions. 45 12.90 [13.4612.39]
A metabolite of phenol degradation (cis, cis-muconic 53 13.48 [14.1112.87]
acid) was observed in Halomonas spp. (Hinteregger and 69 54.17 [85.7634.22]
72 71.92 [79.9464.71]
Streichsbier, 1997). The amounts of cis, cis-muconic acid
81 4100
secreted by Halomonas spp. were related to salt concentra-
tions in the medium (maximum at 2% NaCl and minimum
at 5% NaCl). However, our isolate of P. chrysogenum did

Fig. 5. Toxicity testing by the Microtox assay. Comparison of EC50 values

Fig. 4. Degradation of 200 mg l 1 phenol at different saline concentra- of (K) batch culture samples from phenol degradation by P. chrysogenum
tions (K) 2% NaCl and () 5.9% NaCl. Each point represents the mean at 2% NaCl and an initial phenol concentration of 185 mg l 1 with ()
of triplicates. phenol standard solutions.
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the Microtoxs assay. This corroborates the HPLC data
and suggests the absence of toxic intermediates.
4. Conclusions
The results showed that phenol could be completely
mineralized by the P. chrysogenum CLONA2 isolated from
a salt mine. At 5.9% NaCl, P. chrysogenum CLONA2 was
able to rapidly detoxify phenol. Degradation of phenol in
the presence of glucose suggested that it could proceed via
hydroquinone. The possibilities of using this strain in the
biological treatment of phenol-containing wastewaters are
of interesting approach that could be applied in petro-
chemical efuents. It will be important to test this
possibility at pilot scale, comparing its performance
economically and technically with the standard physico-
chemical treatments.
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