An isolated Candida albicans TL3 capable of degrading phenol at large concentration

Article abstract of DOI:10.1271/bbb.69.2358

An isolated yeast strain was grown aerobically on phenol as a sole carbon source up to 24 mM; the rate of degradation of phenol at 30°C was greater than other microorganisms at the comparable phenol concentrations. This microorganism was further identified and is designated Candida albicans TL3. The catabolic activity of C. albicans TL3 for degradation of phenol was evaluated with the K_s and V_max values of 1.7 ± 0.1 mM and 0.66 ± 0.02 μmol/min/mg of protein, respectively. With application of enzymatic, chromatographic and mass-spectrometric analyses, we confirmed that catechol and cis,cis-muconic acid were produced during the biodegradation of phenol performed by C. albicans TL3, indicating the occurrence of an ortho-fission pathway. The maximum activity of phenol hydroxylase and catechol-1,2-dioxygenase were induced when this strain grew in phenol culture media at 22 mM and 10 mM, respectively. In addition to phenol, C. albicans TL3 was effective in degrading formaldehyde, which is another major pollutant in waste water from a factory producing phenolic resin. The promising result from the bio-treatment of such factory effluent makes Candida albicans TL3 be a potentially useful strain for industrial application.

Full text of DOI:10.1271/bbb.69.2358

Biosci. Biotechnol. Biochem., 69 (12), 2358–2367, 2005
An Isolated Candida albicans TL3 Capable of Degrading Phenol
at Large Concentration
y
2;
San-Chin TSAI,1;2 Li-Duan TSAI,2 and Yaw-Kuen LI
1Department of Medical Technology, YuanPei University of Science and Technology, Hsinchu, Taiwan
2Center of Interdisciplinary Molecular Science and Department of Applied Chemistry,
National Chiao Tung University, Hsinchu, Taiwan
Received June 30, 2005; Accepted September 18, 2005
An isolated yeast strain was grown aerobically on
phenol as a sole carbon source up to 24 mM; the rate of
degradation of phenol at 30 ꢀC was greater than other
microorganisms at the comparable phenol concentra-
tions. This microorganism was further identified and is
designated Candida albicans TL3. The catabolic activity
of C. albicans TL3 for degradation of phenol was
evaluated with the Ks and Vmax values of 1:7 Æ 0:1 mM
and 0:66 Æ 0:02 ꢀmol/min/mg of protein, respectively.
With application of enzymatic, chromatographic and
mass-spectrometric analyses, we confirmed that catechol
and cis,cis-muconic acid were produced during the bio-
degradation of phenol performed by C. albicans TL3,
indicating the occurrence of an ortho-fission pathway.
The maximum activity of phenol hydroxylase and cate-
chol-1,2-dioxygenase were induced when this strain grew
in phenol culture media at 22 mM and 10 mM, respec-
tively. In addition to phenol, C. albicans TL3 was effec-
tive in degrading formaldehyde, which is another major
pollutant in waste water from a factory producing phe-
nolic resin. The promising result from the bio-treatment
of such factory effluent makes Candida albicans TL3 be a
potentially useful strain for industrial application.
a prospective tool to detect pollutants.
Many microorganisms, including bacteria,3–12)
yeasts,13–17) algae18) and aquatic fungi,19) are found to
be capable of degrading phenol at various concentra-
tions. Since 1985, the catabolism of aromatic hydro-
carbons by microbes has been widely discussed. The
typical pathway of phenol degradation in microorgan-
isms occurs via the formation of catechol derivative
following by ring cleavage through the ortho-fission or
meta-fission pathway.20) Both pathways commonly use
phenol hydroxylase, a monoxygenase, as catalyst at first
step of degradation. However, they are different in the
second degradation step (Fig. 1). In general, yeasts and
bacteria utilize catechol-1,2-dioxygenase and 2,3-di-
oxygenase, corresponding to the ortho-fission and the
meta-fission pathway, respectively, to perform the
oxidative reaction of catechol.21,22) Several reports stated
that certain soil yeasts possess a great inductive capacity
for degradation of diverse aromatic compounds of small
molar mass,23,24) but, based on currently available
literature, we found that yeasts were less commonly
reported. We are interested in how to isolate a naturally
occurring yeast possessing a capacity to effectively
degrade phenol at higher concentration for use as a
prospective microorganism in waste water or treatment
of soil. In this work, we characterized the capacity of an
isolated strain, Candida albicans, to degrade not only
phenol but also formaldehyde, another toxic pollutant.
Although many microorganisms exhibit a catabolic
power on formaldehyde,25–28) a strain with effective
operation on both toxic ingredients has been rarely
reported. We discuss here the phenol catabolic pathway
of C. albican that we investigated, and also evaluated
the efficiency of C. albicans TL3 to treat waste water
from a factory producing phenolic resin.
Key words: phenol degradation; phenol hydroxylase;
catechol-1,2-dioxygenase; catechol; cis,cis-
muconic acid
Phenol and phenolic compounds, hazardous pollutants
in the environment throughout the last century, are
present in effluents from many industries including
petrochemical, coal-gasification, ceramic, pharmaceuti-
cal and dye manufacturing industries.1) Physical and
chemical methods to treat organic compounds require
many processing steps, which are costly and typically
produce also other toxic end products.2) Biodegradation
has been considered to be a highly effective method of
decontamination of a fouled environment. The remark-
able ability of microbes to decompose chemicals not
only is useful in pollution remediation but also serves as
Materials and Methods
Chemicals. Chemicals (Sigma, St. Louis, MO, U.S.A.,
or Merck, Darmstadt, Germany), Yeast Nitrogen Base
y
To whom correspondence should be addressed. Department of Applied Chemistry, National Chiao Tung University, 1001 Ta-Hseh Rd, Hsinchu,
Taiwan; Tel: +886-3-5731985; Fax: +886-3-5723764; E-mail: ykl@cc.nctu.edu.tw

Phenol Degradation by Candida albicans TL3
2359
OH
phenol hydroxylase
OH
OH
Meta-fission
Ortho-fission
catechol
catechol 2,3-dioxygenase
OH
catechol 1,2-dioxygenase
COOH
COOH
COOH
CHO
cis, cis-muconic acid
2-hydroxy muconic semialdehy
O
O
CH3
C
+
SCoA
CH3
CH3
C
+
H
COOH
O
C
COOH
COOH
Fig. 1. Two Common Phenol Degradation Pathways, the Ortho- and Meta-Fission, Occur in Microorganisms.
(YNB) without amino acids (Difco, Detroit, MI, U.S.A.),
a glucose oxidase dehydrogenase/phenol 4-aminoanti-
pyrine (GOD–PAP) assay kit (Randox, Antrim, UK)
were obtained from the indicated suppliers. All other
chemicals used were of pure and reagent grades.
same YNB medium with 15 mM phenol. Strains were
obtained after purification by sequential cultures. A
powerful phenol-degradation strain was identified as
Candida albicans by CBS (Centralbureau voor Schim-
melcultures, Boarn, Netherlands) based on its morphol-
ogy, physiological and biochemical characteristics. To
investigate a carbon source, we directly added a suitable
amount of phenol, glucose, or cis,cis-muconic acid to a
YNB culture medium (0.67%). All resulting media were
sterilized by ultra-filtration before use. Unless otherwise
stated, all flask cultures were incubated in a rotary
shaker (150 rpm) at 30 ꢀC.
Media formulation and microorganism screening.
The microorganism used in our work was isolated from
the soil sample of a petrochemical plant in Taiwan. Soil
sample was stirred for 2 h at room temperature in sterile
distilled water. 5 ml of the above sample was inoculated
in 150 ml of YNB medium (0.67% w/v) containing
various phenol concentrations (10, 12, 14, 16, and
18 mM). After 2 weeks of cultivation (150 rpm, 30 ꢀC),
0.1 ml of the resulting culture medium (OD600 of 1.65)
was subjected to spreading on agar plates containing the
Cell growth and Phenol degradation. The isolated
strain was cultured at 30 ꢀC in flask containing YNB
medium (50 ml, 0.67%) with phenol at various concen-

2360
S.-C. TSAI et al.
trations (0–24 mM) and/or glucose (0.2%, w/v). To
examine the prospective application of this microbe, we
used waste water obtained from a local manufacturer of
phenolic resin (Chang Chun Plastics Co., Ltd., Taiwan)
as a carbon source and added it directly to a YNB
culture medium (0.67%). We withdrew samples from
the cultures at various intervals, and estimated the cell
density by measurement of the absorbance of the sample
at 600 nm using a UV–Vis spectrophotometer (Shima-
dzu, UV-1601) and with a colorimetric method, using
4-aminoantipyrene to estimate the residual phenol in
the culture medium.29) The residual glucose and form-
aldehyde were determined by GOD–PAP assay and
Hantzsch reaction,30) respectively. All cultures were
cultivated with an initial cell density 0.02 (OD600) per
milliliter of medium in triplicate; the mean values are
reported, for which values of standard error were less
than 10%.
For measuring the kinetic parameters of the whole-
cell, different phenol concentrations (0.5–5 mM) were
prepared in 1 ml culture medium (50 mM Tris, pH 7.0)
containing the final cell density (stationary phase) of
OD600 of 1. The resulting mixture was incubated at
30 ꢀC with an agitation rate of 150 rpm. The residual
phenol concentration was monitored after the inocula-
tion of microorganism, with which the initial rate of
phenol degradation can be obtained. Data were analyzed
by non-linear regression (Grafit program) using the
Haldane’s equation31) shown as below.
crease at 375 nm),34) respectively. Control reactions
(without substrate or crude enzyme extract) were
performed for each assay. One unit of phenol hydroxyl-
ase is defined as the amount of enzyme that catalyzes the
disappearance of NADPH at 1 mmol minꢃ1. One unit of
catechol-1,2-dioxygenase is defined as the amount of
enzyme that catalyzes the formation of cis,cis-muconic
acid at 1 mmol minꢃ1. The concentration of protein was
determined with a protein assay kit (Bio-Rad) with
bovine serum albumin as standard; the specific activity
is defined in units per mg protein.
Product analysis and identification. To prepare the
metabolites, we added crude enzyme extract (described
in the previous section, 30 ml) to reaction mixture
(970 ml, 50-mM potassium phosphate buffer, pH 7.6,
containing 170-mM phenol, 1-mM ꢀ-mercaptoethanol,
0.1-mM EDTA, 10-mM FAD and 170-mM NADPH). The
resulting mixture was incubated for 10 or 25 min at
25 ꢀC. The reaction was terminated on addition of
methanol (1 ml). After removing the protein precipitant
by centrifugation, we analyzed the supernatant by
HPLC. The suspected product fractions were collected
and used for GC–mass analysis. For analysis by HPLC
(Lab Alliance series 4 pump, detector: GL Sciences UV-
620) we used a reversed phase column (Waters, u
Bondapak C18, 3:9 ꢄ 300 mm; mobile phase, methanol:
1% acetic acid = 80:20 v/v), at a flow rate 1 ml minꢃ1
,
and monitored the compounds of the supernatant at
260 nm. To prepare the enzymatic products of catechol-
1,2-dioxygenase, we added crude enzyme extract (10 ml)
to reaction mixture (990 ml, 50-mM Tris–HCl buffer,
pH 8.3, containing 5-mM ꢀ-mercaptoethanol, 20-mM
FeSO4, and 1-mM catechol) and incubated for 2 or
10 min at 25 ꢀC. The reaction was terminated on addition
of methanol (1 ml). The resulting protein precipitant was
removed by centrifugation. We analyzed the supernatant
by ion chromatography (IC), and collected the product
fractions for use in LC mass and LC tandem mass-
spectrometric analysis. We performed ion-chromato-
graphic analysis (DX-500 with Gp40 gradient pump) on
a column (DIONEX, Ion Pac Asll, 4 ꢄ 250 mm; mobile
2
v ¼ ½Vmax ꢁ ðSÞꢂ=½ðSÞ þ Ks þ ðSÞ =KIꢂ
Where v is the initial rate of degradation, (S) is the
phenol concentration, Ks is the half-saturation constant
(equivalent to Km in enzyme kinetics), KI is the
inhibition constant, and Vmax is the theoretical maximum
degradation rate.
Enzyme activity assays. A cell culture (50 ml) was
harvested from YNB medium (0.67%) containing
phenol at various concentrations when the growth of
C. albicans TL3 approached a stationary phase. After
centrifugation, a cell pellet were washed twice with
distilled water (2 ml) and then resuspended in the assay
buffer (0.5 ml, 50-mM potassium phosphate buffer,
pH 7.6, containing 1-mM ꢀ-mercaptoethanol, 0.1-mM
EDTA and 10-mM FAD). After disruption by sonication,
the cell debris was removed by centrifugation at 13700 g
for 30 min at 4 ꢀC. The supernatant (crude enzyme
extract) was used for assay of enzymatic activity,
metabolite preparation and determination of protein
concentration. Phenol hydroxylase (EC 1.14.13.7) ac-
tivity was assayed spectrophotometrically on monitoring
the disappearance of NADPH in absorbance at 340
nm.32) Catechol-1,2-dioxygenase (EC 1.14.13.1) and
catechol-2,3-dioxygenase (EC 1.13.11.2) activities were
assayed by determining the rate of accumulation of
cis,cis-muconic acid (absorbance increase at 260 nm)33)
and 2-hydroxymuconic semialdehyde (absorbance in-
phase, NaOH solution 0.01 M, flow rate 1.5 ml minꢃ1
)
and monitored the conductivity of compound with an
electrochemical detector (ED40) and an anion self-
regenerating suppressor set at 50 mA.
Mass-spectrometric analysis. For GC–MS (ion trap
detector, Perkin-Elmer Instruments, Turbo Mass Gold
Mass spectrometer, with a MDN-5 ms 30-mm column,
inner diameter 0.25 mm, film thickness 0.25 mm, MDN,
Supelco, PA, U.S.A.), the analytical conditions were
70 ꢀC for 2 min, 70–250 ꢀC at 20 ꢀC per min and 250 ꢀC
for 5 min. The temperatures of the injector, ion source
and transfer lines were 250 ꢀC, 200 ꢀC and 220 ꢀC,
respectively. The mass spectra were recorded on a time-
of-flight mass spectrometer (Q-TOF Micromass) in the
electrospray (ꢃ) mode. The mass analyzer was scanned

Phenol Degradation by Candida albicans TL3
2361
Table 1. Capability of Complete Degradation of Phenol by Various
Yeasts
TL3 in a liquid medium with phenol and/or glucose at
various concentrations. As shown in Fig. 2(a), increas-
ing the concentration (5–24 mM) of phenol in the culture
medium evidently produced a prolonged lag for C. al-
bicans TL3 to adapt to the growth environment and
consequently resulted in a delay to attain the stationary
phase of cell growth. The cell growth approached the
stationary phase within four days with the amended
substrates, except for the case of phenol (24 mM), for
which incubation for ten days was required. The
temporal course of substrate degradation [Fig. 2(b)]
correlated well with cell growth. At the stationary phase,
the biodegradation of phenol was nearly complete
(>99%). When glucose (0.2%) was used as the sole
nutrient, C. albicans TL3 grew rapidly with a log-phase
feature, whereas in a mixed substrate system (15-mM
phenol þ 0.2% glucose) the cell grew with a two-stage
feature, shown as Fig. 2(a). Glucose was consumed
rapidly in the first stage of cell growth, whereas, phenol,
serving as nutrient in the second growth stage, was
nearly unchanged in the first stage [Fig. 2(b)]. Although
C. albicans TL3 can use phenol as a source of energy,
the presence of phenol could inhibit cell growth in a
glucose medium [c.f. the growth curves of 0.2% glucose
with and without phenol in Fig. 2(a)]. Based on the rate
of substrate disappearance, we conclude that C. albicans
TL3 favors utilization of glucose over phenol when both
ingredients exist together. The effects of glucose on
phenol degradation were, however, dependent on the
inoculated microbial strains. For instance, the preference
for glucose metabolism of C. albicans TL3 was similar
to that of Trichosporon cutaneum sp. LE3,14) but
different from that of another strain of T. cutaneum38)
and C. maltosa.39) As discussed in the literature,40) the
presence of glucose and thereafter its metabolites might
inhibit C. albicans TL3 from the transport of phenol or
the synthesis of such a transport system. Most meso-
philic yeasts that are highly active in degrading phenol,
such as Candida tropicalis,15) Candida maltosa13) and
Trichosporon spp.14) are reported to degrade phenol (16–
18 mM) already within 5–6 days at 30 ꢀC, but C. albi-
cans TL3 needed only three days to consume phenol
(20 mM) at the same temperature [Fig. 2(b)]. Some
mesophilic bacteria with great activity to degrade
phenol, such as Burkholderia cepacia PW3 and Pseu-
domonas aeruginosa AT25) were reported to degrade
phenol (22 mM) within 11–15 days at 30 ꢀC, but
C. albicans TL3 required only four days at the same
temperature and concentration of phenol [Fig. 2(b)].
These results show that the rate of degradation of phenol
by C. albicans TL3 is clearly greater than that of other
microorganisms effective in degrading phenol.
Limit of phenol
degradation (mM)
Strain
Reference
Trichosporon cutaneum
Rhodotorula glutinis
< 5:3
5
17
35
Trichosporon dulcitum
Basidiomycetous yeast
Candida tropicalis
15
15
16
18
18
22
24
3
3
15
Trichosporon cutaneum sp. LE3
Candida maltosa
14
13
Candida tropicalis mutant
Candida albicans TL3
36
this work
over a ratio m=z of mass to charge 50–500 u, with a scan
step 2 s and an inter scan 0.1 s/step. The source block
temperature and desolvation temperatures were set at
80 ꢀC and 150 ꢀC, respectively. The rate of flow of the
delivery solvent (10% acetonitrile containing 0.1%
aqueous NH3) was 1 ml minꢃ1
.
Results and Discussion
Identification of the isolated strain and its tolerance
against phenol
Because of the industrial demand of phenol degrada-
tion, many microbes have been screened. Potentially
useful strains might be subjected to random mutagenesis
to improve the degrading power.6,36) Table 1 summa-
rizes the capability of various yeasts to degrade phenol
completely.3,13–15,17,35,36) Most native yeasts can tolerate
phenol at a concentration no greater than 18 mM. With
much effort in mutagenesis, Candida tropicalis was
improved to accept a phenol concentration up to
22 mM.36) Using phenol enrichment, we screened a few
microbial strains with varied ability to utilize phenol as a
carbon source. Among them, Candida albicans was
identified (further designated TL3) and evaluated as
having the greatest potential to degrade phenol. Without
further mutagenic treatment, Candida albicans TL3
tolerates phenol up to 24 mM (ꢅ2400 ppm). This record
is not only the greatest among yeasts but also greater
than most bacteria isolated in the environment, accord-
ing to current literature.3,6,15,37) The ability of C. albi-
cans to degrade phenol that we discovered is first
reported here.
In addition to phenol, potential metabolites such as
catechol and cis,cis-muconic acid were tested as a
source of carbon for C. albicans TL3. The results show
that catechol was effectively digested, but cis,cis-
muconic acid remained intact.
Cell growth and phenol degradation
The kinetic property of phenol degradation catalyzed
by C. albicans TL3 gave a Ks and a Vmax value of
1:7 ꢆ 0:1 mM and 0:66 ꢆ 0:02 mmol/min/mg of protein,
respectively. The Ks and Vmax values are higher than
those for several Pseudomonas strains5,31) and Burkhol-
deria cepacia.5) Phenol also exhibited a weak inhibition
To understand further the catabolic property of
C. albicans TL3, we investigated the microbial growth
with various carbon sources. Figure 2 shows the
temporal course of replicated biomass population and
the simultaneous phenol degradation by C. albicans

2362
S.-C. TSAI et al.
2
1.8
1.6
1.4
1.2
1
(a)
0.8
0.6
0.4
0.2
0
0
50
100
150
200
(b)
250
300
Time, h
30
0.25
25
20
15
10
5
0.2
0.15
0.1
0.05
0
0
0
50
100
150
Time, h
200
250
300
Fig. 2. Time-Course Profiles of Cell Growth (a) and Consumption of Phenol and Glucose (b) of Candida albicans TL3.
The cells were incubated at 30 ꢀC with YNB medium (0.67%) containing phenol at various concentrations: 5 mM (
); 10 mM (
); 15 mM
), and the residual
(
); 20 mM (
concentrations of phenol (
measurements. Data shown are the mean of triplicate experiments with standard deviation within 10%.
); 22 mM (
); 24 mM (
), 0.2% glucose (
), and the mixture of 15-mM phenol þ 0.2% glucose (
) and glucose (
) in the mixture medium. Each data point represents the mean of triplicate independent
with a KI value of 40 ꢆ 8 mM, which is much higher than
those of Pseudomonas strains5,31) and Burkholderia
cepacia ranging from 1–17.8 mM.5)
phase with phenol at various concentrations. The
specific activities of phenol hydroxylase and catechol-
1,2-dioxygenase of the crude enzyme extract were
examined and are summarized in Table 2. In general,
significant activities of phenol hydroxylase and cate-
chol-1,2-dioxygenase are detectable except the case of
using glucose (0.2%) alone as nutrient. The activity of
catechol-2,3-dioxygenase was not detected throughout
these tests. Although the strain required phenol as an
inducer to initiate the biosyntheses of phenol hydrox-
ylase and catechol-1,2-dioxygenase, phenol at a greater
concentration suppressed the biosynthesis of both
enzymes. For instance, phenol hydroxylase and cate-
chol-1,2-dioxygenase activity began to decline when
phenol was employed at concentrations >22 mM and
>10 mM, respectively. When the strain was grown in a
medium containing phenol (15 mM) and glucose (0.1%,
0.2% and 0.4%), the specific activities of phenol
hydroxylase were approximately 70, 34 and 17% of
that of the culture in medium with phenol as sole carbon
Effect of temperature on the growth of C. albicans
TL3
The growth of C. albicans TL3 in phenol (15 mM) at
25, 30, 35 and 40 ꢀC was monitored (data not shown).
After incubation (48 h), the cell cultures at 30 and 35 ꢀC
approached a stationary phase, whereas the cell density
of cultures at 25 and 40 ꢀC were only approximately
33% and 7% of that at 30 ꢀC (or 35 ꢀC), respectively.
The cell growth temperature was therefore controlled at
30 ꢀC for further enzymatic tests.
Characterization of the pathway of phenol degrada-
tion by C. albicans TL3
To assay the activity of phenol-degrading enzyme, we
prepared the crude enzyme extract by ultra-sonication of
the C. albicans TL3 cell cultivated to the stationary

Phenol Degradation by Candida albicans TL3
2363
Table 2. Comparison of Enzyme Specific Activity of Candida albicans TL3ꢇ
Specific activity (unit/mg protein)
Source of carbon
phenol hydroxylase
catechol-1,2-dioxygenase
glucose, 0.2%
phenol, 5 mM
0
0
0:025 ꢆ 0:0008
0:044 ꢆ 0:0021
0:083 ꢆ 0:0046
0:057 ꢆ 0:0027
0:028 ꢆ 0:0022
0:014 ꢆ 0:0018
0:115 ꢆ 0:0063
0:156 ꢆ 0:011
0:129 ꢆ 0:0045
0:270 ꢆ 0:01
0:734 ꢆ 0:037
0:217 ꢆ 0:019
0:174 ꢆ 0:008
0:149 ꢆ 0:012
0:128 ꢆ 0:007
0:107 ꢆ 0:009
0:101 ꢆ 0:005
0:087 ꢆ 0:006
phenol, 10 mM
phenol, 15 mM
phenol, 15 mM þ glucose, 0.1%
phenol, 15 mM þ glucose, 0.2%
phenol, 15 mM þ glucose, 0.4%
phenol, 20 mM
phenol, 22 mM
phenol, 24 mM
ꢇGrowth conditions: YNB medium (0.67%) containing phenol and/or glucose, at 30 ꢀC. Data are expressed as mean ꢆ standard deviation (n ¼ 3).
with NADPH, and with phenol, respectively. Discern-
ible in Fig. 3(d–f), an additional feature with a retention
period 3.1 min evolved when both NADPH and phenol
were added in the assay solution. This feature is
suggested to be catechol, according to spiking catechol
(0.02 mM) in the sample (chromatogram e in Fig. 3).
The area of the feature for a sample incubated 25 min
(chromatogram f) is about triple that of a sample
incubated for 10 min. To identify the enzymatic product,
we collected the suspected product fractions and used
them for GC–mass analysis. The result shows that a
major peak with retention period near 6.31 min in the
GC chromatogram; the corresponding mass analysis
yielded m=z 39, 53, 64, 81, 92, 110 (Fig. 4). This
fragmentation pattern is virtually identical to that of a
pure catechol standard. To analyze the catalytic activity
of 1,2-dihydrogenase, we treated the reaction mixture
containing catechol with the crude enzyme extract. The
resulting mixture was subjected to product analysis by
ion chromatography as described in the method section.
In Fig. 5, a new feature, as compared with both
background chromatograms of catechol and crude
enzyme sample, was observed at a retention period
2.2 min after the 2-min enzymatic catalysis of catechol.
This feature was enhanced when cis,cis-muconic acid
was spiked in the sample (chromatogram b) or when the
duration of reaction was increased to 10 min. (chroma-
togram c). The eluent corresponding to the new peak
area was collected and analyzed with electrospray
ionization LC mass spectrometry (LC/MS) in a neg-
ative-charge mode and tandem mass spectrometry (MS/
MS); the results are shown in Fig. 6(a) and (b). The
signal at m=z 163 is consistent with the molar mass of
monosodium muconate. The tandem mass analysis
exhibited the following signals with m=z 97, 137 and
163, which are identical to the fragment pattern of pure
cis,cis-monosodium muconate. Based on the enzymatic
product and activity analysis, we conclude that an ortho-
fission pathway is involved in the degradation of phenol
by this isolated strain.
Table 3. Effect of Temperature on Specific Enzyme Activity of
Candida albicans TL3ꢇ
specific activity (unit/mg protein)
Growth temperature
(ꢀC)
phenol hydroxylase catechol-1,2-dioxygenase
25
30
35
40
0:087 ꢆ 0:0041
0:083 ꢆ 0:0046
0:064 ꢆ 0:0037
0:061 ꢆ 0:002
0:275 ꢆ 0:012
0:217 ꢆ 0:019
0:158 ꢆ 0:007
0:100 ꢆ 0:003
ꢇThe microbe was grown with YNB medium (0.67%) containing phenol
(15 mM). Data are expressed as mean ꢆ standard deviation (n ¼ 3).
source, respectively. Similarly, the specific activities of
catechol-1,2-dioxygenase were 1.25, 1.46 and 1.70-fold
smaller in a phenol medium containing glucose at 0.1%,
0.2% and 0.4%, compared with that without glucose.
The biosynthetic paths of phenol hydroxylase and
catechol-1,2-dioxygenase in this microbe are hence
likely modulated by one or more additional regulatory
mechanisms such as catabolite repression.
The effect of temperature on enzyme induction was
also investigated. The activities of phenol hydroxylase
and catechol-1,2-dioxygenase of C. albicans TL3 culti-
vated at temperatures 25, 30, 35 and 40 ꢀC are summa-
rized in Table 3. A trend is observable that a lower
temperature seemed to be favorable to induce both
enzyme activities. The optimal growth temperature for
C. albicans TL3 in phenol was near 30–35 ꢀC (data not
shown), but both enzyme activities decreased inversely
with growth temperature. As the temperature factor on
the rate of cell growth and the induction of phenol
hydroxylase and catechol-1,2-dioxygenase were dis-
cordant, both enzymes were unlikely to play a major
role in rate of growth of C. albicans TL3.
To identify the phenol metabolic pathway in C. albi-
cans TL3, we isolated the metabolites of phenol and
analyzed them with a mass spectrometer. Figure 3
shows overlaid HPLC chromatograms with samples
having various compositions (such as NADPH and
phenol) and enzymatic product(s). The chromatograms
labeled a, b and c in Fig. 3 exhibited the analytical
outcomes of samples without both NADPH and phenol,
Although both catechol and cis,cis-muconic acid are
intermediates in the ortho-fission pathway of phenol
degradation, we found that C. albicans TL3 utilized

2364
S.-C. TSAI et al.
Fig. 3. HPLC Analysis of the Enzymatic Product of Degradation of Phenol Catalyzed by Crude Enzyme Extract.
Reactions were terminated at 10 or 25 min on addition of excess methanol. Samples taken from the mixtures were then centrifuged to remove
the precipitant before applying to a column. The resulting supernatants containing varied compositions were analyzed: (a) without addition of
NADPH and phenol; (b) with phenol; (c) with NADPH; (d) 10-min reaction with both NADPH and phenol; (e) with spiking 0.02-mM catechol in
the sample (d); (f) 25-min reaction with both NADPH and phenol. The extract medium of crude enzyme as described in the text.
Fig. 4. GC–Mass Analysis of the Enzymatic Product of Degradation of Phenol Catalyzed by Crude Enzyme Extract.
The sample containing catechol, the anticipated product of phenol hydroxylase-catalyzed reaction, was analyzed and confirmed by electron
ionization mass spectrometry (EI/MS). The feature at m=z 110 corresponds to the molecular ion of catechol (Mþ). All fragments shown are
consistent with those of standard catechol.
Fig. 5. Ion-Chromatographic Analysis of the Product(s) of Catechol Catalyzed by Crude Enzyme Extract.
Reactions were stopped at 2 or10 min on addition of excess methanol. Samples taken from the mixtures were then centrifuged to remove the
precipitant before applying to a column. The figure contains the overlay of chromatograms with sample from (a) 2-min reaction mixture; (b) 2-
min reaction mixture with spiking of 0.015 mM cis,cis-muconic acid; (c) 10-min reaction mixture.

Phenol Degradation by Candida albicans TL3
2365
Fig. 6. Electrospray Ionization Mass Analysis (ESI) of the Product of Catechol Catalyzed by Crude Enzyme Extract.
The sample containing monosodium muconic acid (mw.164), the anticipated product of catechol-1,2-dioxygenase-catalyzed reaction, was
analyzed by ESI/MS (a) and confirmed by ESI/MS/MS (b). The feature at m=z 163 corresponds to monosodium muconate (Mꢃ). The pattern of
fragments shown in (b) is identical to that of standard monosodium muconate.
Fig. 7. Growth of C. albicans TL3 (
Time.
) and the Remaining Phenol (mg/l) (
) and Formaldehyde (mg/l) (
) in Waste Water as a Function of
At the beginning, C. albicans TL3 culture (0.3 ml, OD600 ¼ 1:5) at a stationary phase was added to waste water (49.7 ml) containing phenol
(14.5 mM) and formaldehyde (0.44 mM). Each data point represents the mean of triplicate independent measurements. Data shown are the mean
of triplicate experiments with standard deviation within 10%.
only catechol as a nutrient, unlike Acinetobacter sp.
CNU96137) which can grow in the medium with either
intermediate as a carbon source. Perhaps C. albicans
TL3 lacks a transport system for cis,cis-muconic acid so
that no nutrient can be appropriately uptaken to support
cell growth.
phenolic resin as a carbon source. C. albicans TL3 grew
in the waste water diluted 5-fold. The cell growth and
the residual concentrations of phenol and formaldehyde
at various intervals are shown in Fig. 7. After incubation
for six days with C. albicans TL3 at 30 ꢀC, phenol and
formaldehyde were nearly completely degraded. The
degradation of formaldehyde and phenol correlated well
with the growth of Candida albicans TL3. Although
some strains were reported to function as degraded of
phenol and of trichloroethene,4,31,41) a microbe with a
biodegrading power toward phenol and formaldehyde
concurrently has been scarcely reported. Candida
Application to the treatment of industrial effluent
To evaluate the prospective application of C. albicans
TL3, we obtained a sample of waste water containing
formaldehyde (65 mg/l; i.e. 2.17 mM) and phenol (6750
mg/l; i.e. 71.8 mM) from a local factory producing

2366
S.-C. TSAI et al.
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Acknowledgments
The National Science Council of Taiwan and the
Center of Interdisciplinary Molecular Science of
National Chiao Tung University provided support of
this work. We thank Ms. Yuming Lee for assistance
with the mass-spectrometric analysis.
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