RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 14, 905910 (2000)

Biotreatment of tannin-rich beer-factory

wastewater with white-rot basidiomycete
Coriolopsis gallica monitored by pyrolysis/gas
chromatography/mass spectrometry
Susana Yague1, Mara C. Terron1, Tania Gonzalez1, Ernesto Zapico1, Paola Bocchini2,
Guido C. Galletti2 and Aldo E. Gonzalez1*
1
Centro de Investigaciones Biologicas, CSIC, Velazquez 144, E-28006 Madrid, Spain
2
Analytical Group of Chromatography Mass Spectrometry, Department of Chemistry G. Ciamician, University of Bologna,
Via F. Selmi 2, I-40126 Bologna, Italy

Some fractions of beer-factory wastewaters represent an important environmental concern owing to their
high content of polyphenols and dark-brown color. The capacity of Coriolopsis gallica to preferentially
degrade lignin has been successfully applied in our laboratory to the biotreatment and decolorization of
paper-industry efuents. In this work, the ability of this white-rot fungus to degrade high-tannin-containing
wastewaters is evaluated. Under all the conditions studied, efuent decolorization and chemical oxygen
demand reduction achieved by C. gallica at day 12 of incubation were close to 50 and 65%, respectively. No
adhesion of dark color to the fungal mycelium was observed suggesting that decolorization could be
ascribed to C. gallica degradation systems. Mycelium dry-weight values showed that C. gallica is tolerant to
relatively high tannin content present in the efuent samples. In the sample containing the highest efuent
concentration (60% v/v), dry-weight values suggested an inhibition of fungal growth at day 6 of incubation
and a further adaptation of the fungus to the stressing tannin effect at day 12 of fungal treatment. Pyrolysis/
gas chromatography/mass spectrometry results showed a decrease of polyphenols pyrolysis products,
mainly phenol and guaiacol, with the incubation time. All these results indicate the potential use of C. gallica
in bioremediation of tannin-containing industrial wastewaters and in other applications where a reduction
in polyphenols content is required. Copyright # 2000 John Wiley & Sons, Ltd.
Received 18 February 2000; Revised 20 March 2000; Accepted 22 March 2000

Beer production is industrially carried out by yeast-
mediated fermentation using barley (Hordeum vulgare)
grains as raw material. Barley grains are first cooked to
allow amylase enzymes to act, then a yeast-mediated
fermentation takes place. After the sedimentation of the
yeast biomass, the fermented liquid is passed through
polyvinylpolypyrrolidone (PVPP) filters to remove poly-
phenols in order to obtain the final light beer. This last step
is necessary as phenolic compounds tend to polymerize,
causing colloidal haze, relegating them as undesirable
compounds. The industrial effluent formed and studied here
is the result of washing through the PVPP filters with NaOH
solution, which gives the dark-brown color together with a
high proportion of polyphenols (mainly tannins). These may
cause potential environmental pollution on discharge
together with the other fractions of the final effluent, to
the receiving waters. The biological oxygen demand,
chemical oxygen demand and suspended solids of the final
effluent are removed by conventional anaerobic-aerobic
treatments but the color is hardly removed. For this reason,
it is necessary to continue research into alternative
*Correspondence to: A. E. Gonzalez, Centro de Investigaciones
Biologicas, CSIC, Velazquez 144, E-28006 Madrid, Spain.
Contract/grant sponsor: FAIR Program (EU); Contract/grant number:
CT95-0805 (DG12-SSMA).
Contract/grant sponsor: CICYT; Contract/grant number: BIO96-1131-
CO2-01 and BIO97-0655.
biotechnological processes able to achieved effective color
removal from these effluents.
After lignins, tannins are the second most abundant group
of plant polyphenols. Vegetable tannins are usually divided
into two classes, namely hydrolyzable and condensed (non-
hydrolyzable) tannins. The hydrolyzable tannins are mainly
esters of glucose with hydroxyphenolic acids such as gallic
acid. The condensed tannins are polymers of flavonoid
precursors such as catechin. Polyphenols of cereals and
legumes are predominantly of flavonoid origin and they
consist of a series of polymeric phenols. In addition,
hydrolyzable tannins are apparently absent in these groups
of plants, since neither glucose nor other sugars are found
after hydrolysis.1
Tannins have been extensively studied as harmful or anti-
quality factors mainly in animal nutrition,2 but little is
known about their contaminant effects on the environment.
Tannins inhibit the growth of a number of microorganisms,
resist microbial attack and are recalcitrant to biodegrada-
tion.3 Their toxic effect is mainly due to enzyme inhibition,
action on membranes, and metal-ion and substrate depriva-
tion.4 These compounds have the ability to participate in
non-reversible reactions with proteins, due to the presence
of a large number of phenolic hydroxyl groups. Never-
theless, some fungi, yeasts and bacteria are quite resistant to
tannins and can also degrade them.5,6
White-rot fungi are gaining notable ecological interest

Copyright # 2000 John Wiley & Sons, Ltd.

906 BIOTREATMENT OF TANNIN-RICH BEER-FACTORY WASTEWATER
due to their great capability to degrade a broad diversity of
environmentally persistent xenobiotics and organopollu-
tants.79 The well-known ability of white-rot fungi to
completely mineralize lignin has been investigated for
potential use in upgrading animal feeds,10,11 pulp produc-
tion,12 environmental bioremediation13 and in the treatment
of effluents from the pulp and paper industry.1417 In
addition, these fungi seem to be among the most promising
organisms for detoxification of industrial effluents by a
laccase (E.C.1.10.3.1.) mediated degradation of low mol-
ecular weight phenolics.18
The white-rot fungus Coriolopsis gallica, in particular, is
the result of a previous screening performed in our
laboratory of more than ninety fungal species, in order to
achieve the maximum decolorization of a lignin-containing
paper-industry effluent.19 A laccase enzyme seems to be
involved in this process. Its coding gene has been cloned
and sequenced.20,21 Since the chemical structure of tannins
shows a series of common characteristics with lignin, and it
has been shown that tannins induce the secretion of laccase
by other basidiomycetous fungi such as Trametes hirsuta22
and Fomes annosun,23 the possible application of C. gallica
to the biological decontamination of beer-factory effluents
was considered interesting enough to be evaluated and the
results are presented in this work.
Pyrolysis/gas chromatography/mass spectrometry (Py/
GC/MS) has proved to be a suitable technique to analyze
lignin2426 and vegetable tannins of different origin.2729
Compared with other spectroscopic analytical techniques,
Py/GC/MS is relatively inexpensive, requires little sample
preparation, is easy to operate and overall allows chemical
characterization of the original polyphenolic sample.29 In
addition, pyrolysis simultaneously provides data for differ-
ent classes of compounds (e.g., sugars, phenolic derivatives,
proteins, etc.).
In the present work, Py/GC/MS was applied to the
chemical characterization of high-tannin-containing efflu-
ents from a Spanish beer-factory. Quantitative and qualita-
tive changes in the wastewater composition, occurring after
treatment with C. gallica, were analyzed in order to evaluate
the potential use of this fungus in the biotreatment of this
type of effluent.
EXPERIMENTAL
Efuent
The effluent was provided by a Spanish beer-factory in
Madrid. As mentioned before, it is generated from the
alkaline washings of the PVPP filters used to remove
polyphenols from beer and it represents only a fraction of
the final effluent. A preliminary characterization of this
tannin-containing effluent was carried out at our laboratory
by the determination of pH; color units at 465 nm according
to a procedure previously described,30 chemical oxygen
demand (COD),31 total phenols32 and total tannins.33
Culture conditions and biological treatment
The basidiomycete Coriolopsis gallica IJFM A-241 (In-
stituto Jaime Ferran de Microbiologa, CIB, CSIC, Spain)
was grown on agar plates with modified Czapeck medium34
for seven days at 28 C. Ten plugs (1 cm2) were cut and
inoculated under sterile conditions into culture flasks
containing 200 mL of the same growth medium and four
Rapid Commun. Mass Spectrom. 14, 905910 (2000)
1.5 cm diameter glass beads. After incubation for 24 h at
100 rpm and 28 C in an orbital shaker, an inoculum of 1:10
(v/v) was transferred into flasks containing 75 mL (total
volume) of the same growth medium already mixed with the
corresponding amount of effluent. The final concentration
of the effluent in the growth medium was 20, 40 or 60%
(v/v) in the samples labelled as V1, V2 and V3, respectively.
Effluent pH was adjusted to 5.7 before being added to the
growth medium, and both were autoclaved together at 0.5
atm for 20 min before inoculation.
All media were incubated for 0, 6 and 12 days under the
same conditions of temperature and agitation mentioned
before. Both color units and COD were monitored in the
controls (day 0 of incubation) and in the fungal-treated
samples (day 6 and 12 of incubation). After every
incubation time C. gallica mycelium was filtered and its
dry-weight was determined gravimetrically. All experi-
ments were run in duplicate.
It is important to note that all attempts to determine total
proteins and laccase activity in the extracellular medium
were unsuccessful due to the large interference caused by
the high color of the effluent samples.
Pyrolysis/gas chromatography/mass spectrometry
Sample preparation for Py/GC/MS and gravimetric analy-
sis. Twenty mL of control and fungal-treated efuent
samples were acidied at pH 1 using 6 M HCl. After 24 h
at 4 C the precipitated fraction was recovered by centrifu-
gation (11 950 g, 20 min) and freeze-dried prior to Py/GC/
MS. Dry-weight of the precipitated fraction was determined
in duplicate gravimetrically. In the case of biologically
treated samples, the fungal biomass was removed by
ltration, prior to acidication.
Py/GC/MS conditions
Samples of 1 mg were pyrolyzed at 600 C for 5 s using a
platinum CDS Pyroprobe 1000 heated filament pyrolyser
with a quartz sample holder (Chemical Data Systems,
Oxford, PA, USA), interfaced to a GC/MS system (interface
temperature 150 C), consisting of a Varian 3400 capillary
gas chromatograph (Varian Analytical Instruments, Walnut
Creek, USA) coupled via a transfer line to a Finnigan MAT
800 mass spectrometer ion trap detector (Finnigan MAT,
Palo Alto, CA, USA). The gas chromatograph (Supelco
SPB-5 column: 30 m  0.32 mm i.d.; 0.25 mm film thick-
ness; Supelco, Bellefonte, PA, USA) was programmed from
50 to 250 C at 5 C min1 holding the initial temperature
for 10 min. The injector and transfer line temperatures were
250 and 230 C, respectively. The carrier gas was helium
(1 mLmin1) and the split ratio was set at 1/120. The
electron impact mass spectra (1 scan s1) were obtained at
70 eV from m/z 40 to 350 u.35 The identification of the
pyrolysis products was based on mass spectral interpreta-
tion, and comparison with the National Bureau of
Standard's spectral library and with Py/GC/MS data
reported elsewhere.25 Quantification of samples are ex-
pressed as peak area percentages.
RESULTS AND DISCUSSION
Efuent characterization
The beer-factory effluent that we studied is a dark-brown
liquid, with a very high COD value. According to our
Copyright # 2000 John Wiley & Sons, Ltd.
 BIOTREATMENT OF TANNIN-RICH BEER-FACTORY WASTEWATER
Figure 1. Decolorization (a) COD decrease (b) and dry-weight of the
acid-precipitable fraction (c) in the effluent samples at different days of
treatment with Coriolopsis gallica.
preliminary analysis, it presents a basic pH (13.6),
71538  3525 color units and 29.88  2.3 ppm COD. Total
phenols and total tannins values determined in the effluent
were 1.50  0.05 and 1.08  0.06 g/L, respectively.
Efuent decolorization after C. gallica treatment
Decolorization percentage for the different samples at
different incubation times with C. gallica are shown in
Fig. 1(a). Although in all cases decolorization values at the
end of the incubation period were close to 50%, these were
very different at day 6 of the experiment. In the sample
containing 20% of effluent (V1) a color reduction of 40%
was already reached at day 6 of fungal treatment and being
comparatively lighter up to day 12. On the other hand, a
Copyright # 2000 John Wiley & Sons, Ltd.
907
minimal color decrease was observed in the first six days of
incubation in the sample containing 40% of effluent (V2),
achieving a 51% final color reduction from day 6 to 12.
The lowest final decolorization value (45.7%) was
attained for sample V3, containing the higher proportion
of effluent (60%). In addition, color units increased during
the first six days of fungal treatment giving rise to negative
decolorization values. This color increment may be due to
the pigment production by the fungus, as reported by
Bergbauer and Eggert36 for some white-rot fungi such as
Trametes versicolor. A color increase due to laccase activity
could not be disregarded under these experimental condi-
tions. Brenna and Bianchi37 have reported an increment in
color units in a catechin-containing sample treated with an
immobilized laccase. Moreover, Bollag and co-workers18
have explained fungal adaptation to toxic phenolic com-
pounds by means of the activity of a fungal laccase which is
able to mediate detoxification of pollutant phenolic
compounds catalyzing their polymerization with naturally
occurring phenols making the former less toxic for the cells.
Scalbert5 has also demonstrated that one of the mechanisms
used by microorganisms to detoxify tannins is the synthesis
of tannin-complexing polymers. The color increment that is
observed at day 6 of treatment in the sample containing the
highest proportion of tannins, V3, could be the result of
these polymerization reactions, supporting this statement.
The highest absolute decolorization values versus
cultivation time was achieved by C. gallica in the V3
sample during the last six days of culture varying from
negative values of decolorization (10.7%) at day 6 of
culture to 45.7% of color reduction at day 12. Since no
adhesion of dark color to the fungal mycelium was
observed, the decolorization results can be ascribed to a
fungal degradation mechanism.
Chemical oxygen demand after C. gallica treatment
The chemical oxygen demand results followed a similar
tendency to those of the color-units decrease. In the same
way, the final COD reduction observed in all samples was
close to 65% although the values at day 6 of the experiment
were very different in every case. In sample V1 a 48.8%
COD decrease took place in the first six days of fungal
incubation achieving only an additional smaller reduction in
the rest of the incubation time (Fig. 1(b)).
Most of the COD decrease in V2 was observed in the
second week of the fungal kinetic; although, in this case, an
important reduction (28.5%) was also attained in the first six
days of treatment. Similar trends to the decolorization
results, the highest COD reduction versus cultivation time,
was observed on sample V3 in the last six days of
biotreatment changing from 20 to 65%.
Changes in the acid-precipitable fraction by gravimetric
analysis
The decrease in the dry-weight values of the acid-
precipitable fraction showed a similar tendency to those of
color units and COD reduction results (Fig. 1(c)). The slight
increment in dry-weight values of the precipitated fraction
after the first week of the experiment in V3, compared with
the initial values corresponding to this sample, supports the
statement of a possible phenolic-compounds polymerization
phenomenon postulated above. Similarity for decolorization
and COD results, the maximum weight reduction of the
Rapid Commun. Mass Spectrom. 14, 905910 (2000)
908 BIOTREATMENT OF TANNIN-RICH BEER-FACTORY WASTEWATER

Table 1. Coriolopsis gallica growth, on different concentrations of

efuent, determined gravimetrically as dry-weight of
mycelium expressed in mg
Day
0 6 12
V1 (20% efuent) 0 417 660
V2 (40% efuent) 0 365 723
V3 (60% efuent) 0 158 845

precipitable fraction was also achieved from day 6 to day 12

of fungal treatment in sample V3.

Coriolopsis gallica growth

The variation in the mycelium dry-weight values along the
incubation time is shown in Table 1. A percentage of
industrial effluent of 20% (V1) and 40% (V2) did not seem
to affect the normal growth of C. gallica. On the contrary,
the mycelium dry-weight value, at day 6 of culture, is
notably lower in the sample containing 60% of industrial
effluent (V3) than in the samples V1 and V2, and a lag-time
in the growth can be observed (Table 1). The high
proportion of tannic or polyphenolic substances present in
V3 could explain this lag-time in the C. gallica growth. The
toxic effect of tannic compounds over the growth of several
microorganisms is well known.5,38 Surprisingly, as if certain
adaptation phenomenon take place, once this lag-time is
overcome the fungus enters an exponential growth phase
and the dry-weight values are even higher than in the other
two samples containing lower effluent proportion at day 12
of incubation.
Bearing in mind the above-mentioned lag-time of the
fungal growth in sample V3, it is possible to explain the
decolorization and COD results. Decolorization and COD
reduction can hardly take place if the growth of the fungus is
not optimal, as is the case in the sample V3 during the first
six days of incubation. Nevertheless, once the fungus adapts
to the stress substance, its growth starts and the decoloriza-
tion and COD reduction processes are not only possible but
also achieve the highest efficiency, as shown by the results
attained from day 6 to 12 of treatment in sample V3 (Figs
1(a) and 1(b)).
In our view, the high proportion of tannins present in
sample V3 could be toxic for C. gallica inhibiting its normal
growth during the first six days of culture. Fungal laccase
activity could be gradually secreted to the extracellular
medium and detoxify these compounds polymerizing them.
This is in agreement with the increase in color observed
during the first week of treatment, with the gravimetric
results of the acid-precipitable fraction (Figs 1(a) and 1(c)),
and with several reports claiming the detoxifying effects of
phenoloxidases via polymerization of phenols.5,8
Once these phenolic compounds have been modified and
detoxified, C. gallica can grow normally during the last
week of culture and the decolorization and COD removal of
the effluent can take place, as is the case during the last
week of fungal treatment in sample V3 (Figs 1(a) and 1(b)).
Strong support for all these statements comes from some
recent results obtained in our laboratory showing that the
production of laccase activity by C. gallica increased 20-
fold when 40% v/v of this effluent was added to a Kirk
culture medium (results not shown).
Figure 2. Pyrograms of samples, containing 40% of effluent, before (a)
and after twelve days of fungal treatment with C. gallica (b). For peak
designations see Table 2.
Pyrolysis results
Representative pyrograms of the control and biotreated
effluent samples are shown in Fig. 2. Peak numbers refer to
the compounds listed in Table 2, in which the relative peak
areas are also shown. The most volatile products, eluting up
to scan 200, were not considered since most of them
represent the smallest fragments obtainable by pyrolysis and
are not characteristic markers.
The major pyrolysis products in the samples were
identified as phenol (3) and 4-methylphenol (5). Other
main peaks were guaiacol (6), and the peak produced by the
coelution of 4-vinyphenol and catechol (9). Smaller peaks
corresponding to 4-ethylguaiacol (10) and 3-methylphenol
(4) were also detected. Phenol (3), 4-methylphenol (5),
guaiacol (6) and catechol (9) were observed as pyrolysis
products of several hybrids of Italian Sorghum grains which,
as Hordeum vulgare, belongs to the gramineae plant
family.27 As will be discussed later, these compounds have
been described as typical tannin pyrolysis products, all of
them as 4-vinylguaiacol (12) (also detected in a minor
proportion), have also been reported for lignin, straw and
wood pyrolysis.24,25,39 This is an expected result because
lignin and tannins share a series of common structural
polyphenolic features.40
Phenol (3), guaiacol (6), catechol and 2,6-dimethoxy-
phenol (13) have been reported as typical tannin pyrolysis
products, present in wine samples, that can be ascribed to
the presence of pelargoninidin-, peonidin-, cyanidin- and
malvidin-like moieties, respectively, in the original tannic
substance. On the other hand, the fission of the heterocyclic
ring of these tannins produces relatively smaller amounts of
4-methylphenol, 4-ethylphenol (7), 4-vinylphenol, 4-

Rapid Commun. Mass Spectrom. 14, 905910 (2000) Copyright # 2000 John Wiley & Sons, Ltd.
 BIOTREATMENT OF TANNIN-RICH BEER-FACTORY WASTEWATER 909

Table 2. Pyrolysis products, in control and fungal biotreated samples, of beer-factory efuent at three different concentrations
No. Name Scan MW Relative area*
V1 V1 V2
D0 D6 D12 D0 D6 D12 D0 D6 D12
1 Methylpyrrole 213 81 8.28 6.86 8.33 3.19 3.57 2.20 0.58 2.30 1.14
2 Styrene 292 104 4.25 7.93 7.28 1.69 1.56 3.07 2.23 1.59 2.05
3 Phenol 612 94 34.03 29.37 26.40 36.27 35.02 29.23 27.50 31.05 19.63
4 3-Methylphenol 866 108 3.56 3.02 4.84 2.94 2.80 3.37 1.39 3.31 3.00
5 4-Methylphenol 929 108 27.55 23.83 26.96 23.49 23.48 23.13 20.31 26.07 21.21
6 Guatacol 935 124 4.16 4.63 1.63 5.94 6.20 6.42 5.21 7.75 4.78
7 4-Ethylphenol 1154 122 2.73 3.61 4.38 4.08 4.11 4.21 3.25 2.97 4.29
8 4-Methylguaiacol 1188 138 1.25 1.53 0.53 2.53 2.69 2.31 3.84 4.29 6.06
9 4-Vinylphenol Catechol 120/110 230 7.74 12.02 9.59 13.66 14.85 16.06 24.91 13.89 23.83
10 4-Ethylguaiacol 1361 152 1.44 1.00 0.69 1.65 1.07 1.42 3.00 2.64 2.99
11 Indole 1391 117 2.69 4.59 7.86 1.64 2.20 5.48 1.46 2.35 3.51
12 4-Vinylguaiacol 1426 150 2.21 1.44 1.51 2.56 2.03 2.73 5.53 1.64 6.81
13 2,6-Dimethoxyphenol 1496 154 0.10 0.16 0.34 0.41 0.36 0.78 0.16 0.68
*
Relative areas. The differences from 100 correspond to unknown compounds.
Sample labelling: V1, V2 and V3 = final concentration of the effluent in the growth medium: 20, 40 and 60% (v/v), respectively. D0 = control; D6 and D12 = Days 6 and
12 of treatment, respectively.

methylguaiacol (8) and 4-methylcatechol.29 With the
exception of 4-methylcatechol, all of these compounds
were also found in our samples. In addition, one of the main
pyrolysis products observed, catechol, is a diagnostic
fragment in the pyrolysis of condensed tannins of natural
origin.27
The relative areas of some compounds, such as indole
(11), styrene (2) and methylpyrrole (1), showed a general
tendency to increase with the time of biotreatment with C.
gallica (Table 1). They have been reported as typical
pyrolysis products of amino acid moieties. In particular,
indole has been described as a pyrolysis product of
tryptophan41,42 and styrene of phenylalanine.42 The extra-
cellular enzymes produced by C. gallica could coprecipitate
with the tannins at the acid pH used to obtain the samples for
pyrolysis and may explain the increment of these amino acid
pyrolysis products. Compounds such as phenol, 4-methyl-
phenol, and 4-ethylphenol, that are diagnostic of tannins, are
also obtained in the pyrolysis of amino acids,41 so they can
also be pyrolysis products of the extracellular proteins
produced by the fungus and coprecipitate with tannins.
In all the samples, the relative area of phenol (3)
decreased along the biotreatment time; the only exception
was V3 at day 6 of culture that showed an increment that
may be regarded as the polymerization of tannins mentioned
before. Phenol reduction values were 22.4, 19.4 and 28.6%
at day 12 in samples V1 V2 and V3, respectively. In addition,
a decrease in the relative area values is also observed for
guaiacol (6) at day 12 of incubation in the samples V1
(60.8%) and V3 (8.2%), as for 4-methylguaiacol (8)
(57.6%), 4-ethylguaiacol (10) (52%) and 4-vinylguaiacol
(12) (31.6%) at day 12 in the sample V1.
It is important to note that no adhesion of dark color to the
fungal mycelium was observed. For this reason, the
reduction of tannin pyrolysis products could be ascribed to
the capacity of C. gallica to degrade them. The variations
produced in the relative areas of the pyrolysis products, on
the different samples and days of treatment, did not show a
uniform tendency; therefore, these are somehow indicative
of the occurrence of modifications in the structure of the
precipitated tannins, probably due to the enzymatic
degradation by the fungus.
The microbial degradation of tannins has been revised
recently. Despite the proven toxic effect that tannins
produce in some organisms,43 several bacterial species
and a number of filamentous fungi, especially those
belonging to the Aspergillus and Penicillium genus, have
demonstrated a capacity to grow on the surface of liquids of
tannery pits or tannery wastes44 and to efficiently degrade
tannins. There are several publications regarding the
capability of white-rot fungi to degrade condensed tannins.
Gamble and co-workers45 reported a reduction of 70 and
47% of the initial tannic content from leaves of a perennial
legume after treatment with Ceriporiopsis subvermispora
and Cyathus stercoreus, respectively, showing the ability of
these fungi to preferentially degrade condensed tannins and
their possible use in the treatment of forage crop to improve
its digestibility.
In the recent revision of Bhat and co-workers,43 the
capacity of Penicillium, Aspergillus, Trichoderma, Neuro-
spora, Fusarium and other filamentous fungi to degrade
tannins is ascribed to the enzyme tannase. On the contrary,
the enzymatic system responsible for the documented
ability of white-rot fungi to degrade condensed tannins is,
to the best of our knowledge, still unknown. From some
preliminary experiments carried out in our laboratory, it has
been suggested that C. gallica is not able to produce
tannase, but this basidiomycete was able to grow in Petri
dishes where tannic acid was supplied as the only carbon
source (results not shown). These results indicate that at
least one enzyme other than tannase must be produced in
order to metabolize tannic acid and uses as the only source
of carbon for the growth of C. gallica.
An enzyme preparation secreted by the white-rot
basidiomycete Trametes versicolor transformed catechol
with evidence of ring cleavage and the production of
hydroxy muconic semialdehyde.46 Lorusso and co-work-
ers47 reported a reduction of more than 80% in the initial
tannins content of canola meal using an enzyme preparation
from T. versicolor. In addition, Archambault and co-
workers48 have shown that an enzyme preparation of T.
versicolor transformed tannic acid and catechin. Some
authors have ascribed these transformations to a phenol-
oxidase activity.49,50 This fungus is indeed a known
producer of laccase activity. The enzymatic removal of
phenolic compounds from must and wine has been reported

Copyright # 2000 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 14, 905910 (2000)
910 BIOTREATMENT OF TANNIN-RICH BEER-FACTORY WASTEWATER
on the laboratory scale using immobilized laccase from a
mutant strain of T. versicolor.37
From the results presented here, it can be concluded that
C. gallica has shown to be an efficient basidiomycete in the
decolorization of a beer-industry effluent containing a high-
tannins proportion and in the reduction of its COD. In
addition, despite the well-known antimicrobial properties of
tannins, the results indicate that this microorganism is
tolerant to the relatively high-tannins content present in the
effluent samples that this fungus seems to be able to
detoxify. To the best of our knowledge, this is the first report
of a fungus of the genus Coriolopsis able to decrease the
concentration of tannic compounds in this type of effluents.
The potential of C. gallica to decrease the concentration
of tannins make this fungus interesting in biotechnological
applications such as biotreatment of tannins-containing
industrial wastewaters or to improve the utilization of
lignocellulosic materials by ruminants. Further research to
optimize the decolorization rates of tannin-containing
effluents by C. gallica under different culture conditions,
as well as improving our understanding of the biological
mechanism of tannin degradation in this fungus, is already
in progress in our laboratory.
Acknowledgements
The authors wish to thank the Mahou beer-industry (Spain) for the
facilities to carry out this work; to the FAIR Program (EU) CT95-
0805(DG12-SSMA) and CICYT (Spain) BIO96-1131-CO2-01 and
BIO97-0655 for financial support. An OPIS grant (Ministerio de
Educacion y Cultura, Spain) funded the work of S.Y. in Centro di
Studio per la Conservazione dei Foraggi (CNR Bologna) enabling the
performance of the Py/GC/MS analysis. Jose M. Carbajo is acknowl-
edged for the determination of total polyphenols and total tannins.
REFERENCES
1. Deshpande SS, Cheryan M, Salunkhe DK. Crit. Rev. Food Sci.
Nutr. 1986; 24: 401.
2. Windham WR, Petersen JC, Terrill TH. In Microbial and Plant
Opportunities to Improve Lignocellulose Utilization by Ruminants,
Akin DE, Ljungdahl LG, Wilson JR, Harris PJ (eds). Elsevier: New
York, 1990.
3. Field JA, Lettinga G. In Plant Polyphenols: Synthesis, Properties,
Signicance. Hemingway RW, Laks E. (eds). Plenum Press: New
York, 1992. 673692.
4. Red JD. J. Animal Sci. 1995; 73: 1516.
5. Scalbert A. Phytochemistry 1991; 30: 3875.
6. Saxena RK, Sharmila P, Singh VP. In Biotransformation:
Microbial Degradation of Health Risk Compounds, Singh VP
(ed). Elsevier Science B. V: Amsterdam, 1996; 259270.
7. Bumpus JA, Tien M, Wright D, Aust SD. Science 1985; 228: 1434.
8. Hammel KE. In Degradation of Environmental Pollutants by
microorganisms and their Metalloenzymes, vol. 28. Siegel H,
Siegel A (eds). Dekker: New York, 1982; 41.
9. Field JA, Jong E-de, Feijoo-Costa G, Bont JAM-de. Trends
Biotechnol. 1993; 11: 44.
10. Akin DE, Sethuraman A, Morrison WH III, Martin SA, Eriksson
K-EL. Appl. Environ. Microbiol. 1993; 59: 4274.
Rapid Commun. Mass Spectrom. 14, 905910 (2000)
11. Hatakka AI, Mohammadi OK, Lundell TK. Food. Biotechnol.
1989; 3: 45.
12. Messner K, Srebotnik E. FEMS Microbiol. Rev. 1994; 13: 351.
13. Aust SD. Microb. Ecol. 1990; 20: 197.
14. Archibald F, Paice MG, Jurasek L. Enzymol. Microbiol. Technol.
1990; 12: 846.
15. Terron MC, Martn C, Manzanares P, Galletti GC, Gonzalez AE.
Rapid Commun. Mass Spectrom. 1993; 7: 659.
16. Garg SK, Modi DR. Crit. Rev. Biotechnol. 1999; 19: 85.
17. Breen A, Singleton FL. Curr. Opin. Biotechnol. 1999; 10: 252.
18. Bollag JM, Shuttleworth KL, Anderson DH. Appl. Microbiol.
Biotechnol. 1988; 54: 3086.
19. Calvo AM. PhD Thesis, Alcala University, Madrid, Spain, 1995.
20. Calvo AM, Copa-Patino JL, Alonso O, Gonzalez AE. Arch.
Microbiol. 1998; 171: 31.
21. Zapico E. PhD Thesis, Alcala University, Madrid, Spain, 1999.
22. Arora DS, Sandhu DK. Folia Microbiol. (Prague) 1984; 29: 310.
23. Haars A, Chet I, Huttermann A. Eur. J. Forest Pathol. 1981; 11:
67.
24. Martn F, Saiz-Jimemez C, Gonzalez-Vila FJ. Holzforschung
1979; 33: 210.
25. Ralph J, Hateld RD. J. Agric. Food Chem. 1991; 39: 1426.
26. Terron MC, Fidalgo ML, Galletti GC, Gonzalez AE. J. Anal. Appl.
Pyrolysis 1995; 33: 61.
27. Galletti GC, Reeves JB. Org. Mass Spectrom. 1992; 27: 226.
28. Galletti GC, Antonelli A. Rapid Commun. Mass Spectrom. 1993;
7: 656.
29. Galletti GC, Modafferi V, Poiana M, Bocchini P. J. Agric. Food
Chem. 1995; 43: 1859.
30. CPPA. Technical Section Standard Method H5P. Canadian Pulp
and Paper Association: Montreal, Canada, 1974.
31. Clesceri LS, Greenberg AE, Trusel RR (eds). Standard Methods
for the Examination of Waste and Wastewater. (17th edn).
American Public Health Association: Washington, D C, 1989.
32. Cadaha E, Conde E, Fernandez de Simon B, Garca-Vallejo MC.
Holzforschung 1997; 51: 125.
33. Makkar HPS, Goodchild AV. In Quantication of Tannins: A
laboratory Manual. (2nd edn). International Center for Agricultural
Research in the Dry Areas: Aleppo, Syria. 1996; iv 25 pp.
34. Guillen F, Martnez AT, Martnez MJ. Appl. Microbiol. Biotech-
nol. 1990; 32: 465.
35. Terron MC, Fidalgo ML, Gonzalez AE, Almendros G, Galletti GC.
J. Anal. Appl. Pyrolysis 1993; 27: 57.
36. Bergbauer M, Eggert C. Can. J. Microbiol. 1993; 40: 192.
37. Brenna O, Bianchi E. Biotechnol. Lett. 1994; 16: 35.
38. Zhao J, Kwan HS. Appl. Environ. Microbiol. 1999; 65: 4908.
39. Terron MC, Fidalgo ML, Almendros G, Gonzalez AE. Rapid
Commun. Mass. Spectrom. 1996; 10: 413.
40. William F, Boominathan K, Vasudevan N, Gurujeyalakshmi G,
Mahadevan A. J. Sci. Ind. Res. 1986; 45: 232.
41. Chiavari G, Galletti GC. J. Anal. Appl. Pyrolysis 1992; 24: 123.
42. Stankiewicz BA, Hutchins JC, Thomson R, Briggs DEG, Evershed
RP. Rapid Commun. Mass Spectrom. 1997; 11: 1884.
43. Bhat TJ, Singh B, Sharma OP. Biodegradation 1998; 9: 343.
44. Rajakumar GS, Nandy SC. Appl. Environ. Microbiol. 1983; 46:
525.
45. Gamble GR, Akyn DE, Makkar HPS, Becker K. Appl. Environ.
Microbiol. 1996; 62: 3600.
46. Khan A, Overend R. FEMS Microbiol. Lett. 1990; 66: 215.
47. Lorusso L, Lacki K, Duvnjaj Z. Biotechnol. Lett. 1996; 18: 309.
48. Archambault J, Lacki K, Duvnjak Z. Biotechnol. Lett. 1996; 18:
771.
49. Lacki K, Duvnjak Z. Appl. Microbiol. Biotechnol. 1996; 45: 530.
50. Lacki K, Duvnjak Z. Biotechnology and Bioengineering. 1998; 57:
694.
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