Wat. Res. Vol. 33, No. 4, pp.

919±928, 1999
# 1999 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(98)00288-7 0043-1354/99/$ - see front matter

DECOLOURISATION OF COTTON BLEACHING

EFFLUENT WITH WOOD ROTTING FUNGUS
FU-MING ZHANG1, JEREMY S. KNAPP1* and KELVIN N. TAPLEY2
1
Department of Microbiology, University of Leeds, Leeds, LS2 9JT, U.K. and 2Department of Colour
Chemistry, University of Leeds, Leeds, LS2 9JT, U.K.

(First received January 1998; accepted in revised form July 1998)

AbstractÐThe decolourisation of cotton bleaching e‚uent by a wood rotting fungus was studied. It
was found that fungus No. 7 could remove more than 70% of the colour (initial A400=2.0±2.4) from
the e‚uent within 4 days under agitated conditions. The fungal mycelia could be reused for a prolonged
time and the decolourisation activity of mycelial pellets was quite stable during a long period of cool
storage. Many factors a€ecting the decolourisation process were studied, including: concentration of
glucose, e‚uent, NH+ 4 and Mn(II); initial pH; temperature. The activity of manganese peroxidase
(MnP) appeared to correlate well with the decolourisation rate. After the fungal treatment, an improve-
ment in the treatability of the e‚uent by other microorganisms was observed. # 1999 Elsevier Science
Ltd. All rights reserved

Key wordsÐbiodegradation, decolourisation, white rot fungi, e‚uent treatment

INTRODUCTION
Cotton is a very economically important vegetable
®bre, containing mainly cellulose. The noncellulosic
constituents have to be removed before the ®bre is
used. The purpose of bleaching is to destroy the
coloured material and to confer a pure white
appearance to the ®bres (Hickman, 1995). The cot-
ton is ®rst boiled in strongly alkaline solutions
made with sodium carbonate and sodium hydrox-
ide. This process, which removes the pectins con-
tained in the cotton, is followed by bleaching with a
dilute solution of chloride of lime or another chlor-
ine preparation and then treatment with dilute
acids. After each process, the treated materials are
rinsed with plenty of water (Fresenius et al., 1989).
The amount of water needed in cotton bleaching
plants is about 50±100 m3 water/tonne product. The
total amount of contaminants contained in cotton
bleaching wastes is about 197 kg per tonne of ma-
terial (Fresenius et al., 1989).
Cotton bleaching e‚uent is deep brown in colour
being similarly to the bleaching e‚uents from pulp
and paper industry. It is very dicult to remove the
colour from the e‚uents by conventional waste
water treatment systems. Recently, the potential use
of wood rotting fungi for the treatment of pulp mill
waste e‚uents has been reported (Eaton et al.,
1980; Archibald et al., 1990; Feijoo et al., 1995;
Perez et al., 1997). Phanerochaete chrysosporium
and Coriolus versicolor have been widely investi-
*Author to whom all correspondence should be addressed.
[Fax: +44-233-5638; E-mail: j.s.knapp@leeds.ac.uk].
gated for this purpose. However, there are very few
reports regarding the use of wood rotting fungi for
the treatment of cotton bleaching e‚uents. One rel-
evant study reported that cotton mill black liquor
was treated continuously in a bioreactor with C.
versicolor (Summerwill and Burns, 1993).
In this current paper results from the decolourisa-
tion of cotton bleaching e‚uent with an unidenti-
®ed wood rotting fungus (strain No. 7) is presented.
Preliminary studies were carried out to discover the
optimum conditions for the fungal decolourisation
processes in batch cultures. The activity of enzymes
likely to be involved in the decolourisation pro-
cesses was determined. Samples of cotton bleaching
e‚uent decolourised by fungi were further treated
with activated sludge to investigate if there was an
increase in their biodegradability by other microor-
ganisms following fungal treatment.
MATERIALS AND METHODS
Microorganism
The organism used in these studies was an unidenti®ed
wood rotting fungus, fungus 7, which was isolated from
fruiting bodies of a basidiomycete fungus collected in
woodland around Bolton Abbey (Yorkshire). Fungus 7
was maintained on plates of Sabouraud dextrose agar
(Oxoid, Basingstoke, Hants) and stored at 48C.
Cotton bleaching e‚uent
The e‚uent was kindly provided by the North West
Water Company (U.K.). It was dark brown in colour. The
UV-VIS absorption spectrum of cotton bleaching e‚uent
revealed no speci®c absorbance peak. The intensity of the
colour was found to be pH dependant. The brown colour
showed darker when the pH of the e‚uent was higher

919
920 Fu-ming Zhang et al.

Fig. 1. Decolourisation of cotton bleaching e‚uent with fungus 7 at di€erent initial pH values (top);
and changes of pH with time (bottom).

than 10. The colour changed very little within the range
pH 4.5±7. There was some precipitation produced in the
e‚uent when the pH value was reduced below 2. It was
also found that the changes in e‚uent colour with pH
were reversible. Some physical and chemical properties of
the e‚uent were established: COD = 13400 mg lÿ1; total
dissolved solid = 19500 mg lÿ1; pH = 13.5; concentration
of carbohydrate (as glucose) = 1200 mg lÿ1, absorbance at
400 nm (pH 5, 20 vol% e‚uent) = 2.2. Before being used
in decolourisation, the pH of the original cotton bleaching
e‚uent was adjusted to pH 5 with 1 M hydrochloric acid
and it was usually diluted.
Activated sludge
Activated sludge was obtained from a local e‚uent
treatment plant (Owlwood S.W., Allerton Bywater) which
treats an e‚uent, which is wholly domestic in character. It
was washed three times with sterile distilled water before
being used in the experiment.
Cultivation of fungal mycelial pellets
The mycelial inoculum was prepared by homogenising
fungal mycelium aseptically, using a sterilised Waring
blender (size: 25 ml, at full speed for 30 s). Erlenmeyer
 E‚uent decolourisation by wood rot fungi
¯asks (500 ml) containing 200 ml of malt extract media
(2% w vÿ1, malt extract) were inoculated with 2 ml of the
mycelial inoculum and incubated at 278C on a rotary sha-
ker (100 rev. minÿ1) for 3±4 days. The mycelial pellets were
harvested after cultivation in malt extract media and used
in most of the decolourisation tests.
Decolourisation
Mycelial pellets were separated from the initial growth
media and inoculated to Erlenmeyer ¯asks. Unless other-
wise indicated, nitrogen limited media (NLM) were used
in all decolourisation tests. NLM contained the following
(litreÿ1 of distilled water): glucose 5 g; MnCl2 3 mg;
FeSO4
7H2O 4 mg; MgSO4
7H2O 40 mg; with the solution
adjusted to pH about 5.0 with 1 M HCl. NLM combined
with cotton bleaching e‚uent was autoclaved at 1218C for
15 min. The decolourisation was carried out with agita-
tion, using the same shaking conditions as for the initial
mycelial growth described above.
The treatment of fungally-decolourised cotton bleaching
e‚uent with activated sludge
20% cotton bleaching e‚uent mixed with NLM (based
on 5 g lÿ1 glucose) had been previously decolourised with
mycelial pellets of fungus 7. In the process, about 80% of
the colour was removed. The decolourised e‚uents were
treated with activated sludge (10 ml of activated sludge in
100 ml medium). In a control experiment media containing
20% cotton bleaching e‚uent (without any fungal pre-
treatment) plus glucose (3 g lÿ1) were treated with acti-
vated sludge (10 ml activated sludge in 100 ml media)
under shaken culture conditions at 278C.
Assay of colour in cotton bleaching e‚uent
The colour of cotton bleaching e‚uent was measured
spectrophotometrically at 400 nm and presented as A400.
Samples were pretreated by centrifuge (Micro Centaur,
13,000 rpm for 5 min) to remove suspended particles.
Samples were diluted appropriately with distilled water.
Enzyme assays
Lignin peroxidase (LiP) assay (Tien and Kirk, 1988):
LiP activity was measured by determining the rate of oxi-
dation of veratryl alcohol to veratraldehyde. The unit of
LiP activity is de®ned as the amount of veratryl alcohol
oxidised (mmol minÿ1 mlÿ1) by an enzyme solution at room
temperature.
Manganese peroxidase (MnP) assay (Paszczynski et al.,
1988): guaiacol was used as substrate and its oxidation
gave rise to a chromophore with lmax at 465 nm (molar
extinction coecient = 12,100 M ÿ1 cmÿ1). The unit of
MnP activity is de®ned as the amount of guaiacol oxidised
(mmol minÿ1 mlÿ1 of enzyme solution) at room tempera-
ture. No activity was observed in controls lacking added
manganese sulphate.
Laccase assay (Bourbonnais and Paice, 1990): laccase
activity was determined by oxidation of 2-2'-azinobis-(3-
ethlyl benzothiazoline-6-sulphonate) (ABTS). No laccase
activity was detected in cultures of fungus 7.
Chemical assays
COD was determined by method No. 5220C of APHA
standard methods (closed re¯ux, titrimetric method)
(Greenberg et al., 1992). The proportion of the total COD
derived from cotton bleaching e‚uent was calculated by
subtracting the COD derived from added carbohydrate
(this was calculated on the basis of an experimentally de-
rived relationship between carbohydrate and COD) from
the total COD.
Concentration of total carbohydrate (as glucose) was
assayed spectrophotometrically by the phenol±sulphuric
acid method of Dubios et al. (1956).
921
Data points on graphs represent the mean and standard
deviation, where error bars cannot be seen replicates were
identical or errors were smaller than the data point.
RESULTS
The e€ect of temperature on decolourisation
Decolourisation of cotton bleaching e‚uent with
mycelia of fungus 7 was conducted at di€erent tem-
peratures (20, 27, 30 and 378C) with mycelial pellets
originally grown at 278C. Decolourisation activity
was high at 278C (76% colour removal in 4 days)
and was relatively lower at 20 and 308C (60 and
68% colour removal in 4 days, respectively). Very
little decolourisation occurred at 378C (21% colour
removal in 4 days). This suggests that the optimum
decolourisation temperature for fungus 7 is about
278C.
The e€ect of initial pH on decolourisation
A series of media di€ering in initial pH values
were tested. The results (Fig. 1) show that with cul-
tures starting at pH 7 or 8 little decolourisation
occurred in the ®rst two days, after which decolour-
isation proceeded rapidly with a simultaneous
decrease in pH to ca. 4.5±4.7 by day 5. On day 5
all cultures had a similar pH value, but the pH 5
culture had given 81% decolourisation whereas cul-
tures incubated at pH 7 or 8 only had about 65%
decolourisation. The optimum initial pH for deco-
lourisation was about 5 but decolourisation was
also very good when the solution was initially set at
pH 4.
The e€ect of cool storage time of mycelial pellets on
decolourisation activity
Mycelial pellets of fungus 7, which had been kept
in a cool room (about 48C) for 1, 3, 4 and 6 months
respectively, were used in decolourisation exper-
iments in comparison with fresh mycelia. The
results of the experiment demonstrated that the
decolourisation activities of mycelial pellets kept for
1, 3 and 4 months (68%±74% colour removal in
4 days) were as high as those of fresh mycelia (73%
colour removal in 4 days), although the decolourisa-
tion activity of 6 month old mycelia was markedly
lower (36% colour removal in 4 days).
The e€ect of glucose concentration on decolourisation
NLM with di€erent concentrations of glucose (in
the range 0 to 10 g lÿ1) were used for decolourisa-
tion of cotton bleaching e‚uent. Results (Fig. 2)
showed that in media with 1.0 g lÿ1 or below of
added glucose, there was not only no decolourisa-
tion but the colour was slightly increased. Rates of
decolourisation were similar at 2, 5 and 10 g lÿ1 of
glucose, however at 2 g lÿ1, decolourisation stopped
after 3 days. Therefore the most suitable concen-
tration of glucose tested for decolourisation of cot-
ton bleaching e‚uent was about 5 g lÿ1.
922 Fu-ming Zhang et al.

Fig. 2. E€ect of glucose concentration (co-substrate) on decolourisation of cotton bleaching e‚uent by

fungus 7.

The e€ect of Mn2+ concentration on decolourisation nitrogen on the decolourisation. The mycelial pellets
Di€erent amounts of MnCl2 were added to NLM were reused three times, each batch of mycelium
to show the e€ect of Mn2+ on the decolourisation. received the same concentration of NH+ 4 in each

The mycelial pellets were reused three times, each
set of mycelial pellets receiving the same concen-
tration of Mn2+ in each round. All the three
rounds of experiments showed that the decolourisa-
tion rates in the media of no added Mn2+ were
lower than that in the media with added Mn2+
(Table 1). The e€ect of Mn2+ concentrations (from
2 to 20 mg lÿ1 of MnCl2) on the decolourisation
was very similar. The absence of Mn2+ had more
e€ect on decolourisation potential of mycelium in
the later rounds of decolourisation. For example, in
the 3rd round of decolourisation the amount of col-
our removed in the absence of added Mn2+ was
only 44% of that removed at 2 mg lÿ1 and ca.
42.7% of that removed at 20 mg lÿ1. The strong
stimulation of colour removal by Mn2+ suggests
that manganese peroxidase may be involved in the
decolourisation of the e‚uent.
The e€ect of NH+
4 concentration on decolourisation
Di€erent amounts of ammonium chloride
(NH4Cl) were added to NLM to show the e€ect of
cycle of decolourisation and fresh glucose and
Mn2+ were added at each cycle. The results showed
that the decolourisation rate with media containing
no added NH4Cl was much higher than that of
media with added NH4Cl from the second round of
decolourisation (Table 2). It was obvious that the
addition of NH4Cl was deleterious to the decolouri-
sation and the sustained decolourisation only
occurred under nitrogen-limiting conditions.
E€ect of carbon sources as co-substrate on decolouri-
sation
In this experiment, a range of carbon sources
were tested for their ability to promote the deco-
lourisation of cotton bleaching e‚uent in compari-
son with glucose. The results showed (Table 3) that
starch, maltose and cellobiose were good carbon
sources for the decolourisation of cotton bleaching
e‚uent while sucrose, lactose, xylan, xylose, metha-
nol and glyoxal were poor carbon sources.

Table 1. The e€ect of Mn(II) concentration on the decolourisation of cotton bleaching e‚uenta

Concentration of MnCl2 added in NLM (mg lÿ1)

0 2 5 10 20

1st round colour removal (%), 6 days 78.9 87.1 88.2 88.2 87.1
2nd round colour removal (%), 5 days 54.9 74.6 78.9 79.3 79.3
3rd round colour removal (%), 6 days 33.7 76.3 78.4 77.9 78.9

a
The initial concentration of the e‚uent in NLM was equivalent to about 20% of the original cotton bleaching e‚uent.
 E‚uent decolourisation by wood rot fungi 923

Table 2. The e€ect of NH+

4 concentration on the decolourisation of cotton bleaching e‚uent
a

Concentration of NH4Cl added in NLM (g lÿ1)

0 0.25 0.5 1.0 2.0

1st round colour removal (%), 5 days 85.6 84.0 76.6 74.4 78.8
2nd round colour removal (%), 5 days 80.9 34.1 13.9 11.1 4.5
3rd round colour removal (%), 5 days 76.8 34.2 16.3 15.3 12.6

a
The initial concentration of the e‚uent in NLM was equivalent to about 20% of the original cotton bleaching e‚uent.

Decolourisation of cotton bleaching e‚uent at di€er-
ent concentrations
A series of decolourisation tests was carried out
with the media containing 20, 30, 40 and 50 vol%
of e‚uent. The results of these are shown in
Table 4. The 30 vol% concentration seemed to be
optimum for the decolourisation in terms of the
amount of colour removed, although it was not the
best for not the percentage colour removal. It was
interesting that the amount of colour removal in
5 days was almost identical at 30, 40 and 50 vol%
of e‚uent but was lower at 20 vol%. However, the
of carbon and nitrogen levels in the media on
enzyme production.
Lignin peroxidase (LiP). LiP was produced at
di€erent activities in all cultures with di€erent
media (Fig. 4). The activities of LiP in NLM with
added NH+ 4 were slightly higher than those in
other media. This suggested that LiP could be pro-
duced in cultures of fungus 7 in media with higher
levels of nitrogen.
Manganese Peroxidase (MnP) (Fig. 5). There
was almost no MnP activity detectable in NLM
with added NH+ 4 . MnP activities in normal NLM

percentage colour removal was greatest at 20 vol%
e‚uent.
COD changes during the decolourisation of cotton
bleaching e‚uent by fungus 7
The changes in COD, concentrations of carbo-
hydrate, pH and colour in the media were moni-
tored during the decolourisation of cotton
bleaching e‚uent (at about 20% strength) by fun-
gus 7 (Fig. 3). It was estimated that after decolouri-
sation, about 74% of colour and 41% of COD
derived from cotton bleaching e‚uent was removed
(colour: initial A400=1.9, in 4 days A400=0.5; the
estimated COD derived from the e‚uent was
initially 2900 mg lÿ1 and after 4 days it was
1700 mg lÿ1). Therefore, in the decolourisation
process the mycelia caused both colour removal
and COD decrease at the same time.
Activity of lignolytic enzymes produced by wood rot-
ting fungus 7 in nitrogen limited media (NLM)
For studies on activity of enzymes produced by
wood rotting fungi, mycelial pellets were inoculated
in NLM as used in the decolourisation experiment.
In order to assay enzyme activity easily, the NLM
used here was free of cotton bleaching e‚uent.
Three kinds of media including standard NLM, glu-
cose-free NLM and NLM with added NH+ 4 e‚uent it removed not only the residual carbo-
(1.0 g lÿ1 NH4Cl) were used to investigate the e€ect
were obviously higher than those in glucose-free
NLM. The results would seem to imply that high
levels of nitrogen in media could inhibit MnP pro-
duction and that glucose (a carbon source) was
necessary to maintain high MnP activity.
Laccase. No laccase activity was detected in any
culture of fungus 7.
Treatment of fungally decolourised cotton bleaching
e‚uent with activated sludge
When fungally decolourised e‚uents were treated
with activated sludge, the results (Fig. 6) showed
that COD could be removed much more quickly in
the media with added phosphate and ammonium
(1 g lÿ1 K2HPO4 and 0.5 g lÿ1 (NH4)2SO4 added)
than that in the control (the total percentage COD
removal in 42 h was 90% compared to 55%). The
stimulatory e€ects of phosphate and ammonia ad-
dition were not a surprise in view of the nutrient
de®ciency of the decolourisation medium. The col-
our of the fungally-treated e‚uent was almost
unchanged by the subsequent treatment with acti-
vated sludge, suggesting that activated sludge could
not remove colour from cotton bleaching e‚uent.
This was con®rmed by an experiment in which cot-
ton bleaching e‚uent was treated directly with acti-
vated sludge which showed a ca. 20% increase in
colour occurred over 5 days incubation. When acti-
vated sludge was used to treat fungally decolourised
hydrate (reducing its concentration from 1.75 to

Table 3. E€ect of di€erent carbon sources as co-substrate on decolourisation of cotton bleaching e‚uent (5 g lÿ1 carbon source added,
initial A400 of the media: 1.8±1.9)

Carbon source No carbon Glucose Xylose Cellobiose Xylan Starch Lactose Sucrose Maltose Methanol Glyoxal
(source control)

Colour removal in 5 days (%) 13.9 71.7 19.4 71.7 0 68.9 22.2 0 62.8 13.9 23.3
924 Fu-ming Zhang et al.

Table 4. The results of decolourisation at di€erent concentrations of cotton bleaching e‚uent

Concentration of e‚uent in NLM (%)

20 30 40 50

A400, 0 h 2.0 2.9 3.8 4.8

A400, 5 days 0.37 0.72 1.64 2.7
Colour removal in 5 days (%) 81.5 75.2 56.8 43.8
Decrease of absorbance in 5 days 1.63 2.18 2.16 2.1

Fig. 3. The COD, concentration of carbohydrate (top), pH and colour (bottom) changes during the
treatment of cotton bleaching e‚uent with fungus 7.
 E‚uent decolourisation by wood rot fungi 925

Fig. 4. Activities of LiP produced by mycelial pellets of fungus 7 in NLM, the activities were assayed
with whole culture samples.

0.17 g lÿ1) but also removed COD derived from the
cotton bleaching e‚uent. The COD derived from
the e‚uent was reduced from 850 to 70 mg lÿ1 (ca.
92% removal) in 42 h. In the control experiment,
COD due to the e‚uent was reduced from 1700 to
400 mg lÿ1 (76% reduction) in 5 days. The results
suggested that the fungally-decolourised cotton
bleaching e‚uent was much more easily mineralised
by activated sludge than e‚uent with no prior fun-
gal treatment.
DISCUSSION
Fungus 7 was e€ective in removal of colour from
cotton bleaching e‚uent and could also remove
some COD. In the only similar study that appears
to have been reported (Summerwill and Burns,
1993) a colour reduction of 502 10% was achieved
by C. versicolor, when continuously fed a 20 vol%
dilution of sterile cotton black liquor containing
1% (w vÿ1) glucose as co-substrate with a 3-day
retention time. No mention is made of COD
removal. Thus fungus 7 achieved slightly better col-
our removal, removed some COD and also needed
a lower glucose concentration.
The optimum decolourisation temperature for
fungus 7 was about 278C. Similar temperature
optima (around 28±308C) have been reported for
some other wood rotting fungi, such as C. versicolor
and Lentinus edodes, for decolourisation of pulp

Fig. 5. Activities of MnP produced by mycelial pellets of fungus 7 in NLM.

926 Fu-ming Zhang et al.
Fig. 6. The treatment of fungally-decolourised cotton bleaching e‚uent with activated sludge: change
in colour and COD.
bleaching e‚uents (Archibald et al., 1990; Mehna
et al., 1995). In comparison, most of the decolouri-
sations of pulp bleaching e‚uents with the more
thermotolerant P. chrysosporium have been con-
ducted at 37±398C (Eaton et al., 1980; Messner et
al., 1990; Feijoo et al., 1995).
The pH optimum (in the range 4±5) for decolour-
isation by fungus 7 was similar to those reported
for decolourisation of pulp and paper mill e‚uents
by other white rot fungi. For example Mittar et al.
(1992) suggested a pH range from 3.5 to 4.5 for P.
chrysosporium and Mehna et al. (1995) a range
from 4.5 to 5.0 for T. versicolor strain B7.
The decolourisation activity of mycelial pellets
was quite stable during a long period of cool sto-
rage. Good storage stability is an encouraging prop-
erty as if mycelial pellets are to be used as a
commercial biocatalyst in colour removal processes
the ability to store mycelia for later use will
improve the ¯exibility of the process.
The absolute requirement of fungus 7 for an
easily metabolisable nutrient, such as glucose or
sucrose, for fungal decolourisation is in line with
other published reports (Yin et al., 1989; Archibald
et al., 1990; Mehna et al., 1995) on a variety of
white rot fungi. In decolourisation of cotton bleach-
ing e‚uent with fungus 7, glucose, starch, maltose
and cellobiose are good carbon sources, while
sucrose, lactose, xylan, xylose, methanol and
glyoxal are poor carbon sources. The ability to use
starch was encouraging from a commercial view-
point. The failure to utilise xylan was surprising as
most wood rotting fungi tend to utilise hemicellu-
loses. Archibald et al. (1990) tested a variety of
simple and complex sugars to promote decolourisa-
tion by C. versicolor. It was found that the fungus
readily used nearly all, except some of the ``nonbio-
logical'' L-isomers.
Successful decolourisation of cotton bleaching
e‚uent by fungus 7 was dependent on the glucose
concentration. If the concentration was too low,
decolourisation did not occur (1 g lÿ1) or ceased
early and was incomplete (2 g lÿ1). Rates were simi-
lar at 5 or 10 g lÿ1. Similar results were reported in
a study on the role of glucose in fungal decolourisa-
tion of wood pulp bleaching e‚uents with P. chry-
sosporium (Yin et al., 1989). It was reported that a
critical amount (between 1.05 and 2.1 g lÿ1) of glu-
cose is needed to maintain the decolourisation ac-
tivity. Once the critical amount of glucose was
present in the reactor, the fungus had approxi-
mately the same activity.
The decolourisation rate with media containing
no added NH4Cl was much higher than that of
media with NH4Cl added from the second round
decolourisation. It appears that addition of NH4Cl
was harmful to the decolourisation, which only
occurred under nitrogen-limiting conditions. The
e€ects of ammonia are probably associated with the
e€ects of ®xed nitrogen on induction of lignolytic
enzymes (see below). The fact that the e€ect of
added NH4Cl was only noted in the 2nd and 3rd
cycles of decolourisation may be due to carry over
of preformed enzymes with the inoculum in the 1st
cycle. In contrast with these results, in a study on
decolourisation of pulp mill e‚uent with T. versico-
lor, Mehna et al. (1995) reported that decolourisa-
tion eciency increased with increase in ammonium
 E‚uent decolourisation by wood rot fungi
nitrate concentration and levelled o€ beyond
1.75 g lÿ1. As fungus 7 was clearly able to grow
during prolonged decolourisation experiments it
was presumed that it was able to obtain ®xed nitro-
gen from the cotton bleaching e‚uent.
The initial concentration of e‚uent a€ected the
decolourisation processes. The degradation rate
increased up to 30 vol% and then remained con-
stant or slightly declined, possibly due to inhibition.
In batch culture at 50% e‚uent concentration (in-
itial A400=4.8) only 43% decolourisation occurred.
However in other work (data not shown) higher
concentrations of e‚uent (initial A400=5 to 6) were
decolourised e€ectively in a continuous ¯uidised-
bed bioreactor. Using the same fungus, 70±80%
colour removal was achieved with a 4 day retention
time (Zhang, 1997).
The experiment on enzyme production by fungus
7 in NLM showed that glucose (carbon source) was
necessary to maintain high MnP activity and that
the addition of NH+ 4 could depress MnP pro-
duction but stimulate LiP production. On the other
hand, glucose (or another carbon source) was essen-
tial for the decolourisation processes, but the ad-
dition of NH+ 4 could markedly inhibit the
decolourisation. Comparison of the e€ects of NH+ 4 remove total organic chlorine in bleaching e‚uent.
and glucose levels in media on enzyme production
and their e€ects on e‚uent decolourisation leads to
some tentative conclusions about the role of par-
ticular enzymes in decolourisation by fungus 7. It
seems likely that MnP plays an important role in
the decolourisation of cotton bleaching e‚uent by
fungus 7, while there was no obvious role for LiP
in this decolourisation. Furthermore, the con-
clusions about MnP were also supported by the
results of experiments which showed that the ad-
dition of Mn2+ to NLM increased the rate of deco-
lourisation.
The participation of LiP and MnP in decolouris-
ing kraft bleach plant e‚uent with P. chrysosporium
was reported (Michel et al., 1991). Studies on the
precise role of ligninolytic enzymes in the fungal
decolourisation process with C. versicolor, using iso-
lated enzymes or the whole micro-organism have
been reported (Archibald, 1992; Bourbonnais and
Paice, 1992). Although LiP from P. chrysosporium
has been reported to bleach kraft pulp and to facili-
tate subsequent chemical bleaching (Arbeola et al.,
1992), it seems unlikely that lignin peroxidase was
involved in kraft pulp bleaching by C. versicolor,
since the enzyme was not detected during the
bleaching process (Archibald, 1992). In a paper
pulp e‚uent decolourisation process with C. versi-
color, Manzanares et al. (1995) reported that laccase
and MnP were released into the decolourisation
media. Recently, Perez et al. (1997) used
Phanerochaete ¯avido alba, in which LiP and MnP
were detected, for decolourisation of partially bio-
depurated paper mill waste waters. They found that
both LiP and MnP activities seemed to be impli-
927
cated in the decolourisation process, but LiP ac-
tivity appeared to play a more important role.
Nevertheless, many aspects of the mechanisms and
functions of the enzymes involved in fungal deco-
lourisation processes are still scarcely known.
The fungal decolourisation process could e-
ciently remove most of colour from cotton bleach-
ing e‚uent (by breaking down the chromophoric
molecules) but only eliminated a relatively small
part of COD from the e‚uent. Therefore, a two-
stage-treatment of fungal decolourisation, combined
with activated sludge, was proposed to solve the
problem. Firstly, media with cotton bleaching e‚u-
ent were decolourised with mycelial pellets of fun-
gus 7 giving about 80% colour removal. In the
second stage, the decolourised e‚uents were treated
with activated sludge to eliminate the residual COD
in the media. The results demonstrated that cotton
bleaching e‚uent decolourised by fungus 7 was
much more easily mineralised by activated sludge
than e‚uent with no prior fungal treatment,
suggesting an improvement in treatability of the
e‚uent by other microorganisms, following fungal
treatment. Similarly Yin et al. (1990) reported that
fungal treatment enhanced the ability of bacteria to
Bacteria removed nearly twice as much total or-
ganic chlorine from e‚uent given a fungal treat-
ment compared to untreated e‚uent. These results
suggest a combination of fungal and activated
sludge treatment processes may o€er the most e€ec-
tive means of purifying these recalcitrant e‚uents.
AcknowledgementsÐThe authors would like to acknowl-
edge the ®nancial support from North West Water
Company. FZ is grateful to the Committee of Vice-
Chancellors and Principals for an ORS award and the
University of Leeds for a Tetley and Lupton Scholarship.
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