Process Biochemistry 34 (1999) 31 – 37

Decolorization of polymeric dyes by a novel Penicillium isolate

Zuoxing Zheng a, Robert E. Levin a, Jennifer L. Pinkham b, Kalidas Shetty a,*
a
Department of Food Science, Uni6ersity of Massachusetts, Amherst, MA 01003, USA
b
Department of Biochemistry and Molecular Biology, Uni6ersity of Massachusetts, Amherst, MA 01003, USA

Received 24 November 1997; received in revised form 9 March 1998; accepted 2 April 1998

Abstract

A novel polymeric dye-degrading fungal strain ATCC 74414 was isolated. Taxonomic identification including morphological
and cultural characterization indicated that this isolate was a strain of Penicillium. Strain ATCC 74414 aerobically decolorized
both Poly R-478 and Poly S-119 in liquid media containing 0.01% of polymeric dyes. The decolorization rate was examined in
three distinct liquid media: Schenk and Hildebrandt-K2SO4 medium (SHK), potato dextrose broth (PDB), and half Murashige-
Skoog medium (HMS). Strain ATCC 74414 rapidly decolorized R-478 in SHK medium but the color was subsequently released
from the mycelial mass into the medium after 2–3 days, indicating that the decolorization in SHK medium could be due to
adsorption of Poly R-478 by the mycelia. In contrast, in HMS and PDB media ATCC 74414 decolorized Poly R-478 more
steadily, and the dye was initially adsorbed onto the mycelia and was subsequently decolorized without being released into the
medium. Strain ATCC 74414 also decolorized Poly S-119 steadily in SHK, HMS and PDB media. It appears that the
decolorization process involved initial mycelial adsorption of dye compounds, which was probably followed by biodegradation
through microbial metabolism, and the decolorization may be affected by medium constituents. Although aerobic decolorization
may not necessarily lead to complete mineralization of dyes, these results have suggested the potential of strain ATCC 74414 in
bioremediation of dye-contaminated water and soil. © 1999 Published by Elsevier Science Ltd. All rights reserved.

Keywords: decolorization; Penicillium; polymeric dyes

1. Introduction While many physical and chemical methods including

Synthetic dyes are extensively used in textile dyeing,
paper printing, color photography, pharmaceutical,
food, cosmetic, and other industries [1]. Approximately
10,000 different dyes and pigments are used industri-
ally, and over 0.7 million tons of synthetic dyes are
produced annually worldwide. It is estimated that 10–
15% of the dyes are lost in the effluent during such
dyeing processes [2]. Major classes of synthetic dyes
include azo, anthraquinone, and triarylmethane dyes,
and many of them are toxic or even carcinogenic
compounds with long turnover times [3]. Therefore, the
discharge of highly colored synthetic dye effluents from
those industries can result in serious environmental
pollution problems.
* To whom all correspondence should be addressed. Tel. 1-413-
545-1022; fax: 1-413-545-1262; e-mail: kalidas@foodsci.umass.edu.
adsorption, coagulation, precipitation, filtration, and
oxidation have been used for the treatment of dye-con-
taining effluents, adsorption is the most widely used
method at present due to its convenience and efficiency
[4,5]. The most commonly used adsorbent for color
removal is activated carbon, but it is relatively expen-
sive and the synthetic dyes are not degraded [6]. Biolog-
ical methods, being simple to use and low in cost, have
been the main focus in recent studies on dye biodegra-
dation [7–10]. Many synthetic dyes are generally recal-
citrant to bacterial degradation [11–13], but more
recent studies have demonstrated that many bacteria
are able to degrade azo dyes aerobically and anaerobi-
cally [7,14–17]. Bioremediation of azo dye-contami-
nated wastewater has been proposed as a two-step
process. The first step required is an anaerobic reduc-
tion of the azo bond, resulting in the production of two
aromatic amines. An aerobic digestion of the resulting
aromatic amines by a mixed bacterial population leads

0032-9592/99/$ - see front matter © 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 9 8 ) 0 0 0 6 1 - 2
32 Z. Zheng et al. / Process Biochemistry 34 (1999) 31–37
to their complete mineralization. Aerobic reduction of
azo bond could simplify the bioremediation of azo dyes
since it would obviate the need for a two-step process
[15]. Chivukula et al. [18] reported that the white rot
basidiomycete fungi that can mineralize the wood com-
ponents, lignin, cellulose, and hemicellulose can also
degrade azo dyes. Lignin-degrading basidiomycete fun-
gus Phanerochaete chrysosporium is also capable of
mineralizing a number of nonsulfonated and sulfonated
azo dyes to CO2 [9,12,19]. Dozens of fungal isolates
from soil and environments were found to be able to
decolorize polymeric dye Poly R-478 to varying degrees
[20 – 23]. Ollikka et al. [24] and Spadaro et al. [25]
demonstrated that the lignin peroxidase from P.
chrysosporium plays an important role in azo dye degra-
dation although some contradictory results were also
reported [26,27]. The detailed biochemical pathways
underlying the fungal degradation of azo dyes are not
understood.
In our study on the stimulation of plant secondary
metabolites by plant – microbial interactions, a
serendipitous discovery of a fungal contaminant that
completely decolorized the polymeric dye poly R-478
was made. Polymeric dyes are considered to be good
indicators of ligninolytic activity because dye decol-
orization in 6i6o coincides with the onset of lignin
metabolism in white rot fungal cultures [19]. In addi-
tion, Field et al. [28] demonstrated that screening with
Poly R-478 was a useful tool in selecting promising
polycyclic aromatic hydrocarbon (PAH)-degrading
fungi. Therefore, we isolated the fungus and found that
the new isolate was able to decolorize polymeric dyes
such as Poly R-478 and Poly S-119 with high efficiency.
Poly R-478 is a polyanthraquinone dye, and Poly S-119
is an azo-chromophoric dye. These two polymeric dyes
represent the majority of synthetic dyes. The main
objective of this study was to examine the ability of the
fungal isolate to decolorize polymeric dyes in liquid
systems and to taxonomically identify the new isolate at
the genus level.
2. Materials and methods
2.1. Microorganisms
A fungal strain that decolorized Poly R-478 was
isolated from a plant (anise-Pimpinella anisum) root
culture medium and it was designated as ATCC 74414
by American Type Culture Collection (Rockville, MD).
The other fungi used in this study for subsequent
comparisons included Trichoderma 6iride IF-26, Tricho-
derma harzianum ATCC 24274, Trichoderma pseu-
dokoningii ATCC 26801, Penicillium 6ermiculatum,
Penicillium chrysogenum, Penicillium P-1. All Tricho-
derma spp. were obtained from Ameriean Type Culture
Collection (Rockville, MD), and the Penicillium spp.
were obtained from our laboratory collection.
2.2. Taxonomic determination of the new fungal isolate
Morphological appearance and cultural characteris-
tics were determined with the use of Mycological Agar,
YM Agar, Sabourauds Agar, Wort Agar, Orange
Serum Agar and Czapek Agar media for the prepara-
tion of giant colonies and Henrici micro slides [29].
Details of structures were observed with the use of a
light microscope. Carbon and nitrogen utilization was
determined with the use of yeast nitrogen and carbon
base, washed spores, purified agar and the auxano-
graphic plate technique [30]. Morphological and physi-
ological growth characteristics of the new isolate were
compared to current information on Penicillium [30,31].
2.3. Culture media
All fungi were maintained and subcultured on potato
dextrose agar (PDA) slants or plates prior to inoculat-
ing into liquid media. The liquid media used in this
study were:
1. HMS (half MS salts and hormone free medium), a
revised medium based on Murashige and Skoog
(MS) medium [32]. It consisted of 217 g of MS salts,
5 ml of Nitsch and Nitsch vitamins [33] and 15 g of
sucrose per liter with pH 5·8;
2. SHK medium which was based on Schenk & Hilde-
brandt [34] and revised by Shetty & McKersie [35].
It consisted of 3·2 g of SH salts, 10 ml Nitsch and
Nitsch vitamins, 1·74 g of K2SO4, and 30 g of
sucrose per liter with pH 5·8; the HMS and SHK
media were originally devised for plant tissue cul-
ture; and
3. PDB (potato dextrose broth), a commonly used
commercial medium for fungal culture.
2.4. Incubation conditions
The PDA plates were incubated statically at 25°C for
one week before use. 125 ml Erlenmeyer flasks contain-
ing 50 ml of liquid medium were inoculated with 107
conidia spores and incubated at 25°C in aerobic condi-
tion on a shaker at a speed of 150 rpm for 3–12 days.
2.5. Decolorization assay
Poly R-478 (polyvinylamine sulfonated backbone
with anthrapyridone chromophore, violet color) and
Poly S-119 (polyvininylamine backbone with azo chro-
mophore, orange color) were purchased from Sigma
Chemical Co. The absorbance of polymeric dyes Poly
R-478 and Poly S-119 was measured spectrophotomet-
rically at 520 nm and 472 nm, respectively. Dyes were
 Z. Zheng et al. / Process Biochemistry 34 (1999) 31–37 33

added to the liquid medium as an aqueous solution to

a final concentration of 0·01% (w/v) for decolorization
study. After inoculation and at the indicated intervals
of incubation, 0·5 ml of the extracellular culture
medium was removed and diluted 10-fold with distilled
water before the absorbance was measured. Since in
most cases the mycelia were growing into ball-shaped
biomass in the liquid medium, it was easy to separate
the liquid from biomass without centrifugation or filtra-
tion. If the liquid sample was turbid, it was necessary to
centrifuge and filter it before measuring the absorbance.
The uninoculated medium was used as control while
the medium without dyes was used as blank.

3. Results and discussion
3.1. Decolorization of Poly R-478
Strain ATCC 74414 was able to grow in HMS, SHK
and PDB media and the decolorization was observed in
all three liquid systems. The decolorization patterns of
R-478 in three media, however, were quite different
(Fig. 1). In SHK medium, the decolorization occurred
at a very rapid rate in the first 2 – 3 days, but afterwards
the absorbance began to rise until it reached the initial
level after 10 days of incubation (Fig. 1). We inter-
preted that this initial decolorization was due to the
adsorption of the dye compound to the growing myce-
lial mass, but the adsorption itself was not stable and
the dye compound was released from the surface of
mycelial cells as the fungus grew to a certain stage.
Factors affecting adsorption/desorption of R-478 on
the mycelia of ATCC 74414 will be the subject of future
study.
Surprisingly, the complete decolorization of Poly R-
478 without release from the mycelia by the same
organism occurred in both HMS and PDB media,
although the decolorization rates in these two media
were slightly slower than that in SHK medium (Fig. 1).
Fig. 2. Decolorization of Poly S-119 (0·01%) by Penicillium ATCC
74414 in liquid media SHK, HMS and PDB.
3.2. Decolorization of Poly S-119
When polymeric dye Poly S-119 was added to HMS,
SHK, or PDB medium, strain ATCC 74414 was able to
completely decolorize Poly S-119 in 3–4 days (Fig. 2).
Unlike Poly R-478, Poly S-119 was completely decol-
orized in SHK medium, and no subsequent release
from mycelial mass to the medium was observed. Mi-
croscopic observation clearly showed that the dye (Poly
S-119) was initially adsorbed onto the mycelia (Fig. 3)
and as the culture aged it was degraded from the
mycelia without release into each of the media.
3.3. Decolorization test comparing other related fungi
To compare the ability of decolorization by strain
ATCC 74414 with other related fungi, we selected six
fungal strains to test their ability of decolorization of
Poly R-478 in HMS medium. These fungi included
Penicillium 6ermiculatum, Penicillium chrysogenum,
Penicillium P-1, Trichoderma 6iride IF-26, Trichoderma
harzianum ATCC 24274 and Trichoderma pseu-
dokoningii ATCC 26801. The Trichoderma species were
selected for comparison studies because of the ability of
some Trichoderma to degrade aromatic pollutants [36].
After 4 days of incubation, nearly complete decol-
orization of Poly R-478 was achieved by strain ATCC
74414 while none of the selected strains were able to
decolorize Poly R-478 (Fig. 4).

3.4. Morphological characterization

As has been outlined by Raper & Thom [31], the

Fig. 1. Decolorization of Poly R-478 (0·01%) by Penicillium ATCC
74414 in liquid media SHK, HMS and PDB.
taxonomy of the genus Penicillium is based primarily
on the cultural and morphological characteristics of
colonies grown on standardized agar media. The colony
formed by ATCC 74414 on a PDA medium in a petri
plate exhibited typical Penicillium characteristics. These
34 Z. Zheng et al. / Process Biochemistry 34 (1999) 31–37

included white woolly floccose mycelium turning to

blue –green on formation of conidiospores, velvety and
fairly compact texture, slightly spreading and wrinkled
habit [30]. Microscopic observations established struc-
tural characteristics typical of the genus Penicillium
including dual branching (Fig. 5a) of the conidiophore
(biverticillate), penicillus (Fig. 5b), and elongated sterig-
mata (Fig. 5c). Individual metula bore a cluster of 4–5
sterigmata, each leading to an elongated chain of conid-
iospores. All the above morphological characteristics
observed indicated that the strain ATCC 74414 is a
member of the genus Penicillium [30,31], but its species
designation was not made.

Fig. 4. Decolorization of Poly R-478 (0·01%) in HMS medium by

different organisms after 4 days of incubation. (1) the new Penicillium
isolate; (2) Penicillium 6ermiculatum; (3) Penicillium chrysogenum; (4)
a pigment-producing Pencillium strain P-1; (5) Trichoderma 6iride
IF-26; (6) Trichoderma pseudokoningii ATCC 26801; (7) Trichoderma
harzianum ATCC 24274. The negative results are due to pigments
produced by certain organisms.

3.5. Giant colony characteristics

Strain ATCC 74414 was incubated at 32°C for 8 days

following inoculation of a 1/4 inch diameter zone at the
center of petri plates with various classical fungal cul-
ture media. The characteristics of the giant colonies
formed on each plate (Fig. 6) with different media are
summarized in Table 1. The giant colony characteristics
further confirmed that the strain ATCC 74414 belongs
to the Penicillium genus.

Fig. 3. Decolorization of Poly S-119 by Penicillium ATCC 74414: (a)

decolorization of Poly S-119 (0·01%) in PDB medium on day 4; (b)
cross-section of the mycelial mass of Penicillium which was grown in Fig. 5. Morphological characteristics of Penicillium ATCC 74414: (a)
PDB medium containing 0·01% of Poly S-119 on day 3 (40 ×); and dual branching of the conidiophore (biverticillate) (920 ×); (b) peni-
(c) cross-section of mycelial mass on day 8 (40 × ). cillus structure (510 × ); and (c) sterigmata (1400×).
 Z. Zheng et al. / Process Biochemistry 34 (1999) 31–37 35

Fig. 6. Giant colony characteristics of Penicillium ATCC 74414: (a) YM Agar, (b) Wort Agar, (c) Mycological Agar, (d) Orange Serum Agar, (e)
Czapek Agar, and (f) Sabourauds Agar. Photographs on left are top conidiospore surfaces, and photographs on right correspond to reverse
surfaces (back sides) of the plates.

3.6. Growth parameters and C/N source utilization
On Orange Serum Agar medium, the growth of
ATCC 74414 occurred at 10°C, 20°C, 32°C and 37°C,
but not at 4°C.
In order to test the carbon source utilization, aux-
anographic plates were prepared with washed spores,
yeast nitrogen base and purified agar. The growth
results showed ATCC 74414 was able to utilize a fairly
diverse spectrum of carbon sources. These included
dextrin, dextrose, fructose, galactose, inositol, sorbitol,
sucrose, xylose, melibiose, melezitose, b-galacturonic
acid, mannose, mannitol, maltose, esculin, cellobiose,
arabinose, arabitol and salicin. However, it did not
utilize starch, inulin, dulcitol, adonitol or fucose. It was
able to utilize nitrate as the nitrogen source.
The above decolorization and taxonomic studies sug-
gested that the strain ATCC 74414 could be a unique
Penicillium species. Decolorization of polymeric dyes by
the genus Penicillium may not be surprising in light of
the recent report on ligninolytic Penicillium chryso-
genum [37] and the clear correlation of the ligninolytic
activity of white rot fungi and the degradation of
polymeric dyes [19,24]. However, in our study, the
Penicillium chrysogenum strain that was tested did not
decolorize Poly R-478 and Poly S-119, suggesting that
the ability to decolorize polymeric dyes may not be
common in all Penicillium species.
4. Conclusions
A Penicillium strain ATCC 74414 capable of decol-
orizing polymeric dyes was isolated. It was able to grow
at 10–37°C but not at 4°C on Orange Serum Agar
medium.
The dye decolorization patterns by ATCC 74414
were different in different media. Strain ATCC 74414
was able to completely decolorize polymeric dye Poly
R-478 (0·01%) in HMS and PDB media after 3–4 days
36 Z. Zheng et al. / Process Biochemistry 34 (1999) 31–37

Table 1
Characteristics of giant colony of strain ATCC 74414

Medium Characteristics

YM Agar 80 mm diameter zone of growth;

45 mm diameter central mouse grey zone of conidiospores;
5 mm white peripheral margin of mycelium;
5 mm blue–grey ring of conidiospores just internal to white outer margin of mycelium;
Rear of colony: pale yellow and smooth
Wort Agar Smooth grey and velvet conidial growth;
Hexagonal shape of colony with parallel sides 49 mm distant;
Central zone of 20 mm diameter dark grey;
Rear of colony: pale yellow with six deep wrinkles
Mycological Agar 75 mm diameter zone of growth;
30 mm diameter central dark grey zone of conidiospores;
7 mm white peripheral margin of mycelium;
Mouse grey color of conidiospores;
Rear of colony: pale yellow and smooth
Orange Serum Agar 74 mm diameter zone of growth;
Light grey color of conidiospores;
35 mm diameter central darker zone with concentric rings;
Mycelial mat of radiating wrinkles;
Rear of plate: yellow/buff with 52 spoked wrinkles
Czapek Agar 48 mm diameter zone of growth;
Green (olive drab) color of conidiospores;
Outer margin of 2 mm white mycelial mat of radiating wrinkles;
Rear of colony: light buff and wrinkled
Sabourauds Agar Blue-grey conidial zone beyond central diameter of 45 mm;
Exudate droplets in center zone of 40 mm diameter;
66 mm blue–grey zone and 6 mm white outer peripheral zone of mycelium;
Rear of colony: light tan and smooth

of aerobic incubation at 30°C, while decolorization
occurred in the first 2 – 3 days but subsequent release of
Poly R-478 from mycelia into medium occurred in
SHK medium. The isolate was able to completely de-
colorize Poly S-119 (0·01%) in HMS, PDB and SHK
media after aerobic incubation at 30°C for 4 days.
The decolorization of Poly S-119 by strain ATCC
74414 involved adsorption of the dye compound by
fungal mycelia at the initial stage, followed by
biodegradation through microbial metabolism. Further
studies are required to understand the decolorization
mechanisms including adsorption/desorption kinetics
and mineralization process of polymeric dyes by strain
ATCC 74414.
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