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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.
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 Collection (Rockville, MD), and the Penicillium spp.
azo bond could simplify the bioremediation of azo dyes were obtained from our laboratory collection.
since it would obviate the need for a two-step process
[15]. Chivukula et al. [18] reported that the white rot 2.2. Taxonomic determination of the new fungal isolate
basidiomycete fungi that can mineralize the wood com-
ponents, lignin, cellulose, and hemicellulose can also Morphological appearance and cultural characteris-
degrade azo dyes. Lignin-degrading basidiomycete fun- tics were determined with the use of Mycological Agar,
gus Phanerochaete chrysosporium is also capable of YM Agar, Sabourauds Agar, Wort Agar, Orange
mineralizing a number of nonsulfonated and sulfonated Serum Agar and Czapek Agar media for the prepara-
azo dyes to CO2 [9,12,19]. Dozens of fungal isolates tion of giant colonies and Henrici micro slides [29].
from soil and environments were found to be able to Details of structures were observed with the use of a
decolorize polymeric dye Poly R-478 to varying degrees light microscope. Carbon and nitrogen utilization was
[20 – 23]. Ollikka et al. [24] and Spadaro et al. [25] determined with the use of yeast nitrogen and carbon
demonstrated that the lignin peroxidase from P. base, washed spores, purified agar and the auxano-
chrysosporium plays an important role in azo dye degra- graphic plate technique [30]. Morphological and physi-
dation although some contradictory results were also ological growth characteristics of the new isolate were
reported [26,27]. The detailed biochemical pathways compared to current information on Penicillium [30,31].
underlying the fungal degradation of azo dyes are not
understood. 2.3. Culture media
In our study on the stimulation of plant secondary
metabolites by plant – microbial interactions, a All fungi were maintained and subcultured on potato
serendipitous discovery of a fungal contaminant that dextrose agar (PDA) slants or plates prior to inoculat-
completely decolorized the polymeric dye poly R-478 ing into liquid media. The liquid media used in this
was made. Polymeric dyes are considered to be good study were:
indicators of ligninolytic activity because dye decol- 1. HMS (half MS salts and hormone free medium), a
orization in 6i6o coincides with the onset of lignin revised medium based on Murashige and Skoog
metabolism in white rot fungal cultures [19]. In addi- (MS) medium [32]. It consisted of 217 g of MS salts,
tion, Field et al. [28] demonstrated that screening with 5 ml of Nitsch and Nitsch vitamins [33] and 15 g of
Poly R-478 was a useful tool in selecting promising sucrose per liter with pH 5·8;
polycyclic aromatic hydrocarbon (PAH)-degrading 2. SHK medium which was based on Schenk & Hilde-
fungi. Therefore, we isolated the fungus and found that brandt [34] and revised by Shetty & McKersie [35].
the new isolate was able to decolorize polymeric dyes It consisted of 3·2 g of SH salts, 10 ml Nitsch and
such as Poly R-478 and Poly S-119 with high efficiency. Nitsch vitamins, 1·74 g of K2SO4, and 30 g of
Poly R-478 is a polyanthraquinone dye, and Poly S-119 sucrose per liter with pH 5·8; the HMS and SHK
is an azo-chromophoric dye. These two polymeric dyes media were originally devised for plant tissue cul-
represent the majority of synthetic dyes. The main ture; and
objective of this study was to examine the ability of the 3. PDB (potato dextrose broth), a commonly used
fungal isolate to decolorize polymeric dyes in liquid commercial medium for fungal culture.
systems and to taxonomically identify the new isolate at
the genus level. 2.4. Incubation conditions
3. Results and discussion Fig. 2. Decolorization of Poly S-119 (0·01%) by Penicillium ATCC
74414 in liquid media SHK, HMS and PDB.
3.1. Decolorization of Poly R-478
3.2. Decolorization of Poly S-119
Strain ATCC 74414 was able to grow in HMS, SHK
and PDB media and the decolorization was observed in When polymeric dye Poly S-119 was added to HMS,
all three liquid systems. The decolorization patterns of SHK, or PDB medium, strain ATCC 74414 was able to
R-478 in three media, however, were quite different completely decolorize Poly S-119 in 3–4 days (Fig. 2).
(Fig. 1). In SHK medium, the decolorization occurred Unlike Poly R-478, Poly S-119 was completely decol-
at a very rapid rate in the first 2 – 3 days, but afterwards orized in SHK medium, and no subsequent release
the absorbance began to rise until it reached the initial from mycelial mass to the medium was observed. Mi-
level after 10 days of incubation (Fig. 1). We inter- croscopic observation clearly showed that the dye (Poly
preted that this initial decolorization was due to the S-119) was initially adsorbed onto the mycelia (Fig. 3)
adsorption of the dye compound to the growing myce- and as the culture aged it was degraded from the
lial mass, but the adsorption itself was not stable and mycelia without release into each of the media.
the dye compound was released from the surface of
mycelial cells as the fungus grew to a certain stage.
3.3. Decolorization test comparing other related fungi
Factors affecting adsorption/desorption of R-478 on
the mycelia of ATCC 74414 will be the subject of future
To compare the ability of decolorization by strain
study.
ATCC 74414 with other related fungi, we selected six
Surprisingly, the complete decolorization of Poly R-
fungal strains to test their ability of decolorization of
478 without release from the mycelia by the same
Poly R-478 in HMS medium. These fungi included
organism occurred in both HMS and PDB media,
Penicillium 6ermiculatum, Penicillium chrysogenum,
although the decolorization rates in these two media
Penicillium P-1, Trichoderma 6iride IF-26, Trichoderma
were slightly slower than that in SHK medium (Fig. 1).
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).
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 the recent report on ligninolytic Penicillium chryso-
genum [37] and the clear correlation of the ligninolytic
On Orange Serum Agar medium, the growth of activity of white rot fungi and the degradation of
ATCC 74414 occurred at 10°C, 20°C, 32°C and 37°C, polymeric dyes [19,24]. However, in our study, the
but not at 4°C. Penicillium chrysogenum strain that was tested did not
In order to test the carbon source utilization, aux- decolorize Poly R-478 and Poly S-119, suggesting that
anographic plates were prepared with washed spores, the ability to decolorize polymeric dyes may not be
yeast nitrogen base and purified agar. The growth common in all Penicillium species.
results showed ATCC 74414 was able to utilize a fairly
diverse spectrum of carbon sources. These included
dextrin, dextrose, fructose, galactose, inositol, sorbitol, 4. Conclusions
sucrose, xylose, melibiose, melezitose, b-galacturonic
acid, mannose, mannitol, maltose, esculin, cellobiose, A Penicillium strain ATCC 74414 capable of decol-
arabinose, arabitol and salicin. However, it did not orizing polymeric dyes was isolated. It was able to grow
utilize starch, inulin, dulcitol, adonitol or fucose. It was at 10–37°C but not at 4°C on Orange Serum Agar
able to utilize nitrate as the nitrogen source. medium.
The above decolorization and taxonomic studies sug- The dye decolorization patterns by ATCC 74414
gested that the strain ATCC 74414 could be a unique were different in different media. Strain ATCC 74414
Penicillium species. Decolorization of polymeric dyes by was able to completely decolorize polymeric dye Poly
the genus Penicillium may not be surprising in light of 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
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