ELSEVIER

Biodegradation of
triphenylmethane dyes
Wamik Azmi, Rajesh Kumar Sani, and Uttam Chand Banerjee
Biochemical EFlgineeriFlg Research and Process Developmment Center, Institute of Microbial
Technology, Chandigarh, India
Biodegradation of triphenylmethane dyes by bacteria, actinomycetes, yeasts, and fungi are discussed in detail.
The disadvantages of physical and chemical treatment processes of dye wastewater are also discussed. Biological
treatment processes have many advantages over the chemical and physical treatment processes such as
possibilih/ of degradation qf dye molecules to carbon dioxide and water and formation of less sludge in addition
to being environmently friendly. This group of dyes is toxic depending on the concentration used. Toxicity of
triphenylmethane dyes is discussed with respect to different organisms. Some aspects of biodegradativr products
of this group qf dyes are also mentioned. 0 1998 Elsevier Science I nc.
Keywords: Biodegradation: decolorization; triphenylmethane dyes; bacteria; actinomycetes; yeast; fungi
Introduction
Triphenylmethane dyes are used extensively in textile
industries for dying nylon, polyacrylonitrile modified nylon,
wool, silk, and cotton. Some of the triphenylmethane dyes
are used as medicine and biological stains. Paper and leather
industries are also major consumers of triphenylmethane
dyes. This group of dyes are also used for coloring plastics,
gasoline, varnish, fats, oil, and waxes. Food and cosmetic
industries also use different types of triphenylmethane dyes.
Dyes are recalcitrant molecules difficult to degrade biolog-
ically. From the treatment point of view, the degradation of
dyes has received considerable attention. Conventional
wastewater treatement processes are suitable for stabiliza-
tion of nonxenobiotic compounds whereas these processes
do not work well with the xenobiotic compounds. Environ-
mental regulations in most of the countries now have made
it mandatory to decolorize the dye wastewater prior to
discharge. Increasing concerns about color in the effluents
are leading to the worldwide efforts to develop more
effective color removal processes. The characteristics of dye
house wastewater is given in Table 1. A large number of
methods are available to treat the wastewater containing
dyes (Table 2). Presently, most of the processes used for the
treatment of dye wastewater are chemical processes which
are costly, produce large amount of sludge, and are less
Address reprint requests to Dr. U. C. Banerjee, Institute of Microbial
Technology. Sector 39A. Chandigarh 160036, India
Received 25 July 1996: revised 22 July 1997; accepted 22 July 1997
efficient. Nowadays, biological processes are getting more
and more attention since it is cost effective, environmently
friendly, and does not produce large quantities of sludge.
The decolorization may be defined as the removal of color
only possibly by changing the chromophoric group to a
nonchromophoric one while biodegradation is the break-
down of the substrate (dye) molecule by the biological
process. Nelson and Hites in 1980 reported the deposition
of Crystal Violet and Malachite Green in the sediment and
water of Buffalo river in New York (U.S.) They found
aniline dyes in the aquatic environment. From a study of
Black et aL3 in 1980, it was shown that aniline dyes could
be mutagenic and carcinogenic to biota. These chemicals
have been suggested to be responsible for the promotion of
tumor growth in several bottom-feeding species of fish.4
Triphenylmethane dyes are some of the most widely
used dermatological agents. Gentian Violet has been used in
oral consumption for the treatment of pinworms and in
topical applications in humans and domestic animals; it has
been shown to be effective in controlling fungal growth
under varying conditions.5 Gentian Violet has also been
added to poultry feed to control fungus, thus exposing the
human population directly or indirectly to Gentian Violet
through its extensive medicinal and commercial use. The
cytogenic toxicity of Gentian Violet in Chinese hamster
CHO cells in vitro has been studied. It was stated that this
compound is a mitotic poison as well as a clastogen in vitro.
Its clastogenic properties were confirmed in five other
different mammalian cell types. Unless in viva studies prove
otherwise. Gentian Violet and Crystal Violet may be re-
garded as biohazardous substances.
Enzyme and Microbial Technology 22:185-191, 1998
0 1998 Elsevier Science Inc. All rights reserved.
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Table 1 Characteristics of dye house wastewater
Characteristics
Maximum
(mg I-)
Minimum
(r-w I-)
Average
(mg I-)
PH
11 7.3
Alkalinity 3,160 340 1,375
Total solids 7,130 700 3,030
TDS 6,180 680 2,720
ss 950 20 310
BOD 850 20 220
COD 1,910 55 850
This review reports the microbial decolorization and
degradation of triphenylmethane dyes. Toxicity of these
dyes and their biodegradation products are discussed in
detail.
Treatment of triphenylmethane dyes
Chemical processes are extensively used for the decolori-
zation of dyes by different dye manufacturing and dying
industries. Matsui et ~1.~ reported the ozonization of Mala-
chite Green, a model compound of triphenylmethane dye.
Reactions of dyes with ozone were studied from the
treatment point of view. Such reactions are looked at in
terms of reduction in the chemical oxygen demand (COD)
and the decolorization; however, the ozonization mecha-
nism has scarcely been touched. Porter and Spears in 1970
reported the photofading of triphenylmethane dyes. The
photoreaction of Malachite Green in water was reported to
give 4-dimethylarnino benzophenone, the carbinol base of
Malachite Green and p-dimethylamino phenol. The photo-
reaction of Crystal Violet has been reported to give p-
dimethylamino phenol, 4-4-bis dimethylamino benzophe-
none, the leuco and demethylated derivative of Crystal
Violet.8.9 Textile and dy - e stuff industrial wastes having
different dyes are generally treated by physicochemical
methods. These methods include adsorption, chemical pre-
cipitation and flocculation, oxidation by chlorine, hydrogen
peroxide and ozone, electrolysis, reduction, electrochemical
treatment, and ion-pair extraction, but none have been found
to be very suitable as they produce a large quantity of
sludge. All these methods possess significant differences in
color removal results, volume capability, operating speeds,
and capital costs. For example, activated charcoal is
extremely effective for removing color, but is capable of
treating small effluent volume, operates at slow speeds, and
has high capital costs. Membrane technology, ozone treat-
ment, and coagulation and flocculation permit good color
removal in large effluent volumes. Membrane technology is
fast, but the capital cost for implementing this technology is
high. Ozone treatment operates at moderate speeds and also
requires high capital investment. Coagulation and floccula-
tion techniques operate moderate to fast speeds and require
a somewhat lower capital investment, but the biological
methods have many advantages such as the possibility of
degradation of dye molecules to carbon dioxide and water
and the formation of less sludge. Also it is environment
friendly. Ogawa et al. in 1981 reported the use of an
activated sludge system for the treatment of dye wastes. Dye
industries use the activated sludge processes to treat efflu-
ent; however, some dyes are toxic to the microbes and
lessen their purifying function. The acclimatized microbes
showed negative inhibition at a low dye concentration.
Negative inhibition was tested by inoculating the triphenyl-
methane dyes (Methyl Violet) with the acclimatized mi-
crobes in the medium. They have also reported the elimi-
nation of Methyl Violet and New Fuchsin up to 90-100%
by continuous culture of activated sludge.
Idaka et al. I2 in 1985 examined the fate of Congo Red,
Orange II, and Crystal Violet in activated sludges which
were previously acclimatized with the medium and dyes.
They also studied the effect of dye concentration on
acclimatized sludges. They observed that with the increase
in dye concentration, the color loss was decreased. Ogawa
et al. I3 reported the inhibition of growth and respiration of
activated sludges of various dyes and also examined their
inhibitory characteristics. These tests were performed with
unacclimatized sludges. The authors also used sludges
acclimatized to different triphenylmethane dyes which had
adaptability to different concentrations of dyes. With other
dyes, they used Methyl Violet and Crystal Violet and
observed the respiratory inhibition by these dyes. It was
observed that the inhibition level of the unacclimatized
microbes by Methyl Violet increased with the dye concen-
tration. In comparison to the other dyes, Methyl Violet
showed greater inhibitive action. The acclimatized microbes
showed negative inhibition at a low dye concentration and a
positive inhibition at a high concentration. The inhibition of
microbial growth by certain basic dyes was reported by
Ogawa et al. They used Bacillus subtilis as the test
organism and observed that both the mean growth rate of
the cell population at the logarithmic phase and the cell
concentration at the stationary phase decreased with the
addition of dyes. It is also reported that triphenylmethane
dyes (Crystal Violet and Methyl Violet) strongly inhibited
cell growth. The elucidation of inhibition mechanism by the
dyes on chemical composition of cells was needed. For this
purpose, they determined the nucleic acid content of the
cells harvested both at the logarithmic and stationary
phases. They found that the RNA content decreased with
increasing concentration of Methyl Violet; this tendency
was more remarkable in the logarithmic than stationary
phase. The nucleic acids content ratio, i.e., (RNA)/(DNA),
Table 2 Various methods for the treatment of dye waste-
water35
Physical Chemical Biological
Adsorption Neutralization Stabilization pond
Sedimentation Reduction Aerated lagoon
Floatation Oxidation Trickling filter
Flocculation Electrolysis Activated sludge
Coagulation Ion exchange Anaerobic digestion
Foam fractionation Wet air oxidation Bioaugmentation
Polymer Ozonation
flocculation
Reverse osmosis/
Ultrafiltration
Ionization radiation
Incineration
186 Enzyme Microb. Technol., 1998, vol. 22, February 15
decreased with increasing concentration of Methyl Violet,
so they concluded that the dyes act more preferentially to
lower the protein synthesis than inhibit cell division. Due to
the inhibitive action, cell shape also varied. A report
published by Inouye and Honda about the toxicity of
wastewater to acclimatized sludge was examined. These
reports described the mixtures of various kinds of organic
and inorganic compounds in the sludge.
Crystal Violet has an antibacterial actionI against Esch-
erichia coli, Staphylococcus aureus, Streptococcus faecalis.
and Bacillus subtilis. The Crystal Violet at a concentration
of l-6 x lO-6 M was used. The effect of dye, measured as
minimum inhibitory concentration in terms of retardation of
growth, increased as the pH rose from 6 to 8. Out of the four
species, E. coli was the most resistant to Crystal Violet. The
mode of action of Crystal Violet is the formation of an
unionized complex of bacteria with dye. Gram-negative
organisms such as E. coli have high isoelectric points and
contain less acidic components than Gram-positive bacteria
which usually have lower isoelectric points, so the former
combine with Crystal Violet less readily and are more
resistant to dye.
Degradation by bacteria
There are few reports on the biodegradation of triphenyl-
methane dyes by bacteria. In 1981, Yatome et al. reported
the biodegradation of triphenylmethane dyes by Pseudomo-
nas pseudomallei 13 NA. At this time, the information
related to the biodegradation of dyes other than azo dyes
was insignificant. Four triphenylmethane dyes such as Basic
Fuchsin, Methyl Violet, Crystal Violet, and Victoria Blue
were used for biodegradation studies. They demonstrated
that the decolorization of triphenylmethane dyes was infe-
rior to that of azo dyes. They also showed that Methyl
Violet and Crystal Violet were appreciably decolorized
while Basic Fuchsin and Victoria Blue were not decolorized
under experimental conditions. To determine whether de-
colorization was dependent on the chemical structure of the
dyes, they attempted to correlate the decolorization of dyes
with molecular weight and the octanol-water partition
coefficient. The octanol-water partition coefficient of an
organic compound gives an indication of the permeability of
the organic compound through the cell membrane. The
half-decolorization time of triphenylmethane dyes had no
appreciable correlation with the molecular weight. In gen-
eral, the decolorization of the dyes by P. pseudomallei is not
related to molecular weight and the octanol-water partition
coefficient of the dyes. It was also observed that the
half-decolorization time for both Methyl Violet and Crystal
Violet at a dye concentration of 1 X lo-* M was 54 h.
The viable cell count from the incubation mixture with
Crystal Violet was found to be a function of incubation
time. Under the experimental conditions, the viable cell
count was constant up to loo-140 h. Viable cell count did
not decrease during the incubation period. The ultraviolet
(UV) spectrum of the reaction products of Crystal Violet
with intact cells extracted in n-butanol showed the degra-
dation of Crystal Violet. The shift of the peak and increase
in absorbance were indicative that the reaction products are
different from the original substrate.
In 1991, Yatome et al.* reported the degradation of
Crystal Violet by B. subtilis IF0 13719. Besides Crystal
Violet. two other triphenylmethane dyes (Prarosaniline and
Victoria Blue) were degraded by growing cells of B. subtilis
IF0 13719. With the low cell growth, Crystal Violet was
remarkably decolorized by B. subrilis after 8 h. The com-
pound was totally decolorized in 24 h when the cell growth
was higher. They observed that at a very low level of dye
concentration (below 7 X 10d6 mol I- 1, the decolorization
of Crystal Violet occurred. The growth of the bacterium was
completely inhibited at a dye concentration of 1.5-2.0 X
lo-- mol I-. Other dyes like Basic Auramine 0, Basic
Fuchsin, and Victoria Blue were decolorized in cultures of
B. subhlis while cell growth was poor. They also tried other
bacteria for decolorization studies. E. coli, although grow-
ing remarkably, did not decolorize Crystal Violet. Two
other cultures, Pseudomonas cepacia and Pseudomonas
cruciviae, also showed similar results. Roth et al. isolated
twenty-one hydrophobic oleophilic bacterial strains which
showed decolorization activity; the most active strain was
Mycobacterium. Crystal Violet and Malachite Green dyes
were attacked by all active strains. Cells immobilized on the
glass beads showed similar activity. Cell growth was
inhibited when dyes were present but resumed when the
dyes were transformed.
A German patent* states a new process by which the
xenobiotic dyes of triphenylmethane series are degraded
using Covnebacterium and Mycobacterium sp. The process
is rapid and simple and may be performed over a wide range
of temperature ( 15-40C) over l-12 h. No additional
substrate supplements are needed. Both Crystal Violet and
Malachite Green are removed from wastewater or soil
extracts. For decolorization, Mycobacterium sp. was precul-
tured at 32C in a mineral salt medium containing 1%
methanol as the sole energy and carbon source. After 72 h,
cells were harvested and suspended in tap water at a cell
concentration of 10 ml-. An aqueous solution ( 10.5 ml)
of Malachite Green (20 mg ml-) was inoculated with 1.5
ml cell suspension at 24C. The dye concentration was
reduced to 53% of the initial value within 2 h. At 32C, the
solution was completely decolorized after 22 h. the pH
remained constant at 6.8. and the number of cells did not
increase.
Degradation by actinomycetes
The first report of biodegradation of triphenylmethane dyes
by actinomycetes was published by Yatome et al. * in 199 1.
With two actinomycetes, Nocardia corallina and Nocardia
globerula, the decolorization of Crystal Violet was ob-
served. They also showed that decolorization activity of
actinomycetes is intracellular since there was no activity in
the culture filtrate. The dyes were completely decolorized in
24 h. They also detected a degradation product of the
Crystal Violet digestion as Michlers Ketone (MK) by N.
globerula.
In 1993, another report was published by Yatome et al.*
regarding the degradation of triphenylmethane dyes by
actinomycetes. They have shown that Crystal Violet was
degraded to Michlers Ketone by N. corallina; however, N,
corallina could not decolorize Auramine 0, a typical
Biodegradation triphenylmethane dyes: W. Azmi et al.
Enzyme Microb. Technol., 1998, vol. 22, February 15
187
Papers
Table 3 Decolorization of triphenylmethane dyes by N. coral-
lina IAM 121212
Dyes
Half Maximum
decolorization decolorization
A
maxa
time (min)
(%)
Crystal Violet 590 50 98.3
(BV3)
Methyl Violet 590 60 71.8
(BVI 1
Ethyl Violet 600 480 59.8
(BV4)
Basic Fuchsin 555 30 70.0
(BB9)
Victoria Blue B 620 20 33.0
(BB26)
a The solvent used for the determination of X,,, was n-butanol
dimethylmethane dye. N. corallina decolorized Methyl
Violet, Ethyl Violet, Basic Fuchsin, and Victoria Blue to a
much greater extent (Table 3). but Crystal Violet was the
compound most remarkably decolorized. The decoloriza-
tion rate was dependent upon the initial concentration of N.
corallina in the medium. The decolorization rate was also
dependent upon the growth phase of the precultured cells.
The rates at early log phase (lo-20 h incubation), mid-log
phase (20-30 h incubation), and late log phase (30-40 h
incubation) were 10.6, 4.4, and 2.2 nmol mg- min-,
respectively. The decolorization of Crystal Violet was
observed only at a low concentration (5 pmol 1-i) whereas
the cell growth was completely inhibited at 7 pmol 1-l. The
decolorization rates of Crystal Violet in LB, Bennett, and
minimal medium were 10.6,6.9, and 2.8 nmol mg- mini,
respectively. With cell homogenates and extracellular ex-
tracts of N. corallina, the decolorization of Crystal Violet
was not observed even after prolonged incubation. The
decolorization activity was not observed in washed cells of
N. corallina when the cells were incubated in buffer but the
activity was regained when the cells were incubated in LB
medium. The product of biodegradation was Michlers
Ketone.
Degradation by yeasts
Biodegradation of Crystal Violet by oxidative red yeasts is
reported by Kwasniewska.22 He described the role of yeasts
or yeast-like fungi in the removal of environmental contam-
inants (Table 4). He showed that oxidative yeasts such as
Rhodotorula sp. and Rhodotorula rubra were capable of
degrading Crystal Violet in the liquid broth. The growth
media contains (g 1-l) glucose, 10; Peptone, 10; Ox-bile,
15; and Crystal Violet, 0.01. No antibiotics were added in
the media. The final concentration of test Crystal Violet in
the growth media was at the level of 10 ppm. Crystal Violet
degradation was measured in terms of primary degradation
by following the disappearance of Crystal Violet from the
flask broth. After four days of incubation, the absorbance of
the supematant at 600 nm became nonmeasurable. This
indicated the complete biodegradation of Crystal Violet by
both the oxidative yeasts. In order to test the adsorbance of
the dye by the cells, the cells after biodegradation were
sonicated in 70% (v/v) ethanol. The extract did not show
any absorbance at 600 nm. It was also observed that the
fermentative yeast S. cerevisiae did not degrade Crystal
Violet in the liquid medium even after a prolonged incuba-
tion of 30 days. There may be little decrease in optical
density due to the adsorption of the dye to the cells. The
inability of the fermentative yeast S. cerevisiae to biode-
grade Crystal Violet was apparently not due to any toxic
effect from the dye. The yeast culture was found to grow
equally well in both the control and test flasks in terms of
cell dry weight and cell morphology as seen in the phase
contrast microscope. There was a linear degradation of
Crystal Violet by the two oxidative red yeasts between the
2nd and 4th days of incubation. This indicated the presence
of an enzyme system for the degradation of Crystal Violet.
Degradation by fungi
In 1988, Bumpus and BrockZ3 reported the biodegradation
of Crystal Violet in ligninolytic (nitrogen-limited) culture of
the white rot fungus Phanerochaete chrysosporium. In the
culture broth, the Crystal Violet disappeared with the
appearance of its three products by the sequential N-
demethylation of the parent compound. Demethylation of
Crystal Violet was also performed with a H,O,-generating
system in the extracellular fluid. The purified ligninase also
catalyzed the N-demethylation of Crystal Violet. It proved
that the lignin-degrading system is responsible for the
biodegradation of Crystal Violet. Besides Crystal Violet,
other triphenylmethane dyes are also degraded by this
fungus. They also reported that the nonligninolytic culture
of P. chrysosporium could also degrade the triphenylmeth-
ane dyes. This clearly suggested that in addition to the
lignin-degrading system, another mechanism exists in this
Table 4 Microorganisms reported for the decolorization of
triphenylmethane type of dyes
Microorganisms References
BACTERIA
Bacillus subtilis
Corynebacterium sp.
Mycobacterium sp.
Pseudomonas pseudomallei
Rhodococcus sp.
ACTINOMYCETES
Nocardia sp.
Nocardia corallina
Nocardia globerula
YEASTS
Rhodotorula rubra
Rhodotorula sp.
FUNGI
Coriolus versicolor
Cyathus bulleri
Cyathus stercoreus
Cyathus striatus
Funalia trogii
Laetiporus sulphureus
Phanerochaete chrysosporium
Piptoporus betulinus
Pleurotus ostreatus
18
20
19,20
17
19
19
18, 21
18, 21
22
22
29,30
28
28
28
29
29
23, 29, 30, 32, 33
30
30
188 Enzyme Microb. Technol., 1998, vol. 22, February 15
fungus which also degrades the dye. Adsorption to fungal
mycelium may account for decolorization, but it is only
22% of the total decolorization observed. Six different
triphenylmethane dyes were degraded by ligninolytic cul-
ture of P. chrysusporium. Involvement of the lignin-degrad-
ing system was confirmed by results showing the purified
lignin peroxidase decolorizing these dyes. Brilliant Green,
Malachite Green, and Ethyl Violet all contain N-alkyl
groups, therefore, the initial oxidation of all these dyes
proceeds via N-demethylation in a manner similar to that in
Crystal Violet. Pararosaniline, Cresol Red, and Bromophe-
no1 Blue contain no alkyl groups; thus, oxidation of these
dyes occurs by a mechanism different from that observed
for Crystal Violet. These clearly indicate that the lignin-
degrading system of P. chrysosporium is relatively nonspe-
cific. This nonspecific nature is due to the presence of lignin
peroxidase in the organism. The initial oxidation of a wide
variety of organic compounds is catalyzed by lignnin
peroxidases.-2h These enzymes have been shown to be
able to catalyze a wide variety of reactions including
benzylic oxidation, phenol dimerization, carbon-carbon
bond cleavage, hydroxylation, and o-demethylation?~
Decolorization of three triphenylmethane dyes (Crystal
Violet. Bromophenol Blue, and Malachite Green) by three
birds nest fungi, Cyathus bulleri, Cyathus stercoreus. and
Cyathus striatus, was reported by Vasdev et aLz8 Among
the three organisms, C. bulleri was found to be most
efficient in decolorization. The authors also reported the
decolorization activity in extracellular fluid of the culture
filtrate. The rate of Bromophenol Blue decolorization was
very fast compared to the other dyes. This may be due to the
less complex structure of Bromophenol Blue compared to
the other two dyes tested. They obtained lactase and
ligninase activity in C. bulleri and also observed lactase
activity to be on the higher side than that of ligninase during
the dye decolorization period. Under nitrogen-sufficient
conditions and without addition of H,O,, the culture filtrate
of C. bufkri was able to decolorize three triphenylmethane
dyes (Crystal Violet, Malachite Green, and Bromophenol
Blue) in a relatively simple medium. They reported the
decolorization of Crystal Violet by ultrafiltered and dia-
lyzed extracellular culture filtrate from C. bulleri. This
could be due to the presence of active lactase in ultrafiltered
and dialyzed extracellular fluid. The faster dye decoloriza-
tion by whole cultures of fungus (96-100% in 4 days),
compared with extracellular fluid (90% in 10 days), could
be the result of the combined activity of some cell-
associated and extracellular ligninolytic enzyme. C. bulleri
has been found to be capable of decolorizing the Crystal
Violet up to 90 pM wheras P. chrysosporium has been
shown to decolorize the dyes to a much lesser extent (12.3
1_LM)."j
In 1995, Yesilada2 reported the decolorization of Crystal
Violet by different fungi. He used three white-rot fungi such as
Coriolus versicolor, Fun&a trogii, and Phanerochaete chry-
sosporium and one brown-rot fungus, Laetiporus sulphureus.
Yesilada also reported the oxidation of Crystal Violet by
commercial horseradish peroxidase. A significant rate of oxi-
dation was observed only when H,O, was present. Without
H,Oz, the enzyme had no effect on the decolorization of dye,
thus suggesting that a H,O,-dependent enzyme is probably
involved in the oxidation of the dye.
Knapp et al. reported the decolorization of several dyes
by a group of wood-rotting fungi. Fruiting bodies of
basidiomycetes fungi growing on the rotting wood were
used for the decolorization experiment. Brilliant Green and
Crystal Violet were completely decolorized by white-rot
fungi. The ability of white-rot fungi to degrade a diverse
array of xenobiotic compounds is often attributed to the
use in the wide range of dye waste treatment. Das et al.
studied the Crystal Violet decolorization in a column
bioreactor using P. chrysosporium. The decolorization was
performed in a glass column bioreactor (31 cm X 5 cm)
with an eight tier stainless steel inoculum holder through
which the dye containing medium was recirculated by a
peristaltic pump. Crystal Violet was passed through the
column at a concentration of 0.002% with a recycling rate of
20 ml mini at 30C. The data revealed that almost 92%
decolorization is there in 82.4 h in recycled medium as
compared to 64% in shake flasks in 17 days. With glucose,
the peak decolorization is reached in about 3 days as
compared to 4-6 days with sucrose. Ollikka et al. purified
the various isoenzymes of lignin peroxidase (LIP) from P.
chr?jsosporium and studied the decolorization efficiencies
on several dyes including two triphenylmethane dyes (Bro-
mophenol Blue and Methyl Green) by crude lignin peroxi-
dase and by three purified isoenzymes. The three isoen-
zymes are LIP 4.65, LIP 4.15. and LIP 3.85; however.
Ollikka et al. also reported that the various isoenzymes of
lignin peroxidase LIP 4.65 (HZ). LIP 4.15 (H7). and LIP 3.85
(H,) are different from each other with respect to their
N-terminal amino acid sequence and the degree of glycosy-
lation. The specific activities of LIP 4.65, LIP 4.15. and LIP
3.85 were 26, 39, and 31 U mg-, respectively. For
decolorization of dyes, isoenzymes were purified by pre-
parative isoelectric focusing.
In the presence of 2 mM veratryl alcohol, the crude lignin
peroxidase was able to partially decolorize the dyes. The
best decolorization was obtained for Bromophenol Blue
(93%). The dyes were then studied with isolated isoen-
zymes, LIP 4.65, LIP 4.15, and LIP 3.85. of lignin peroxi-
dase for decolorization. The dye decolorization in the
presence of 2 mrvt veratryl alcohol with the isoenzymes was
similar to that with the crude lignin peroxidase. The
decolorization ability of the purified isoenzymes was
greatly decreased when veratryl alcohol was not present in
the reaction mixture. This suggests that veratryl alcohol acts
as a mediator in the reaction but the omission of veratryl
alcohol from the reaction mixture had almost no effect on
the ability of the crude lignin peroxidase to decolorize the
dyes. This may be due to the fact that the fungus itself
synthesizes veratryl alcohol as a product of secondary
metabolism. With the increase in the veratryl alcohol
concentration the rate of decolorization of Methyl Green
increased. In the presence of 1 mM veratryl alcohol, the
crude lignin peroxidase was able to decolorize Methyl
Green almost completely (initial concentration, 29 PM). The
effect of other enzymes present in the crude enzyme on
decolorization can not be excluded. but the effect of
manganese peroxidase should be minor because the assay
Biodegradation triphenylmethane dyes: W. Azmi et al.
Enzyme Microb. Technol., 1998, vol. 22, February 15 189
Papers
pkl,a
CRYSTAL VI OLET
(Cb)zNh 1 -(-&q,, ---, NO METABOLI TES
\--/ u
MI CHLERS KETONE
tC%,2N& OH
o-DI blETHYLAMI NOPHENOL
Figure 1 Degradative pathway of Crysral Violet by N. corallin$
conditions for lignin peroxidase do not favour the enzymatic
activity of manganese peroxidase.
Biodegradation products
Since the researchers had given main emphasis on the
decolorization studies, very few reports are available on the
biodegradation products or intermediates of triphenylmeth-
ane dyes. Yatome et al. studied the degradation of Crystal
Violet with Pseudomonas pseudomallei and demonstrated
the reaction products of Crystal Violet and Methyl Violet by
thin layer chromatography (TLC). The dichloromethane
extract of the products of Crystal Violet digestion using
methanol and dichloromethane (3:97 v/v) as a developing
solvent produced a spot not for Crystal Violet (Rf O.l), but
for some unknown product (Rf 0.6). The results of the
above experiments indicated the probability of biodegrada-
tion of triphenylmethane dyes. Roth et al. l9 were not able to
identify the biodegradation products or intermediates of the
Crystal Violet digestion. They concluded that the decolori-
zation of Crystal Violet and Malachite Green by Mycobac-
terium sp. was irreversible probably due to the degradation
and formation of leuco-derivatives and then to unidentified
fluorescent- and UV-absorbing intermediates. Yatome et
al.* reported that the major degradation product of Crystal
Violet by growing cells of B. subtilis IF0 13719 was 4,4
his dimethylamino benzophenone (Michlers Ketone MK).
The same product was again obtained by Yatome et al.
with Nocardia corallina. The biodegradation product was
identified by TLC and gas chromatography-mass spectrom-
etry (GC-MS). The product was stable since it was not entirely
degraded within 24-48 h by N. corallina (Figure I).
Bumpus and Brockz3 reported the biodegradation and
+ FURTHER METABOLI TES
mineralization of Crystal Violet by white-rot fungi. The
disappearance of Crystal Violet as well as metabolite
formation was monitored by high-Performance Liquid
Chromatography (HPLC). They used methylene chloride
extract for HPLC analysis. It was observed that the initial
degradation starts with the sequential demethylation of
Crystal Violet. Three degradation products were identified
as N,N,N,N,N-peI lta-, N,N,N,N-k&i-, and N,N,N-t&l-
ethylpararosaniline. Biodegradation appears to continue be-
yond that as they obtained two additional unidentified
colored Crystal Violet metabolites during HPLC analysis,
but finally, Crystal Violet degraded to a colorless product.
Future work
It may be recommended for generating large-scale data to use
the biological decolorization system as the alternative method
of dye wastewater treatment. Decolorization and degradation
should accompany the reduction of biochemical oxygen de-
mand (BOD) and chemical oxygen demand (COD) of the
wastewater. It may be necessary to find out the enzymatic
system responsible for the decolorization and degradation of
dyes. Genetic modification of the enzymatic system of differ-
ent microorganisms responsible for decolorization and degra-
dation may be studied to make the system more active.
Enrichment studies should be done by either the soil slung
method or soil column method with triphenylmethane or other
dyes to isolate organisms responsible for utilizing dyes as the
sole source of carbon and energy. This will lead to the
degradation or mineralization of dyes. All the earlier reports
mainly deal with decolorization. No report is available regard-
ing the toxicity status of the decolorized dyes. It is necessary to
estimate the toxicity with respect to decolorization, because
190 Enzyme Microb. Technol., 1998, vol. 22, February 15
Biodegradation triphenylmethane dyes: W. Azmi et al.
decolorized materials may remain toxic unless the products are
retained by the cells.
Conclusions
It may be concluded from the above discussions that it is
possible to decolorize or degrade triphenylmethane dyes by
biological means. There are very few reports on the biological
decolorization and degradation of textile and dye-stuff indus-
trial wastes containing triphenylmethane dyes. All the reports
so far available are on a small scale; no large-scale data on
biological decolorization and degradation is available. Differ-
ent types of organisms such as bacteria, actinomycetes, yeast,
and fungi were reported to decolorize and degrade different
types of triphenylmethane dyes. Reports on complete miner-
alization is very much scanty. There is no report on reduction
of biochemical and chemical oxygen demand after decoloriza-
tion. Biological treatment is the only way for ultimate control-
ling of pollution generated by textile and dye-stuff industries;
however, more and more research and development works are
needed to develop a viable alternative process for the treatment
of dye wastewater.
Acknowledgments
Wamik Azmi and Rajesh Kumar Sani gratefully acknowl-
edge the Senior Research Fellowship from the Council of
Scientific and Industrial Research (CSIR), India.
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