Artículo en PDF
Cómo citar el artículo
Número completo
Más información del artículo
Página de la revista en redalyc.org
Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Rev. Int. Contam. Ambient. 24 (1) 5-11, 2008
BIOTECHNOLOGICAL TREATMENT FOR COLORLESS DENIM AND TEXTIL WASTEWATER
TREATMENT WITH LACCASE AND ABTS
Myrna SOLÍS-OBA
1
, Javier ALMENDÁRIZ
2
, Gustavo VINIEGRA-GONZÁLEZ
2
1
Centro de Investigación en Biotecnología Aplicada, Instituto Politécnico Nacional, carretera estatal Santa Inés
Tecuexcomac-Tepetitla, km 1.5, Tepetitla de Lardizábal, Tlaxcala, México,
C.P. 90700, msolis@ipn.mx
2
Departamento de Biotecnología, Universidad Autónoma Metropolitana, Unidad Iztapalapa, Av. San Rafael
Atlixco 186, Col. Vicentina 09340, México D.F.
(Recibido mayo 2007, aceptado octubre 2007)
Key words: denim, discoloration, ABTS, indigo, laccase enzyme, dye oxidation, recycling
ABSTRACT
The ABTS
(
2,2´-[3-etil benzotiazolin-6-sulphonic]acid)-Laccase system was applied for
discoloration of both commercial denim fabric and a synthetic indigo solution.
After treat-
ment, the denim fabric looked like an aged one, although Fbers did not exhibit any damage
and the synthetic indigo solution was bleached. Additionally, an analysis of both the denim
discoloration residual water and the synthetic indigo solution biodegradation, showed that
in the two cases the residual water was easily biodegraded in either an aerobic or anaerobic
mode, while the control indigo solution required 5 days to be biodegraded in an aerobic mode,
and it became unaltered in anaerobic mode. The oxidized ABTS or the laccase could not
bleach denim or the synthetic indigo solution in the same experimental conditions as when
using the system ABTS-laccase. This is explained considering that, during this process, as
when using the mediator recycles several times between the laccase and the indigo, in such
way that in a 30 min period, one molecule of ABTS oxidized 800 indigo molecules; also,
the observed discoloration rate was two orders of
magnitude higher than the rates observed
when either the oxidized mediator or the enzyme by itself were applied.
Palabras clave: denim, decoloración, ABTS, oxidación, pigmentos, reciclaje
RESUMEN
El sistema ABTS
(
ácido 2,2´-[3-etil benzotiazolin-6-sulfónico])-lacasa se empleó para
decolorar tanto tela de mezclilla como una solución preparada de índigo. Después del
tratamiento, la mezclilla adquirió un aspecto de usada, sin que las Fbras mostraran
evidencia de daño, mientras que la solución de índigo se decoloró. Adicionalmente, el
análisis de la biodegradación de los desechos acuosos de la decoloración de la mezcli-
lla y la solución decolorada control de índigo mostraron que, en ambos casos el agua
residual fue fácilmente biodegradada tanto aeróbica como anaeróbicamente, mientras
que la solución control de índigo requirió de 5 días para biodegradarse aeróbicamente
y
no sufrió ningún cambio en el sistema anaeróbico. El ABTS oxidado ó la lacasa por sí
solos no decoloraron la mezclilla ni la solución de índigo bajo las mismas condiciones
experimentales en que se empleó la combinación de ambos ABTS-lacasa. Esto se explica
porque, durante la reacción de decoloración, el mediador se recicla varias veces entre la
M. Solís-Oba
et al.
6
enzima y el colorante, de tal forma que en 30 minutos, una molécula de ABTS oxida 800
moléculas de índigo, además la velocidad de decoloración fue dos órdenes de magnitud
mayor que la velocidad cuando se empleó el ABTS ó la enzima en forma individual.
INTRODUCTION
Synthetic colors are widely used in industry, it is
estimated that color products in the market are more
than 10,000 species; and the worldwide industrial
production is about 7x10
5
metric tons (Tan
et al.
1999).
Also, according to Balan and Monteiro (2001)
5-10 % of the used color in industrial process goes
unaltered to residual wastewaters.
The most frequently used colors correspond to
azo, anthraquinone and indigo types; these com-
pounds are classifed like hazardous wastes because
in aerobic conditions they are recalcitrant to deg-
radation, and in anaerobic conditions they evolve
into highly toxic amines (Podgornik
et al.
2001).
Treatment of wastewater polluted with color can
be approached by physical or chemical procedures;
although any of those options usually turns out to
be expensive, inefficient, and creates secondary
products which can barely be disposed of. Biologi-
cal processes have shown to be less expensive and
more eFfcient in wastewater treatment (Cheng
et al.
2003). Enzymes produced by white fungi like the
lignine peroxidase or laccase, have been successfully
applied in discoloration of some colors (Nyanhongo
et al.
2002). Campos
et al.
(2001) have reported that
the indigo molecule becomes oxidized by laccase
enzyme producing isatin, an easily hydrolysable
compound that evolves up to anthranilic acid, which
is a toxic compound for aquatic organisms, and so far
to the environment (Instituto Nacional de Seguridad
Higiene en el Trabajo 2003). Bourbonnais and Paice
(1990) proposed that laccase substrate ranges could
be augmented by including a mediator; the last is a
compound of low molecular weight willing to be oxi-
dized by the enzyme, and once oxidized it becomes
able oF interact with the fnal substrate.
The ABTS
molecule is the most studied mediator for laccase,
since it exhibits high stability in ionic state, also its
redox chemistry is well known; the only minus is
that the mechanism of interactions between ABTS
and laccase is not well defned (Collins
et al.
1998).
Potthast
et al.
in 1995 have found evidence suggest-
ing that ABTS acts as an activator or co-oxidant of
the enzyme; they explain that the ABTS transfers one
electron to the enzyme to activate it, working as a
co-oxidant agent and not like an electronic mediator
of the substrate (Potthast
et al.
1995).
Denim market has a high proportion of
“look like
aged products”, this appearance is reached through
several process, one of the most popular is the stone
wash in which the fabric receives attrition by cal-
careous stones in a bathtub machine; this process
produces damage for both the fabric and the machine,
also the removal oF fne stones retained by the Fabric
increase process expenses (Olson
et al.
1993). An
alternative method is applying cellulose enzyme in
the bath, but usually the enzyme produces the back
staining of indigo dye (Cavaco-Paulo 1998). Another
popular option is applying bleach for denim discol-
oration, but bleach is a strong oxidant which usually
damage fbers and to control the discoloration process
becomes troublesome (Tzanov
et al.
2003) plus a
high possibility of generating toxic halogenated
compounds that are unwanted side products.
This work was based on using both ABTS and
laccase enzyme to set up an eFfcient treatment system
for discoloration of both denim and indigo solutions,
with the advantage of lowering costs, improve pro-
cess control and lower environmental threats, since
the ABTS and laccase produce biodegradable re-
sidual wastewater products. The mechanism followed
between laccase and ABTS during the indigo oxida-
tion is also explained. The process of discoloration
described here is in process to be patented.
MATERIALS AND METHODS
Commercial formula for laccase enzyme
The laccase enzyme used in this study was a
commercial one named Deni Lite II S, donated by
Novozymes México S.A. de C.V., the distributor
of the product. This item is a laccase (EC 1.10.3.2)
produced by submerged fermentation of a genetically
modifed
Aspergillus
microorganism. It is a light grey
powder product available in standard strength 120
laccase units/g (Novo Nordisk 1999). Its formulae in-
cludes laccase enzyme, 10 phenothiazolin, propionic
acid as mediator and a non-ionic surfactant as buffer.
Deni Lite II chemical composition was characterized
by Greca
et al.
(2001).
BIOTECHNOLOGICAL COLORLESS DENIM AND TEXTIL WASTEWATER
7
Laccase purifcation
The enzyme was purifed as Follows: mixing one
g of Deni Lite II and 10 cc of distilled water, the
mixture was shaken for 30 min at room temperature;
after that, it was centrifuged at 5000 rpm for 10 min.
The solid phase was discarded and the liquid one was
slowly added to a ±ask containing acetone at 4 °C,
this mixture was placed in a cooled bath for 1 h. Later
the mixture was centrifuged at 10,000 rpm for 15 min,
the solid obtained in this stage was the enzyme frac-
tion, which should be washed out with cold acetone
several times, before placing it onto a glass dish and
letting the solvent to evaporate. ²inally, the purifed
enzyme was dissolved into an acetate buffer solution
at pH=5 and stored at 8 °C.
Enzymatic activity determination
The laccase activity was measured at
λ
= 420 nm
in a spectrophotometer UV-Visible Beckman DU 640
using ABTS as a carrier, absorbance increment was
measured considering a coeFfcient
ε
=36000 mM
-
1
cm
-
1
(Bourbonnais
et al.
1998). A laccase unit is con-
sidered equivalent to the amount of enzyme required
to oxidize 1
µ
mol of ABTS in a 1 min period.
Laccase immobilization
This stage was accomplished following the pro-
cedure reported by Ho and Liao (1983).
Oxidized ABTS preparation
ABTS was oxidized by adding 40 cc of 5 mM
ABTS solution to 10 g of immobilized laccase, left
at rest for 7 days at room temperature. After this
period, the solution was fltered and stored at 4 °C
for later use.
Denim discoloration using laccase and ABTS
A sample of commercial denim fabric was cut
in small pieces, 7 of these were placed in each of 3
±asks named series 1, 2, and 3. The frst was added
with 40
µ
mol of oxidized ABTS; the second was
added with 40 laccase units; and the third one was
added with 40
µ
mols of oxidized ABTS plus 40 lac-
case units. The fnal volume in all ±asks was set up
to 200 cc, and then incubated at room temperature.
A piece oF Fabric was taken out From each ±ask at
the following times: 0, 2, 6, 12, 30, 36, and 48 h.
Each fabric piece was analyzed with a Hunter Lab
colorimeter D25-PC2, taking the direct measurement
of the following parameters Lightness (L), Hue (H)
and Chrome (C). The parameter L has values from
0 to 100, where 100 corresponds to white color and
0 to black color. The parameter H is measured as an
angle over a circle divided in 100 parts; while the C
parameter is a measure of fabric opacity.
Denim discoloration wastewater treatment
Residual wastewater biochemical oxygen demand
(BOD) was analyzed in order to estimate its potential
biodegradation in aerobic conditions; the analysis
was done following the BOD Hach standard proce-
dure technique. At this stage 3 samples were prepared
as Follows: the frst one was flled with wastewater
From denim discoloration bath; the second was flled
with indigo solution; and the third one was flled with
a mixture of ABTS and laccase. The concentration
of the solutions was adjusted to getting a chemical
oxygen demand (COD) of 1 g l
-
1
. Each bottle was
inoculated with aerobic microorganisms and incu-
bated For 5 days at 25 °C. BOD was analyzed every
day during fve days.
Biodegradability in anaerobic regimen was measured
using 3 samples as follows: sample 1 was discoloration
denim wastewater, sample 2 was an indigo solution, and
sample 3 was a positive control solution prepared with
sodium acetate; the last was chosen because the experi-
mental microorganisms were grown in that media. The
concentration of each solution was adjusted up to COD
equal to 1 g L
-
1
. Initial dissolved oxygen was washed
out by a nitrogen gas ±ow. During 72 h, a 3 % sodium
hydroxide solution was used in order to wash out and
quantify the methane production.
Indigo discoloration rate in presence oF oxidized
ABTS and laccase enzyme
Three reaction mixtures were prepared as fol-
lows
:
a)
0.1 µ
Mol indigo and 10 mMol of oxidized ABTS.
b)
0.1 µ
Mol indigo and 2 laccase units.
c)
0.1 µ
Mol indigo
, 1 µ
Mol oxidized ABTS and 2
laccase units
.
The indigo discoloration rate was estimated by
measuring variations in absorbance, taking as refer-
ence
λ
=610 nm (indigo maximum response) and
λ
=420 nm (oxidized ABTS maximum response).
Cycling oF ABTS
A 2 ml sample was prepared in the spectrophotom-
eter cell by adding 1 laccase enzyme unit, and 2.5
µ
mol
oF oxidized ABTS; also, every three minutes a fxed
concentration of 200
µ
mols indigo was added to the
cell, until 10 indigo additions. Absorbance lectures
at
λ
=420 and
λ
=610 were taken, and from a plot of these
lectures versus time, an estimation of the amount of
oxidized ABTS as well as indigo concentration was
obtained, before and after each addition.
M. Solís-Oba
et al.
8
With ABTS
With Laccase
With ABTS
and laccase
0 h
2 h
6 h
12 h
20 h
30 h
36 h
48 h
Fig. 1.
Denim incubated with oxidized ABTS, laccase and the mixture of both
RESULTS
Laccase enzyme
The purifed enzyme had a specifc activity oF
0.5 U mg
-
1
, and a Michaelis-Menten constant of
9.83 x 10
–5
mM a 25
o
C (K
M
), it is the concentration
of substrate in which the initial velocity is the half
of the maxima velocity (Lehninger 1978). The im-
mobilized laccase produced a mixture of ABTS and
ABTS
+1
(Solís-Oba
et al.
2005), since the reduced
form of ABTS did not react with indigo, it was not
necessary to separate the chemical species.
Oxidized ABTS and laccase enzyme for denim
discoloration
Figure 1
shows a photography of denim pieces
after incubation with either oxidized ABTS, laccase
and a mixture of ABTS and laccase (see procedure in
Denim discoloration using laccase ABTS). A careful
analysis of each piece revealed that the piece treated
with the mixture was the only one exhibiting discol-
oration, and its appearance was like an aged piece,
without
any damage oF the fbers, and Fabrics were
not opaque with this treatment. During the test, dis-
coloration was fairly gradual and uniform (no color
spots), so Far it can be aFfrmed that denim discolor
-
ation treatment can be done without problems and
that in this way the process control was easy.
Colorimetric testing for discolored denim
Each denim treatment was evaluated by three
parameters: Lightness (L), Hue (H), and Chrome
(C). The results are resumed in
±gure 2
and
±gure 3
showing the parameters L and H, respectively. Only
denim treated with ABTS and laccase changed in
the parameters L and H because denim was bleached
with those reagents. There were not changes in the C
parameter with any of the three processes.
ABTS and laccase
Laccase
Time (h)
0
10
20
30
40
40
(L)
35
30
35
50
ABTS
Fig. 2.
Colorimetrical analysis of denim treated with ABTS,
laccase and the mixture or both. Variation in the L pa-
rameter
ABTS and laccase
Laccase
Time (h)
0
10
20
30
40
272
H
267
262
257
50
ABTS
Fig. 3.
Colorimetrical analysis of denim treated with ABTS,
laccase and the mixture or
both. Variation in the H pa-
rameter
BIOTECHNOLOGICAL COLORLESS DENIM AND TEXTIL WASTEWATER
9
Biodegradability test for residual denim discolor
-
ation wastewater.
Figure 4
plots the analytical values of BOD from:
a) Denim discoloration residual wastewater. For
this sample BOD/COD was 0.696 in the frst day,
and at the 5
th
day the ratio BOD/COD was 0.764,
this result indicates that the sample was easily
biodegraded aerobically.
b) Synthetic indigo solution. This sample was un-
altered the frst day, but started to change during
the second day. The ratio BOD/COD at the 5
th
day was 0.67, and even though this value could
indicate biodegradability, dark blue color in the
sample was unaltered.
c) ABTS and laccase mixture. With this option the
ratio BOD/COD reached at the
5
th
day a value of
0.31, so the observed BOD/COD in a) was attrib-
uted to the easy biodegradation of indigo oxidation
products not to the ABTS or the laccase.
The 72 hrs methane production was analyzed
considering the following experiments (
Fig. 5
):
a) Denim discoloration wastewater. - Methane produc-
tion reached 75 % of the theoretical value. This result
indicates that the denim wastewater was susceptible
of biodegradation under anaerobic conditions
b) Indigo solution.- Methane production was null,
this condition is attributed to toxic compounds
production, as it is referred by some authors (Tan
et al.
1999,Wong and Jang, 1999)
c) Sodium acetate solution reached a 90 % of the
theoretic value.
Indigo oxidation rate
The oxidation rates for indigo considering the
following treatments were:
a) Using oxidized ABTS. The calculated indigo
oxidation rate was k=4x10
-
6
mol min
-
1
.
b) With laccase.
The oxidation rate was estimated
as k= 8x10
-
5
mol min
-
1
. This value is 20 times the
one obtained with the mediator, which means that
the enzyme by itself provokes a faster oxidation
for indigo.
c) Using the mixture of oxidized ABTS and lac-
case. This procedure reaches an oxidation rate of
6x10
-
4
mol min
-
1
, so far this process is actually
faster compared to the previous ones; even when
it was used a [Indigo]/ [oxidized ABTS]= 100.
ABTS cycling quanti±cation
Figure 6
shows the set of results obtained by
adding 10 indigo dosages to the spectrophotometric
cell; after each indigo addition, a fast indigo solu-
tion discoloration was observed. In this process the
oxidized ABTS was rapidly reduced, and in absence
of indigo, it exhibits accumulation up by the laccase,
until the next indigo addition; this explanation was
deducted from the periodic oscillations present in the
plot of oxidized ABTS, in which the valleys match the
times of indigo addition. It is estimated that in a 30
min period, 1 ABTS
µ
mol was capable of oxidizing
an amount of 800 indigo
µ
mols having laccase in the
reaction medium.
DISCUSSION
The results found in this work are:
a) denim tested
with ABTS and laccase was the only one discolored,
b) the indigo oxidation rate was higher using ABTS
0
0
200
400
600
BOD (mg/L)
800
1000
1
2
3
Day
Denim discoloration wastewater
Synthetic indigo solution
ABTS and laccase mixtures
4
a)
b)
c)
5
6
Fig. 4.
BOD values of: a) Denim discoloration residual waste-
water, b) Synthetic indigo solution and c) ABTS and
laccase mixture
14
12
10
8
6
4
2
0
0
12
24
Time (h)
Denim discoloration wastewater
Sodium acetate solution
Indigo solution
Methane production (mL)
36
48
60
c)
a)
b)
72
84
Fig. 5.
Methane production of: a) Denim discoloration waste-
water, b) Indigo solution and c) Sodium acetate solu-
tion
M. Solís-Oba
et al.
10
and laccase than using the oxidized ABTS or laccase
alone, and c) 1 ABTS μmol oxidized 800 indigo
μmols in the presence of laccase, and give enough
evidence that the ABTS was cycling between laccase
enzyme and indigo, working as an electronic media-
tor of the substrate.
The cycling process followed is explained in the
next scheme:
Laccase + O
2
Laccase + H
2
O
Oxidized ABTS
Oxidized indigo
ABTS Indigo
Indigo
It was demonstrated that the ABTS mediator ac-
complishes a cycling between laccase enzyme and
indigo color; then the required amount of mediator is
very small compared
to the amount of indigo which
can be oxidized by the mediator. With this argument
it can be viable to include expensive mediators like
ABTS in residual colored wastewater treatment or
denim bleach. Even though ABTS is an expensive
mediator, the fact that it is able to accomplish several
cycles between laccase an dye, makes it possible to
consider its inclusion in the process.
The ABTS-laccase system can be applied for denim
industrial discoloration as well as an option for indigo
polluted wastewater treatment. In denim discoloration
the combination ABTS-laccase offers several advan-
tages over the actual commercial process:
a) Denim Fbers are not attacked during discolor
-
ation.
b) Avoiding the use of stones will produce savings
because machine life will be extended, plus
cleaning expenses due to stone recovery are
eliminated.
c) Environmental impact will be reduced because
the ABTS-laccase system will not produce halo-
genated byproducts.
d) Indigo oxidation byproducts are easily biode-
graded in either aerobic or anaerobic systems;
therefore, the residual wastewater treatment is
conducted in a simple way, reducing threats to
the environment.
ACKNOWLEDGEMENTS
The authors thank Dra. Margarita Teutli, Ing.
Jorge Humberto Barrera Macías and Ing. Edgar Gó-
mez Camarillo for their technical help. Myrna Solis
had a scholarship from CONACyT.
REFERENCES
Balan D. and Monteiro R. (2001). Decolorization of
textile indigo dye by ligninolytic fungi. J. Biotechnol.
89,
141-145.
Bourbonnais R. and Paice M. (1990). Oxidation of non-
phenolic substrates. An expanded role for laccase in
lignin biodegradation. FEBS Lett. 267, 99-102.
Bourbonnais R., Leech D. and Paice M. (1998). Electro-
chemical analysis of the interactions of laccase media-
tors with lignin model compounds. Biochem. Biophys.
Acta. 1379, 381-390.
Campos R., Cavaco A., Robra K., Schneider M. and
Gubitz G. (2001). Indigo degradation with laccases
from
Polyporus sp. and Sclerotium rolfsii.
Textil Res.
J. 71, 420-424,.
Cavaco-Paulo A. (1998). Mechanism of cellulase action in
textile processes. Carbohydrate Polym. 37, 273-277.
Cheng Ch., Jane-Yii W., Dar-Jen L., Sz-Chwun J. and
Kuo-Hwang (2003). Decolorization of the textile dyes
by newly isolated bacterial strains. J. Biotechnol. 101,
57-68.
Collins P., Dobson A. and Field J. (1998). Reduction of
the 2,2 -Azino-bis (3-Ethylbenzthiazoline-6-Sulfonate)
cation radical by physiological organic acids in the
absence and presence of manganese. Appl. Environ.
Microbiol. 64, 2026-2031.
Greca M., Costa-Ferreira M. and Pessoa M. (2001). Decol-
orization of an anthraquinone-type dye using a laccase
formulation. Biores. Technol. 79, 171-177.
Ho G. and Liao Ch. (1983). Activation of a siliceous car-
rier for enzyme immobilization. United States Patent
4, 384, 045.
Instituto Nacional de Seguridad Higiene en el Trabajo (2003).
Fichas internacionales de seguridad química, ICSC 1295.
Ministerio del trabajo y asuntos sociales España.
Lehninger A.L.
Bioquímica
. (1978). Primera reimpresión,
Omega, España.
Novo Nordisk (1999). Deni Lite II S Product Sheet, En-
zyme Business. Denmark.
0.10
Indigo (mM)
0.08
0.0020
0.0016
0.0012
0.0008
Oxidized ABTS (mM)
0.06
0.04
0.02
0
0
5
10
Time (min)
Oxidized ABTS
Indigo
15
20
25
30
Fig. 6.
ABTS cycling between laccase and indigo
BIOTECHNOLOGICAL COLORLESS DENIM AND TEXTIL WASTEWATER
11
Nyanhongo G., Gomes J., Gubitz G., Zvauya R., Read
J. and Steine W. (2002). Decolorization of textil dyes
by laccase from a newly isolated strain of
Trametes
modesta
. Water Res. 36, 1449-1456.
Olson A., Gladfelter E. and Burch W. (1993). Decolor-
izing dyed fabric or garments, United States Patent
5, 268, 002.
Podgornik H., Poljansek I. and Perdih A. (2001). Trans-
formation of indigo carmine by
Phanerochaete
chrysosporium
ligninolytic enzyme. Enzyme Microb.
Technol. 29, 166-172.
Potthast A., Rosenau T., Chen.L. and Gratz J. (1995).Se-
lective enzymatic oxidation
aromatic methyl groups
to aldehydes. J. Org. Chem. 60, 4320-4321.
Solis-Oba M., Ugalde-Saldivar V., González I. and
Viniegra-González G. (2005). An electrochemical-
spectrophotometrical study of the oxidized forms of
the mediator 2,2’-azino-bis-(3-ethylbenzothiazoline-
6-sulfonic acid) produced by immobilized laccase, J.
Electroanal. Chem. 579, 59-66.
Tan N., Prenafeta-Boldú F.X., Opsteeg J.L., Lettinga G.
and Field J.A. (1999). Biodegradation of azo dyes in
cocultures of anaerobic granular sludge with aerobic
aromatic amine degrading enrichment cultures, Appl
Micobiol Biotech. 51, 865-871.
Tzanov T., Andreaus J., Guebitz G. and Cavaco-Paulo A.
(2003). Protein interactions in enzymatic processes in
textiles, Electronic J. Biotechnol. 6, 146-154.
Wong Y. and Jian Y. (1999). Laccase-catalyzed decolor-
ization of synthetic dyes. Water Research, 33, 3512-
3520.
logo_pie_uaemex.mx