Clean Techn Environ Policy (2015) 17:1443–1456

DOI 10.1007/s10098-014-0869-6

ORIGINAL PAPER

Degradation of azo dyes by laccase: biological method to reduce

pollution load in dye wastewater
J. Kanagaraj • T. Senthilvelan • R. C. Panda

Received: 21 February 2014 / Accepted: 20 October 2014 / Published online: 1 November 2014
Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Laccases are the oldest with low substrate Keywords Azo dyes  Laccase enzyme  Degradation 
specificity enzymes used for degradation of various com- FT-IR and mass spectra analyses  Pollution reduction
pounds, especially, dyes. In the present investigation, the
cell-free extract of laccase enzyme is applied to degrade
azo dyes used in leather processing. The enzyme degrades Introduction
the azo dyes rapidly at optimum growth conditions of pH
7.0, temperature 37 °C and incubation duration of 72 h. Biotechnological approaches provide alternative process to
Better production of the enzyme was achieved with dex- ensure cleaner, green and eco-friendly environment. Vari-
trose, yeast extract, acetone, copper sulphate and orange ous enzymes have been used to reduce pollution load of
peel. The molecular weight of laccase was found to be different chemicals used in industries. Laccases are one of
63 kDa. The percentage of degradation is found to be the widely used multi-copper enzymes, which catalyze the
96.4 % for CI Acid black 210 and 92.2 % for the CI Acid reaction with one-electron oxidation of many phenolic
black 234. Ultraviolet–visible spectral analysis indicates compounds with concomitant reduction of oxygen to water.
the presence of peaks in the visible region confirming the Laccases are biologically important enzymes that come
complete degradation of the dye sample. Fourier transform- under oxidase group and are also useful to reduce the
infrared spectroscopy analysis shows the transformation of environmental pollution (Yaropolov et al. 1994; Call and
N=N into either N2 or NH3 and then into biomass. The Mucke 1997; Mayer and Staples 2002). It is widely found
mass spectra analysis shows conversion of azo dye to final among higher plants, insects, a few bacteria and fungi
product through various intermediates with their respective (Zille et al. 2005). Laccases are found in plants like cab-
molecular weights. Chemical oxygen demand (COD) and bages, turnip, potatoes, pears, apples and other vegetables
biochemical oxygen demand (BOD) analysis reveal the (Leonowicz et al. 2001). The enzyme plays a significant
reduction of pollution load more than 92 % in the experi- role in lignifications whereas fungal sources of laccase help
mental process while maintaining BOD/COD ratio to about in delignification, sporulation, pigment production, fruiting
30 %. body formation and plant pathogenesis (Rodriguez-Couto
2012). Bacterial sources of the enzyme are Streptomyces
Electronic supplementary material The online version of this lavendulae, Streptomyces cyaneus and Marinomonas
article (doi:10.1007/s10098-014-0869-6) contains supplementary mediterranea etc. The enzyme appears more in fungi than
material, which is available to authorized users. in the higher plants. Laccase from Monocillium indicum
was the first enzyme to be characterized from Ascomycetes
J. Kanagaraj (&)  T. Senthilvelan
Leather Processing Division, CSIR-CLRI, Adyar, which showed peroxidase activity (Claus 2003). Pycnop-
Chennai 600020, India orus cinnabarinus produces laccase that degrades lignin
e-mail: [email protected] compounds whereas Pycnoporus sanguineus yields phenol
oxidase that breaks phenolic compounds. Literature reveals
R. C. Panda
Chemical Engineering Division, CSIR-CLRI, Adyar, that laccases represent an interesting group of oxidative
Chennai 600020, India enzymes owing to their great potential for biotechnological

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1444 J. Kanagaraj et al.

and environmental applications among various enzymes biosorption of Indosol Orange RSN dye using peanut husk
(Jolivalt et al. 1999; Arora and Sharma 2010). biomass along with native, polyethyleneimine treated and
Enzyme activity has been detected in the bacterium Na-alginate immobilized biomass separately. Different
Azospirillum lipoferum, raising the possibility that laccases important process parameters like pH, contact time, bio-
are not restricted to eukaryotes. In recent times, bacterial sorbent dose, initial dye concentration and temperature
laccases have shown to successfully oxidize dyes in various were optimized during batch study and the results con-
industries. Production of bacterial laccases for biotechno- cluded that low pH and low biosorbent dose were found to
logical applications will be advantageous because they are be feasible conditions for the maximum biosorption of dye.
sustainable and can be produced in a short time in inex- It was inferred that maximum dye removal (8.8 mg/L) was
pensive media (Dube et al. 2008). Biodegradation using achieved with 3 cm bed height and 1.8 mL min-1 flow
this enzyme has been practiced widely in treating effluents rate using 70 mg/L of initial dye concentration. In another
from dye, textile, leather and petrochemical industry. work (Gunasekaran and Kanmani 2014; Chen et al. 2014),
Efficiency of the enzyme for the degradation of dyes can be it has been found that gas chlorination in decolourization/
increased by its combination with laccase mediators (Call degradation of textile dyeing wastewater was optimized
and Mucke 1997; Abadulla et al. 2000). The enzyme finds and process kinetics of the chlorination had been studied
application in dye degradation especially for azo dyes that and standardized. The percentage of colour removal and
are very difficult to degrade as it contains aromatic poly- COD degradation were in the range of 89–95 %. Ozonation
phenolic components. was used to treat tannery dye Acid Black 52 where
Several dye industries use azo dyes for its uniqueness chemical oxygen demand (COD) was found to be lower
and quality. These dyes are considered as one of the (Sadhukhan et al. 2014; Yu et al. 2014). Experimental
important and largest class of synthetic dyes having results were analysed by regression analysis and analysis of
–N=N– groups that are widely used in industry due to their variance by statistical methods. The optimum operating
relatively simple synthesis method and are available in conditions for achieving maximum colour and COD
almost unlimited number and types of substituent (Van- removal were found to be at pH: 1.96, dye concentration:
devivere et al. 1998; Park et al. 2005; Stolz 2001; Christie 1,159 mg/L, contact time: 10.6 min (min) and at pH: 4.8,
and Shanmugam 2012). They are labelled as the xenobiotic dye concentration: 1,159 mg/L, contact time: 17 min,
compounds that are very recalcitrant to biodegradation respectively (Vedaraman et al. 2013; Hildebrand et al.
process (Palma et al. 1999; Velu et al. 2011; Chacko and 2014). Use of biomass for removal of industrial dyes
Subramaniam 2011). The azo groups are generally con- (Orange II) by biosorption technique has been investigated.
nected to benzene and naphthalene rings, but can also be The results suggest that the transport of the inorganic
attached to aromatic heterocyclic or aliphatic groups (Ni- phosphorus, as demonstrated through the cellular bioac-
gam et al. 1996; Kanagaraj et al. 2008). The azo dyes used cumulation in the polyphosphate form, was related to the
in the leather industry not only causes pollution problem process of removal of the colour of Orange II, with the
but also are toxic in nature (Kanagaraj and Mandal 2012; reduction of 60 % of the dye solution (Silva et al. 2013;
Kanagaraj and Panda 2011). Hence, removal of these dyes Ozdemir et al. 2014). Among these, degradation of dye
from effluent is inevitable. There are various methods using biological method is an eco-friendly option and has
available for the removal of dyes such as membrane fil- the added advantages over chemical methods as the earlier
tration, coagulation flocculation, precipitation, flotation, method prevents transfer of pollution from one part of the
adsorption, ion exchange, ion pair extraction, ultrasonic environment to another. As dyeing is one unit operation in
mineralisation, electrolysis, advanced oxidation (chlorina- leather processing, huge amount of dyes are used that
tion, bleaching, ozonation, fenton oxidation and photocat- emanates effluent as waste stream. Biological method also
alytic oxidation) and chemical reduction (Kanagaraj et al. provides cleaner environment and economical compared to
2012; Moosvi et al. 2005). Literature reveals recent trends other methods (Claus 2004). Various researchers have
on the degradation of various dyes in wastewater by reported the decolourization of dye using biological
modern process intensification techniques like, sonication, method (Diamantidis et al. 2000; Han et al. 2005).). In the
photocatalysis and ozonation very effectively. Pre-treat- present investigation, the laccase enzyme (cell-free extract)
ment of the dye wastewater followed by above methods obtained from bacterial strain of Micrococcus luteus was
increased the biodegradability of the effluents while further exploited for the degradation of prominent azo dyes used in
biological degradation using acclimatized sludge biomass the leather processing operation. The oxidative enzymes
resulted in COD removal efficiencies to 94, 91 and 82 %, that can cleave phenolic compounds easily will be suitable
respectively, for photocatalysis, sonication and ozonation for the present investigation. The azo group present in dyes
(Meenatchisundaram et al. 2014; Chakraborty et al. 2013). could be cleaved fully by means of oxidative enzymes. As
According to another report (Sadaf and Bhatti 2014), the wood (decaying) contains many phenolic substances in its

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Degradation of azo dyes by laccase 1445

constituents and the environment will be helpful in grow- (g/L): starch-18, yeast extract-2.5, H2PO4-1.2, NaHPO4-
ing/producing the strain that is capable of generating lac- 0.06, MgSO4-0.4, CaCl2-0.011, FeSO4-0.012, MnSO4-
case enzyme with better activity that will be able to cleave 0.0009 and CuSO4-0.004. This experiment was carried out
azo bond completely in azo dyes. Micrococcus luteus not at pH 7 and temperature at 37 °C for 120 h at 80 rpm in
only can grow rapidly in aerobic environment but also have incubator shaker (Sivakumar et al. 2010). The culture was
the ability to resist the unfavourable environments like low then centrifuged at 10,0009g for 10 min at 4 °C. The
pH, filamentous fungi. The bacterial isolates that were supernatant collected was used as crude enzyme solution
harvested in the nutrient agar medium were lysed by and was assayed for enzyme activity. The molecular weight
freezing in liquid nitrogen and thawing the suspension was determined by sodium dodecyl sulphate-polyacryl-
before further freezing. The lysates were centrifuged and amide gel electrophoresis (SDS-PAGE) analysis.
the supernatant was carefully separated from cell debris.
This gives crude extract of enzyme which is called cell-free Optimization of laccase production media
extract and this restores specificity towards the particular
substrate for dye degradation (Das and Charumathi 2012). The medium for production of laccase was optimized for
Hence, the cell-free extract was used. various parameters that affect the growth. These parame-
ters are pH, temperature, incubation time, carbon source,
nitrogen source, organic solvents, inducers and natural
Materials and methods substrates (orange peels, lemon peels, grape peels, wood
chips and rice husk). The optimum pH for enzyme pro-
Materials duction was found by varying the pH of the laccase pro-
duction media from 5 to 9 while other parameters such as
Two different dyes namely CI Acid Black 210 and CI Acid carbon source, nitrogen source were unaltered. Similarly,
Black 234 were obtained from BASF chemicals Pvt Ltd., the temperature range was maintained from 27 to 42 °C,
Chennai, India. All microbiological media and chemicals and incubation period from 0 to 96 h.
used in this study were purchased from Hi-Media chemi-
cals and Sigma-Aldrich, Mumbai, India. Effect of various carbon sources, nitrogen sources
and solvent on laccase production
Isolation and screening of laccase producing bacteria
Various carbon sources such as dextrose, sucrose, maltose,
The laccase producing bacteria was isolated from soil glycerol and mannitol at concentration of 2 % (w/v) were
samples obtained from decaying wood environment by considered separately for optimization study. Different
serial dilution technique. The isolated organism was grown nitrogen sources (0.2 %) like peptone, beef extract,
in Petri plates containing nutrient agar medium supple- ammonium chloride, ammonium nitrate, ammonium sul-
mented with guaiacol as a substrate for production of lac- phate, various organic solvents (3 %) like xylene, acetone,
case enzyme. The nutrient agar medium is composed of chloroform, methanol, ethanol, various inducers (40 lM)
peptic digest of animal disuse-10.00 g/L, beef extract- such as phenol, copper sulphate, ammonium tartarate,
10.00 g/L, sodium chloride-5.00 g/L, agar-15.00 g/L and gallic acid, catechol and natural substrates of orange peels,
final pH (at 25 °C) adjusted to 7.2 ± 0.2. The soil sample lemon peels, grape peels, wood pulp were screened and
collected from decaying wood was further serially diluted optimized by maintaining the other parameters constant. In
and inoculated in nutrient agar plates using spread plate each experiment, the optimum conditions deduced from the
technique. The plates contained 0.02 % of guaiacol solu- previous experiments were considered for study. Then, the
tion as a substrate for screening of laccase producing enzyme was partially purified by ammonium sulphate, ice
organism. Nearly thirty-five colonies were found in Petri cold ethanol method by using the standard procedure
plates out of which four prominent cultures were taken for (Khalid et al. 2007).
research work. The bacterial culture was further sub cul-
tured and stored in nutrient agar medium at -4 °C for Laccase assay
further use. Staining technique and biochemical tests were
carried out for identification of organism. Activity of laccase enzyme is an important parameter that
helps to monitor the degradation of dye. Laccase assay was
Production of laccase enzyme carried out using a quartz cuvette with a total reaction
volume of 1 mL. The activity of the enzyme was assayed at
Enzyme production was carried out in 250-mL Erlenmeyer every 24 h interval for 6 days by measuring the decrease
flasks containing 100 mL of the following liquid medium in the optical density for the azo dye with a Shimadzu

123
1446 J. Kanagaraj et al.

UV—1601 PC UV–Vis spectrophotometer. This method S) is absorbed by the body of microbial species and then gets
was based on the oxidation of ABTS, (2,20 -azino-bis (3- oxidized (degradation rate, rsu) by the enzyme (E) to give a
ethylbenzothiazoline-6-sulphonic acid) at 420 nm with an polar intermediate (M?). The polar radical then further
absorbance coefficient value (36,000 M-1 cm-1). The oxidized to give non-substrate dye (D) that decomposes
reactive mixture composed of 1.5 mL sodium acetate finally into degraded products (P). To study kinetic behav-
buffer (1 mM, pH 5.0), 1.5 mL ABTS (0.5 mM) and iour of dye adsorption and degradation by the enzyme (lac-
1.5 mL culture medium (Hough et al. 2001). Activity of case), a dye concentration of 0.02 % L was taken for
one unit of enzyme is defined as the amount of enzyme that conducting the experiments. The system is studied under the
oxidizes 1l mole of ABTS per minute at 37 °C. The activation of laccase. As the system is well mixed in a con-
activities were expressed in U/mL (Ozdemir et al. 2008). tainer (volume, V),
Accumulation ¼ inputoutput
Dye degradation þ disappearance by reaction

Dye degradation experiments were carried out using dyes As it is a batch system, Accumulation is the disap-
such as CI Acid Black 210 and C.I Acid Black 234 under pearance by reaction
azo category. 1 g of dye was taken in Erlenmeyer flasks ds
V ¼ Vrsu ; ð2Þ
containing 100 mL distilled water. From this, 20 mg of dt
each dye was taken in separate conical flasks where 5 mL ds
of enzyme solution (with activity of 2.75 U/L) was added or ¼ rsu : ð3Þ
dt
(to all the flasks). Cell-free crude extract enzyme was used
Michaelis–Menten growth kinetics can be used to
for degradation of dye sample. Before carrying out the
explain the enzymatic kinetics of the reaction. The kinetics
study, the enzyme was completely centrifuged at
of the reaction is given by a pseudo first-order kinetics
10,000 rpm for 10 min at 4 °C and the supernatant was
k1
taken after filtering the solution with Whatman no. 1 filter E0 þ ½Cd  ,½Cd E0  ! k2 P; ð4Þ
paper. The filtered crude enzyme sample does not contain k1

any live cells in their fluid. The dye degradation study was dCd Vm ½Cd  k2 ½E0 ½Cd 
carried out in shaking condition using shaker incubator at  ¼ kCd ¼ rsu ¼ ¼ : ð5Þ
dt Km þ ½Cd  k1 þ kk2 þ ½Cd 
120 rpm speed and at 37 °C for 96 h. Control was per- 1

formed without adding enzyme to the dye sample for Thin layer chromatography (TLC)
treatment with other conditions kept similar to that of
experimental sample. The % degradation was measured by The parent sample and degraded dye sample were extracted
UV–Vis spectrophotometer by monitoring at specific twice with an equal volume of n-butanol and were subjected
nanometre for each dyes, and then calculated as follows: to TLC. The decanted extracts were concentrated on a rotary
C0  C1 vacuum evaporator (Buchii RII4, Switzerland). Then the
Reduction % ¼  100; ð1Þ samples were resolved on silica gel HF254 TLC plates using
C0
hexane/ethyl acetate/methanol (5:3:2 v/v) as developing
where C0 is initial absorbance and C1 stands for final solvent. The obtained chromatograms were observed under
absorbance. UV light (254 nm) and by exposure to iodine vapours.
The kinetics and mechanism of biodegradation of dye
(10–15 % of used dye in the process goes as wastage) will be UV–Vis spectrophotometric analysis of dye
helpful in explaining (i) decolourization, detoxification of degradation
industrial effluents to stream/environment by microbial species,
(ii) effects of various factors on the dye degradation and (iii) The resultant degraded samples were scanned in the range
scaling up procedures for the system for commercial exploita- of 200–800 nm using UV 1601 spectrophotometer (Shi-
tion at different scales. Thus, the present modelling aims at madzu, Japan) for analysing the cell-free supernatants
investigating the kinetics and mechanism of this enzymatic (5 mL). Spectral shifts caused by the biotransformation of
degradation process through mathematical modelling. dye were observed and noted down.

Mathematical modelling Mass spectrometry

Enzymatic kinetic studies were undertaken to observe the The biotransformed samples were subjected to mass
factors affecting rate of degradation. The present mecha- spectra for finding out the presence of intermediate
nistic model considers that the dye (substrate concentration, metabolites in the degradation process. For this, the

123
Degradation of azo dyes by laccase 1447

biodegraded and control dye samples were dissolved in laccase enzyme, the enzymatic activity was visualized on
acetonitrile solution in 20 ppm range. Further, the samples plates as a reddish-brown zone. The bacterial strain was
were analysed by mass (ESI) spectrum for identification of further sub cultured in nutrient agar plates after screening.
intermediate products of biodegraded dye samples. Mass Then the strain was characterized by staining techniques
spectra were recorded using a Thermo Finnigan LCQ followed by biochemical tests. The gram staining result
Advantage MAX 6000 ESI Mass spectrometer (Senthilv- revealed that the isolated organism belongs to gram posi-
elan et al. 2014). tive cocci, characteristically in tetrads and usually larger
(than staphylococcus aureus) in shape and with yellowish
Fourier Transform Infra Red spectroscopy (FT-IR) pigmented colony morphology.
analysis of biodegraded dye samples The various biochemical tests (Shraddha et al. 2011)
such as Indole production test, Methyl red test, Voges-
In order to find the functional peaks of biodegraded and Proskauer test, Citrate utilization test, Urease test, Triple
control samples, they were subjected to FT-IR spectral sugar iron agar test, Mannitol motility test, catalase test and
analysis. The samples were dried and mixed with potas- oxidase test were carried out for the identification of
sium bromide (1:20; 0.02 g of sample with KBr at a final morphological, physiological and biochemical character-
weight of 0.4 g). The powdered samples were then ization of isolated microorganism. For further confirma-
grounded, desorbed at 60 °C for 24 h and pressed to obtain tion, it was sent to Institute of Microbial Technology
IR-transparent pellets. The spectral absorbance of the (IMTECH), Chandigarh, India and was identified as
samples was recorded using an FT-IR spectrum 2000 Micrococcus luteus (Cameselle et al. 2000). The identifi-
Perkin-Elmer spectrophotometer. The observations were cation was done by verifying molecular analysis of bacte-
collected within the scanning range of 400–4,000 cm-1. rial strain using 16s ribosomal DNA (rDNA) gene
The FT-IR was first calibrated for background signal sequencing by the help of standard gene library.
scanning with a control sample of pure KBr, and then the Table 1 shows the results of biochemical tests for the
experimental sample was scanned (Olukanni et al. 2006). characterization of the bacterial culture. The isolated
organism is found to be catalase test-positive, modified
Estimation of biochemical oxygen demand (BOD) oxidase test- positive and positive for cytochrome C (blue
and chemical oxygen demand (COD) colour was formed). Other biochemical tests such as Indole
production test, Methyl red test, Voges-Proskauer test, Cit-
The biodegraded experimental and control samples were rate utilization test, Urease test, Triple sugar iron agar test
collected from different stages of degradation and were and Mannitol motility test revealed the negative results.
analysed for the pollution loads such as BOD, COD using
the standard methods proposed by APHA (Eaton et al. Measurement of enzyme activity and protein content
1995). The results are expressed in parts per million (ppm)
of the sample. Crude enzyme from the nutrient broth of the Micrococcus
luteus was obtained as cell-free extract without any purifi-
cation. The cell debris was removed from extracellular fluid
Results and discussion of the culture by centrifugation and the supernatant was

Screening of bacteria for laccase production

Table 1 Biochemical tests for the characterization of bacterial
culture
Numerous colonies were identified during primary
S. no. Biochemical tests Results
screening, from which individual colonies were picked out
and streaked in different plates. Finally, about 35 colonies 1. Indole production test Negative
were obtained that were subjected to guaiacol hydrolysis, 2. Methyl red test Negative
out of which one isolate was found to possess capacity to 3. Voges-Proskauer test Negative
produce laccase. The similar result was reported by Do- 4. Citrate utilization test Negative
minguez et al. (2011). 5. Urease test, Negative
6. Triple sugar iron agar test Negative
Biochemical characterization of bacterial culture 7. Mannitol motility Negative
8. Catalase test Positive
For screening of laccase producing bacteria, 0.02 % of 9. Modified oxidase test Positive
guaiacol was supplemented in nutrient agar plates. Due to 10. Cytochrome C (blue colour was developed) Positive
oxidative polymerization of guaiacol by the action of

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1448 J. Kanagaraj et al.

Table 2 Purification of Enzymes

S. no. Enzyme fraction Total Total Total Specific
volume activity protein activity
(mL) (U) (mg) (U/L)

1. Crude extract 500 140 188 0.75

2. 90 % Ice cold 10 25.2 2.6 9.7
ethanol method
3. Ammonium sulphate 50 53.7 4.6 11.6
precipitation
method

taken for further experiment. The cell-free supernatant is

called crude enzyme. Cell-free extract refers to extracellular
enzyme without any biomass, otherwise, it is the crude
enzyme that does not contain any biomass. The activity of the
crude enzyme and partially purified enzyme was assayed.
The total activity and specific activity of the enzymes were
tabulated. The total protein content of the crude extract was Fig. 1 Enzyme activity of Micrococcus luteus at different conditions
188 mg, ethanol precipitated enzyme was 2.6 mg and of pH, temperature and time
ammonium sulphate precipitated enzyme was 4.6 mg
(Table 2). It can be seen from Table that an increase in carbon sources, dextrose supported good growth and
activity to 15.5-fold is attributed to the purification of the laccase production. Similarly, nitrogen sources like yeast
enzyme. Using ice cold ethanol method activity was found to extract, peptone, ammonium sulphate, ammonium chlo-
increase up to 10.7 over that of crude extract. The partially ride and ammonium nitrate at 1 % conc. were tested for
purified activity was increased to 15.5-folds over crude laccase production in Micrococcus luteus. Among them,
extract (Champagne and Ramsay 2010). Yeast extract supported the maximum laccase production
(Souza-Ticlo et al. 2006). Acetone supported the maxi-
Study of Laccase production at different cultivation mum laccase production among five different solvent
conditions sources like xylene, acetone, ethanol, methanol and
chloroform (Dominguez et al. 2005). Among various
The laccase enzyme activity was studied for various param- inducers, used copper sulphate showed maximum enzyme
eters such as pH, temperature and incubation time to find out activity as compared to other inducers such as phenol,
the optimum conditions. The results are presented in Fig. 1. It ammonium tartarate, gallic acid, and catechol. Among
is evident from the figure that the pH significantly influenced various natural substrates, orange peel showed maximum
the extracellular protein content and laccase activity. The enzyme activity as compared to other natural substrates
enzyme shows maximum activity at pH 7. Laccase activity such as lemon peels, grape peels, wood chips and rice
was observed at broad range of temperature from 27 to 40 °C. husk (Table 3). Values of enzyme activities are presented
The maximum laccase production was observed at 37 °C. The with standard deviation. Enhancement in production of
laccase production is also monitored at various incubation laccase can be done using natural substrate (agro wastes)
periods from 0 to 96 h. The time course of laccase production which is easily available and economically feasible.
was monitored. Claus (2004) reported that the maximum Orange peel contains chemical components of volatile oil,
laccase production is observed at pH 7 in 72 h. In the present numerous flavonoids, vitamins and phenolic acid. Phe-
experiment, similar results were observed except that maxi- nolic acids are aromatic compounds containing phenolic
mum production was achieved at 96 h. ring in their structure which plays a major role in
inducing laccase production. Orange peel yields better
Effect of laccase production on carbon, nitrogen, laccase production due to its broad substrate specificity
organic solvents, various inducers and natural towards aromatic compounds (Ellen et al. 2008).
substrates
Analyses of molecular weight of the enzyme
The production of laccase enzyme is enhanced using
various carbon sources such as mannitol, maltose, glu- SDS-PAGE analysis is carried out to find out the molecular
cose, dextrose and sucrose. Among the five different weight of the laccase. The results are presented in Fig. 2.

123
Degradation of azo dyes by laccase 1449

Table 3 Effect of enzyme activity on different carbon sources, It is seen from the Fig. 2 that the enzyme obtained by
nitrogen sources, organic solvents, inducers and natural substrates partially purified ammonium sulphate and 80 % ice cold
S. no. Enzyme activity (U/L) ethanol method followed by dialysis generates the bands in
the lane 1 and 2 corresponding to the molecular weight of
1. Carbon sources
laccase enzyme as 63 kDa.
Dextrose 2.55 ± 0.50 Literature reports that similar results have been obtained
Sucrose 2.17 ± 0.30 by the other researchers also. The molecular weight and pH
Maltose 1.75 ± 0.10 of the laccase enzyme varies according to sources e.g.
Mannitol 2.35 ± 0.30 plant, insect and microorganism (bacteria and fungi).
Lactose 1.98 ± 0.15 Normally, the laccase enzymes contain molecular weight
2. Nitrogen sources ranging from 50-130 kDa (Xu 1999; Baldrian 2006; Ben-
Peptone 2.15 ± 0.30 field et al. 1964). Molecular weight of enzyme depends
Yeast extract 2.6 ± 0.40 upon its carbohydrate content that constitute up to 45 % for
Ammonium chloride 1.75 ± 0.10 plant laccase and 10–30 % for fungal laccase, involving the
Ammonium nitrate, 2.29 ± 0.30 process of glycosylation (Rogalski and Leonowicz 2004).
Ammonium sulphate 1.9 ± 0.15 The carbohydrate compounds such as hexamine, glucose,
3. Organic solvents fructose, galactose, arabinose and mannose are found in the
Xylene 2.0 ± 0.30 composition. Mannose is one of the important carbohydrate
Acetone 2.3 ± 0.40 compounds that involves in the glycosylation process
Chloroform 1.8 ± 0.15 which is very important for enzyme secretion, activity,
Methanol 2.25 ± 0.30 copper retention and lignin formation (Xu 1999; Vasdev
Ethanol 1.65 ± 0.1 et al. 2005; Morozova et al. 2007). The carbohydrate
4. Inducers portion of the laccase enzyme ensures the conformational
Phenol 2.3 ± 0.40 stability of the globule and protects it from proteolysis and
Copper sulphate 2.75 ± 0.50 inactivation by radicals.
Ammonium tartarate 2.1 ± 0.40
Gallic acid 1.8 ± 0.10 Biodegradation of azo dyes
Catechol 2.45 ± 0.50
5. Natural substrates Five millilitre of the crude enzyme was added to conical
Orange peels 2.4 ± 0.40 flasks containing 20 mg of two different dye solutions,
Lemon peels 2.0 ± 0.20 namely, CI Acid Black 210 and CI Acid Black 234. These
Grape peels 1.8 ± 0.15 were degraded by laccase enzyme. The rate of dye degra-
Wood chips 2.1 ± 0.20 dation was monitored at different time intervals (0, 24, 48,
Rice husk 1.6 ± 0.10 72 and 96 h) of time at specific wavelengths. The per-
centage of degradation was found to be 96.4 and 92.2 %,
respectively, while the control sample showed degradation
of 83 % (Fig. 3) after the end of 96 h. It has been found
that Acid Black 210 gives 96.4 % of degradation which is
the highest among the two dyes studied (Arias et al. 2003).

Results on mathematical modelling

It is assumed that dye molecules get absorbed on the sur-

face of microbes and then degrades. The experimental data
on concentration of remaining dye during degradation are
normalized (represented as q). After simulation, the results
are presented in Fig. 4a. The variable, q is defined as
q ¼ c0cc
0
. Let t be incubation time, CE be enzyme concen-
tration (mL), m be partial reaction order with respect to
enzyme, Cd be concentration of dye in effluent (mg/L) and
Fig. 2 SDS-PAGE. M marker, lanes 1 and 2 crude extract of laccase, n is partial reaction order. If k be its specific reaction
lanes 3 and 4 partially purified laccase enzyme rate then.

123
1450 J. Kanagaraj et al.

A plot (Fig. 4a) of 1/q against c/q for the CI Acid Black
210, shows straight line indicating that the growth obeys
Eq. 9. From this curve, slope (k = 0.042) can be obtained
for adsorption isotherm. Straight line nature of the graph
reveals that the adsorption isotherm follows Langmuir
mechanism. From Eq. (9), we get m = 1. Using this
model, theoretical values were calculated to find q after
which they are plotted as shown in Fig. 4b. This figure
shows that error between theoretical and experimental
values are very less confirming suitability of the model for
the specific dye degradation process. From this Fig. 4b, the
value of correlation coefficient, R2 is found to be 0.97.

TLC analysis
Fig. 3 Biodegrdation of dyes at various time durations using laccase
enzyme
The formation of intermediates in this degradation study of
dye by laccase is further supported by TLC analysis
(Fig. S1). The chromatogram of n-butanol extracted sam-
ples of degraded dye showed the movement of dye band
corresponding to parent dye. The distance travelled by the
degraded dye is higher than the parent dye. The retention
factor (Rf value) is also found higher in the experimental
sample compared to parent dye. The Rf values for the dyes
studied here are 0.89, 0.70 for CI Acid Black 210 and CI
Acid Black 234, respectively, in comparison with Rf values
of control samples as 0.32 and 0.40, respectively. The
bands observed in degraded dye indicate the formation of
metabolic intermediates that are different from the parent
dye (Galai et al. 2009). It can be seen that the higher Rf
value in the experimental sample interprets the presence of
degraded intermediate products compared to the control
sample. However, further confirmation of the formation of
intermediate metabolites has been achieved and discus-
sions are presented in a later section.

Mass spectra analysis

Mass spectra analysis was carried out for analysis of lac-

Fig. 4 a Plot of 1/q against c/q for the adsorption of dye by biological
means showing validation of Langmuir isotherm. b Plot of 1/q against case degraded dye samples. The dye residue was collected
c/q: comparison between experimental and theoretical model for the from the experimental conical flasks by adding equal vol-
dye degradation/decolourization process ume of ethyl acetate solution to the flaks. The moisture was
removed from the residues using anhydrous Na2SO4 and
dCd was further placed in rotary evaporator for drying. After
¼ kCEm Cdn ð6Þ
dt that the crystals obtained was collected and dissolved in
For n = 1, we get, acetonitrile solvent in the range of 15–20 ppm before
Cd ¼ Cd0 expðkCdm tÞ ð7Þ analysis of mass (ESI) (McMullan et al. 2001). The Mass
spectra were recorded in the range of 200–1,000 m/z. The
 Cdm
Cd mass spectra analysis was carried out using a Thermo
ln ¼ kt ð8Þ
Cd0 Finnigan LCQ Advantage MAX 6000 ESI Mass spec-
   trometer (Senthilvelan et al. 2014).
1 1 Cd0
m¼ ln ln ð9Þ The mass spectra analysis of dye samples, before and
ln½Cd  kt Cd
after degradation is important for finding out the

123
Degradation of azo dyes by laccase 1451

Fig. 5 Mass spectrum showing the structure of CI Acid black 210

Fig. 6 Mass spectrum showing the structure of CI Acid Black 234
and its by-product after the treatment of enzymes: a before degra-
and its by-product after the treatment of enzymes: a before degra-
dation. Mass spectrum showing the structure of CI Acid black 210
dation. Mass spectrum showing the structure of CI Acid Black 234
and its by-product after the treatment of enzymes: b after degradation
and its by-product after the treatment of enzymes: b after degradation

intermediates in the degradation process. The dye that respect to their molecular weight and the resulting inter-
undergoes degradation process results in generating inter- mediates are presented in Figs. 5, 6 and 7. It is seen from
mediate metabolites. These metabolites are identified with the Figs, that CI Acid black 210 dye is degraded into

123
1452 J. Kanagaraj et al.

Fig. 7 A Structure of CI Acid

Black 210 and its intermediates
a H
N
O
S OH NH2
(a–e) found out from mass N O
N N N
spectrum after the treatment of N N
enzymes. B Structure of CI Acid H2N NH2 O
O S
Black 234 and its intermediates S OK
KO O O
(a–e) found out from mass
spectrum after the treatment of
enzymes C.I. Acid Black 210
Mol. wt. 893.02

NH2 SO3H

NH2

H2N NH2 NH2 NH2

benzene-1,2,4-triamine benzene-1,4-diamine 4-aminobenzenesulfonic acid
Mol.wt 123.16 Mol.wt 108.07 Mol.wt 173.17
(a) (b) (c)

OH NH2
H2N NH2 NH2

HO3S SO3H
3,4,6-triamino-5-hydroxynaphthalene-2,7-disulfonic acid
aniline

Mol.wt 349.34 Mol wt 93.13

(d) (e)

C.I. Acid Black 234

Mol. wt. 860.8

H2N NH2
NH2
OH NH2
OH NH2
2,7,8-triaminonaphthalen-1-ol 7,8-diaminonaphthalen-1-ol
Mol. wt. 189.21 Mol. wt. 174.2
(a) (b)

NH2 NH2 SH

H2N
H2N NH2 NH2
benzene-1,4-diamine benzene-1,2,4-triamine 4-aminobenzenethiol

Mol. wt. 108.14 Mol. wt. 123.16 Mol. wt. 125.19

(c) (d) (e)

123
Degradation of azo dyes by laccase 1453

various intermediates (Fig. 7) such as benzene 1,2,4-tri-

amine, benzene 1,4-diamine, 4-amino benzene sulphonic
acid, 3,4,6-triamine 5-hydroxy naphthalene- 2,7-disul-
phonic acid and aniline with respective mass of 123.16,
108.07, 173.17, 349.34 and 93.13. Similarly, CI Acid Black
234 undergoes degradation process and produces the
intermediates such as 2,7,8-triamino naphthalene-1-ol with
mass of 189.21 and 7,8-diamino napthale-1-ol with mass of
174.2, benzene-1,4-diamine with mass of 108.14, benzene
1,2,4-triamine with mass of 123.16 and 4-amino benzene-
thiol with mass of 125.19. It is evident from the mass
analyses report (Figs. 5, 6) that all the above dyes in the
presence of laccase undergo degradation phenomenon and
result in various intermediate metabolites (Pandey et al.
2007).
Laccase works for cleavage of –N=N– bonds in azo dye
compounds. The plausible mechanism of degradation of
azo dyes has been suggested. The resulting phenoxy radi-
cals further undergoes structural changes to form amines
and carbonyl compounds. The resulting amines are further
degraded by the enzyme both aerobically and anaerobi-
cally. The 1-(3-chloro-4-methoxyphenyl) ethanone under-
goes further structural modification to form 2-chloro-1,4-
Fig. 8 FT-IR spectrum of the degraded dye samples (a control
dimethoxybenzene and 3-chloro-4-methoxybenzaldehyde
sample Acid black 210 at zeroth hour; b control sample Acid black
(Font et al. 2003). The action of laccase on dye molecule 210 after 96 h and c experimental dye Acid black 210 after 96 h)
depends on dye’s chemical structure, differences in elec-
tron distribution and charge density and also steric hin-
drances, which plays major role for extending the Similarly, no new peaks appeared in the region between
degradation rate (Chivukula and Renganathan 1995; Pasti- 1,340 and 1,250 cm-1 corresponding to –NH2 suggests that
Grigsby et al. 1992). Efficiency of degradation of dye the azo linkage could be transformed into N2 or NH3. The
molecules by laccase depends on the presence of hydroxyl presence of aromatic amine in the degraded sample indi-
groups and their strong electron donating moieties in ortho cates the effect of laccase activity. It has been reported that
and para location of the azo bond of the dye molecule the azo compounds with hydroxyl or amino groups were
(Kandelbauer et al. 2004). more likely to be degraded than those with methyl, meth-
oxy, sulfo or nitro groups. Usually, the presence of sul-
FT-IR analysis of enzyme degraded dye samples phonates in reactive dye structures results in low levels of
colour removal. However, this is not applicable to direct
FT-IR spectra analysis is very useful in detecting the dyes that usually exhibit high levels of colour removal
compounds that are transformed from azo linkages into independent of the number of sulphonate groups in the dye
biomass. FT-IR spectra of the degraded dyes are presented structure, reinforcing the idea that steric hindrance and the
in Fig. 8. The bands located within the range of number of azo bonds are responsible for the different
1,610–1,630 and 1,312 cm-1 are due to the presence of azo degradation times (Whiteley 2007).
linkages of –N=N–s on aromatic structure and –N=N–
stretching in a substituted compound, respectively. These Pollution-load (BOD and COD) reduction
peaks decreased during the treatment of laccase, confirm-
ing the previous UV–Vis results about azo linkage dis- Experimental and control samples are subjected to their
ruption. The peak observed at 1,642 cm-1 is due to the pollution-load profile, namely, BOD and COD content. The
conjugation of C=C and C=O groups, suggesting that this samples were periodically withdrawn from 0 to 96 h and
peak could belong to a carbonyl group in a carboxylic acid, the results of analysis are presented in Table 4. These
ketone, ester or conjugated aldehyde groups attached to an values indirectly indicate amount of degradation. The table
aromatic ring. The fact that no new peak appeared between shows the % BOD and COD reductions of the dye along
3,300 and 3,500 cm-1 is attributed to the absence of azo with ratio of BOD/COD. From this table, it can be seen that
bond and OH group in position relative to the azo linkages. CI Acid Black 210 dye gives BOD reduction of 94.01 %

123
1454 J. Kanagaraj et al.

Table 4 Estimation of BOD and COD values and BOD/COD ratio

Dyes BOD (different durations (h)) COD (different durations (h)) BOD/COD ratio (different durations (h)) (%)

0 24 48 72 96 % Reduction 0 24 48 72 96 % Reduction 0 24 48 72 96

CI AB 210 (ppm) 802 653 378 184 48 94.01 2,451 1,964 1,076 564 160 93.47 32.72 33.24 35.13 32.62 30.00
CI AB 234 (ppm) 858 689 402 197 64 92.54 2,530 2,020 1,260 670 210 92.17 33.90 34.10 31.90 29.40 34.7
Control (ppm) 766 657 571 458 230 69.97 2,342 2,112 1,896 1,464 760 67.54 32.70 31.10 30.11 31.28 30.47

CI AB 210 CI Acid Black 210, CI AB 234 CI Acid Black 234

while CI Acid Black 234 shows a BOD reduction of various intermediates such as benzene 1,2,4-triamine, ben-
92.54 %. Control sample is prepared without adding the zene 1,4-diamine, 4-amino benzene sulfonic acid, 3,4,6-tri-
enzyme which gives a BOD reduction of 69.97 %. COD amine 5-hydroxy naphthalene-2,7-disulfonic acid and aniline
analysis of experimental dye sample exhibits 93.47 % with respective mass of 123.16, 108.07, 173.17, 349.34 and
reduction of COD for CI Acid Black 210 dye and 92.17 % 93.13. The mechanism behind the bioprocess is formulated
for CI Acid Black 234 dye whereas the control samples with a comprehensive mathematical model confirming
give a COD reduction of 67.54 % which are commensurate Langmuir isotherm of dye adsorption. According to the
with the results of other researchers (Kandelbauer et al. mechanism, the polarized dyes combine with the enzymes and
2004). Maximum dye degradation and reduction of BOD formulate intermediates, which then degrade into various
and COD concentrations of these dyes has been achieved in products. TLC results reveal the degraded dyes show higher Rf
96 h after which solution has become colour-less that values as compared to parent dyes. BOD showed a reduction
confirms completion of degradation. However, data from of 94.01 and 92.54 % for CI Acid black 210 and CI Acid black
Table 4 reveal that after 96 h also some recalcitrant 234, respectively, whereas COD exhibited a reduction of
organic carbons are present that were contributing towards 93.47 and 92.17 % for the above dyes. The respective ratios of
these values. Results on further reduction of BOD & COD BOD/COD are maintained at about 30 %. From overall
were not significant beyond 96 h indicating that residual investigation, it can be concluded that laccase can be sug-
(recalcitrant organic carbon) present in the solution may gested for commercial use for degradation of azo dyes.
contribute towards the values of BOD and COD. It can also
be observed that BOD/COD ratio in different durations of
sampling is maintained at about 1:3 or 30 %. The experi-
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