World Journal of Microbiology & Biotechnology 20: 545–550, 2004.
2004 Kluwer Academic Publishers. Printed in the Netherlands.
545
Decolourization and detoxification of methyl red by aerobic bacteria from
a wastewater treatment plant
O. Adedayo1, S. Javadpour2, C. Taylor3, W.A. Anderson1,* and M. Moo-Young1
1
Department of Chemical Engineering, University of Waterloo, Ontario, Canada N2L 3G1
2
Hormozgan University of Medical Science, Iran
3
Department of Biology, University of Waterloo, Ontario, Canada N2L 3G1
*Author for correspondence: Tel.: +1-519-888-4567 Ext. 5011, Fax: +1-519-746-4979,
E-mail: [email protected]
Received 11 August 2003; accepted 6 January 2004
Keywords: Azo dyes, biodegradation, Pseudomonas, textile wastewater, Vibrio
Summary
Bacterial cultures from a wastewater treatment plant degraded a toxic azo dye (methyl red) by decolourization.
Complete decolourization using a mixed-culture was achieved at pH 6, 30 C within 6 h at 5 mg/l methyl red
concentration, and 16 h at 20–30 mg/l. Four bacterial species were isolated that were capable of growth on methyl
red as the sole carbon source, and two were identified, namely Vibrio logei and Pseudomonas nitroreducens. The
Vibrio species showed the highest methyl red degradation activity at the optimum conditions of pH 6–7, and 30–
35 C. Analysis by NMR showed that previously reported degradation products 2-aminobenzoic acid and N,Ndimethyl-1,4-phenylenediamine were not observed. The decolourized dye was not toxic to a monkey kidney cell line
(COS-7) at a concentration of 250 lM.
Introduction
Synthetic dyes have a wide application in the food,
pharmaceutical, textile, leather, cosmetics and paper
industries (Rafii et al. 1997; Hildenbrand et al. 1999;
Claus et al. 2002; Selvam et al. 2003a) due to their ease
of production, fastness, and variety in colour compared
to natural dyes. More than 100,000 commercially
available dyes are known and close to one million tons
of these dyes are produced annually worldwide (McMullan et al. 2001; Selvam et al. 2003b).
Azo dyes are the largest group of these dyes used in
industry (Ramalho et al. 2002; Maximo et al. 2003),
representing more than half of the annual production
(Stolz 2001). Azo dyes are characterized by the presence
of one or more azo groups AN@NA (Manu & Chaudhari 2002), which are responsible for their colouration
and when such a bond is broken (degraded) the
compound loses its colour (O’Neill et al. 2000; Chagas
& Durrant 2001). They are the largest and most versatile
class of dye (Manu & Chaudhari 2003), but have
structural properties that are not easily degradable
under natural conditions (Rajaguru et al. 2000) and
are not typically removed from water by conventional
wastewater treatment systems (Wamik et al. 1998). Azo
dyes are designed to resist chemical and microbial
attacks (Chagas & Durrant 2001; Ramalho et al. 2002),
and to be stable in light and during washing (Rajaguru
et al. 2000). Many are carcinogenic (Hidenbrand et al.
1999) and may trigger allergic reactions in man (Waldmann & Vakilzadeh 1997). It is estimated that over 10%
of the dye used in textile processing does not bind to the
fibres and is therefore released to the environment
(Weber & Stickney 1993; Reisch 1996). Some of these
compounds pose a serious threat (van der Zee et al.
2001; Martins et al. 2002) because of their carcinogenic
potential (Chung et al. 1992; Hidenbrand et al. 1999;
Chagas & Durrant 2001) or cytotoxicity (Waldmann &
Vakilzadeh 1997). Several physicochemical techniques,
such as carbon adsorption, have been used for treating
wastewater-containing dyes (Panswed & Wongehaisuwan 1986; Yoshida et al. 1991; Zhou & Zimmermann
1993; Churchley 1994; Yeh & Thomas 1995; Sokmen
et al. 2001; Okazaki et al. 2002; Da Silva & Faria 2003;
Verma et al. 2003; Zhang et al. 2004) but the methodologies have serious limitations (Quezada et al. 2000;
Selvam et al. 2003a,b), and may lead to the generation
of toxic by-products. The new environment-friendly
regulations concerning textile products ban marketing
of textiles dyed with azo dyes capable of reductively
splitting carcinogenic aromatic amines (Pielesz et al.
2002), and they also ban the discharge of coloured waste
(Maximo et al. 2003). These laws, coupled with the
problems resulting from physicochemical treatment of
azo dyes, have generated interest in the wider use of
biodegradation (Banat et al. 1996; Bustard et al. 1998;
McMullan et al. 2001), which is the predominant natural
mechanism in soil. Early work on biodegradation was
546
more focused on anaerobic conditions (Larsen et al.
1976; Chung et al. 1978; Rafii & Cerniglia 1993) and
decolourization of dye resulting from breaking of the
azo-bond was observed as the first step in microbial
degradation (Brown & Laboureur 1983; Brown &
Hamburger 1987; Weber & Wolfe 1987; Haug et al.
1991; Zaoyan et al. 1992), but it may also result in the
release of toxic intermetabolites (Isik & Sponza 2003).
However, the dyes may be degraded by aerobes
(Thurnheer et al. 1988; Dickel et al. 1993). Aerobic
decolourization of azo dyes has been confirmed by a
number of investigators (Cripps et al. 1990; So et al.
1990; Govindaswami et al. 1993; Jian & Bishop 1994;
Wong & Yuen 1996; Coughlin et al. 1997; Tepper et al.
1997; Critina et al. 2003), but in most cases the toxicity
of the dye degradation products has not been assessed.
The objective of this work was to study the aerobic
degradation of an azo dye (methyl red) by a consortium
of bacteria from activated sludge, to isolate and identify
some of the most active bacterial species, and to
evaluate their performance in vitro with a view to
understanding their potential for treating wastewater,
especially considering the potential for toxicity reduction of the dye solution.
Materials and methods
Chemicals and media
Dimethylaminoazobenzene-o-carboxylic acid (methyl
red CI13020) crystal, 2-aminobenzoic acid and N,Ndimethyl-1,4-phenylenediamine (obtained from Fisher
Scientific FairLawn, NJ) were used as standard compounds in this investigation. Biolog kits (GN2 MicroPlate) for microbial identification were obtained from
Biolog, Inc. (Hayward, CA). The agar media (Nutrient,
Sabouraud dextrose and Malt extract agar) were
obtained from Difco laboratories (Detroit, MI).
Microorganism preparation and enumeration
Microorganisms were obtained from activated sludge
from the municipal wastewater treatment plant in
Waterloo, Ontario, Canada. The sludge was mixed to
allow for proper distribution of the microbes and samples
from the supernatant liquid were plated out using
nutrient, malt extract and Sabouraud dextrose agar (for
bacteria, yeast and fungi, respectively) to confirm viability and determine the microbial population of the sludge.
Acclimation procedure
An adaptation procedure was used to acclimatize the
microbes (Ogawa et al. 1981) to an increasing concentration of methyl red as the carbon source. The singleflask procedure of adaptation (Watson 1993) without
transfer of microorganisms was employed but with a
little modification. Methyl red solution was added to a
O. Adedayo et al.
250 ml Erlenmeyer flask containing (45 ml) of minimal
medium (MM) (Wong & Yuen 1996; Rajaguru et al.
2000) and (5 ml) activated sludge. The flask was
maintained in a rotary shaker at 180 rev/min at room
temperature of 25 C. Methyl red was added over the
next 5 days to bring the concentration of the dye in the
flask to 1 mg/l at first, and gradually to a final
concentration of 5 mg/l as the main carbon source. A
subculture from the flask was prepared for further tests
while prepared slants were kept at 4 C for reference.
Degradation of methyl red by bacteria
Degradation of azo dye was determined using the
modified method of Wong & Yuen (1996). Aliquots
from the test solution were centrifuged at about
2500 · g for 5 min. The absorbance of the culture
supernatants was measured at 430 nm (which is the
absorbance maximum for the orange colour of methyl
red) using a Spectronic 20 spectrophotometer.
Effect of pH on biodegradation
Colonies of an overnight growth were suspended in
normal saline to obtain an optical density of 0.6 at
610 nm wavelength. One milliliter of the cell suspension
was used to inoculate 250 ml Erlenmeyer flasks containing 50 ml of MM and 5 mg methyl red/l. The pH of
the medium was adjusted to 6, 7, 8, or 9 with
hydrochloric acid or sodium hydroxide. The cultures
were incubated at room temperature in a rotary shaker
running at 180 rev/min.
Effect of temperature on biodegradation
The biodegradation procedure was carried out in 250 ml
flasks containing sterile MM with methyl red. The pH of
the medium was adjusted to 6 and each flask was
inoculated with an equal cell density of the activated
sludge microbes. All flasks were maintained in a rotary
shaker at 180 rev/min but at a controlled temperature.
Isolation and identification of methyl red-degrading
bacteria
One milliliter of the bacterial culture from activated
sludge was spread on an agar plate containing MM to
which 15 g of agar/l and 5 mg of methyl red/l were
added. The plate was incubated at 30 C for 48 h. The
colonies formed were purified, Gram stained and
examined for oxidase response. Identification was carried out using Biolog GN2 microplates, and all reagents
and accessories for Biolog identifications were used
according to the instructions of the manufacturer.
NMR analysis of decolourized methyl red
The decolourized methyl red was repeatedly extracted
with an equal volume of dichloromethane (Wong &
547
Methyl red decolourization and detoxification
Cytotoxicity testing
Culture of COS-7 cells
COS-7, an African green monkey kidney fibroblast-like
cell line transformed with a mutant of SV40 that codes
for wild-type T antigen, was used to test the cytotoxicity
of methyl red on cultured cells. COS-7 cells were cultured
in Dulbecco’s Modified Eagle’s medium (Sigma) with
0.584 g/l of L -glutamine, 4.5 g/l of glucose, and 0.37%
sodium bicarbonate. The culture media was supplemented with 10% fetal bovine serum (Gibco BRL) and
100 units of penicillin/streptomycin (Gibco BRL). The
cells were maintained in culture at 37 C in a humidified
atmosphere of 5% CO2 and 95% air. The cells were subcultured every 3–4 days by detaching the adherent cells
with a solution of 0.25% trypsin and 1 mM EDTA
(Sigma). The detached cells were dispensed at a split ratio
of 1:10 into a new culture dish with fresh media.
Survival assay for determination of cytotoxicity
COS-7 cells were seeded onto 24-well tissue culture plates
(Corning) and allowed to grow for 24 h. Fresh culture
medium containing either methyl red or decolourized
methyl red, at concentrations of 1, 50, or 250 lM, was
then added to the cells. As a control for bacterial toxins,
solutions that had been incubated with bacteria but
without methyl red and then filter sterilized were also
added to the cells at an equivalent volume. After exposure
to the cells for varying time periods up to 48 h, the
medium containing the dead, floating cells was removed
and the adherent cells were detached from the plate using
a solution of trypsin-EDTA. After detachment, the
medium containing the dead cells was placed back into
the well with the detached cells and mixed. An aliquot of
cells was removed and incubated for at least 2 min with an
equal volume of trypan blue. The percentage of surviving
cells was then determined by counting the number of live
and dead (stained blue) cells on a hemacytometer. At least
200 cells were counted per sample.
5.4 · 105 c.f.u./ml for bacteria, fungi and yeast, respectively. It is readily seen that bacteria were predominant
in terms of cell numbers.
Four bacterial isolates that decolourized methyl red
on MM were isolated from activated sludge and coded
(MRd ¼ methyl red decolourizer). The bacteria were
screened on the basis of the Gram reaction and oxidase
test response. Two strains that were able to degrade
methyl red within 6 h were isolated for further identification using Biolog GN2 micro plates. They were
identified as Pseudomonas nitroreducens and Vibrio
logei, with similarity values of 0.931 and 0.673, respectively, and probabilities of 100% each. According to this
method, identification is acceptable when similarity
values greater than 0.5 are obtained. The other two
isolates with slower degradation kinetics could not be
conclusively identified.
Effects of conditions on methyl red biodegradation
The mixed-culture totally decolourized 5 mg/l methyl
red within 6 h at pH 6 and 7 but exhibited 82% and
65% decolourization of the dye at pH 8 and pH 9,
respectively (Figure 1). At the lowest concentration of
methyl red, the rate of decolourization was slow within
the first 2 h, but increased threefold at pH 6 and 7 and
about fivefold at pH 8 and 9 after 4 h. The microbial
degradation of dye at higher concentrations of methyl
red was optimal at 30–35 C (Figure 2). The microbes
degraded the dye poorly within the first 5 h, but had
improved activity over the last 10 h where complete
decolourization of dye was recorded.
Vibrio logei and Pseudomonas nitroreducens both
decolourized higher concentrations of methyl red faster
than the mixed-culture (Figures 3 and 4). Biodegradation of the dye was poor within the first 4 h but
100
Degradation(%)
Yuen 1996). The chloromethane extracts were pooled
and evaporated to 10 ml at 40 C in a rotary evaporator, and then transferred to a test tube which was left to
dry overnight. The aqueous fraction of the degraded
methyl red was freeze-dried and kept as such for
subsequent analyses.
Solutions of standard methyl red, 2-aminobenzoic
acid, N,N-dimethyl-1,4-phenylenediamine and the dichloromethane and aqueous extracts were prepared,
using
either
deuterochloroform
or
deuterodimethylsulphoxide as the solvent. The 1H NMR
spectra were obtained on a Bruker Ac-300 (300 MHz)
NMR Spectrometer.
90
pH 6
80
pH 7
70
pH 8
60
pH 9
50
40
30
20
10
0
Results and discussion
The supernatant from the mixed activated sludge sample
had a microbial composition of 3.7 · 108, 5.0 · 103, and
0
2
4
6
Time(h)
Figure 1. Effect of pH on the degradation of methyl red by a mixed
culture from the activated sludge supernatant.
548
O. Adedayo et al.
100
100
Photobacterium
90
90
Pseudomonas
25 ˚C
80
30 ˚C
Mixed culture
80
35 ˚C
70
60
50
Degradation ( %)
Degradation (%)
70
40
30
20
60
50
40
30
10
20
0
0
10
5
15
10
Time (h)
0
Figure 2. Effect of temperature on the degradation kinetics, using the
mixed culture.
0
10
5
15
Time (h)
increased fivefold for Vibrio logei in 8 h. Other strains
were not as active.
Figure 4. Decolourization of 30 mg/l methyl red by the isolated and
mixed culture bacteria, at pH 6 and 30 C.
Toxicity test
Tests showed that the bacterial component released to
the medium during biodegradation was not cytotoxic,
and the COS-70 cells died as a result of the toxicity of
methyl red (Figure 5). However, the decolourized/
degraded methyl red was non-toxic to the monkey
120
100
90
100
80
Survival (%)
Degradation (%)
70
60
50
80
60
40
Decolorized Methyl
40
Red
Bacteria Only
30
20
20
Photobacterium
Methyl Red
Mixedculture
10
Pseudomonas
0
0
0
0
10
5
15
1
50
250
Concentration (µM)
Time (h)
Figure 3. Comparison of the decolourization of 20 mg/l methyl red, at
pH 6 and 30 C, by the mixed culture, with the two isolated and
identified bacteria.
Figure 5. Survival of COS-7 cells exposed for 48 h to methyl red or
degraded methyl red at various concentrations. ‘Bacteria only’ is a
control to test for the effect of bacterial components released to the
medium.
549
Methyl red decolourization and detoxification
(Jian & Bishop 1994; Coughlin et al. 1997; Tepper et al.
1997).
Further development of bioprocessing strategies for
optimization of the azo dye-degrading potentials of
Vibrio logei and Pseudomonas reducens may contribute
to detoxification of these dyes in industrial wastewater
treatments.
120
100
Survival (%)
80
60
Acknowledgements
This work was supported in part by the Natural Sciences
and Engineering Research Council of Canada.
40
Decolorized methyl
red
20
Methyl red
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0
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12
24
Time (h)
36
48
Figure 6. Survival kinetics of COS-7 cells treated with 250 lM methyl
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