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Decolorization of Textile Azo-Metal Complex Dyes by a Halophilic Bacterium

Isolated from Çamaltı Saltern in Turkey

Article  in  CLEAN - Soil Air Water · February 2011

DOI: 10.1002/clen.201000150

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Clean – Soil, Air, Water 2011, 39 (2), 177–184 177

Armağan Demirci1
Mehmet Burçin Mutlu1
Research Article
Alaettin Güven2
Elif Korcan3 Decolorization of Textile Azo-metal Complex Dyes
Kıymet Güven1
by a Halophilic Bacterium Isolated from Çamaltı
1
Faculty of Science, Department of Saltern in Turkey
Biology, Anadolu University, Eskiş¸ehir,
Turkey
2
The decolorization of some of azo-metal complex dyes used in textile industry was
Faculty of Science, Department of
Chemistry, Anadolu University,
investigated in this study. The halophilic prokaryotes isolated from a solar sea-saltern
Eskiş¸ehir, Turkey (Çamaltı) in Turkey were screened for resistance to five commercial azo and mixture of
3
Faculty of Arts and Science, azo-metal complex dyes. Only one bacterium was found to be resistant against two of
Department of Biology, Afyon dyes, namely Lanaset Navy R and Lanaset Brown B. The bacterium was identified as
Kocatepe University, Afyon, Turkey
Halobacillus sp. C-22 according to 16S rRNA gene sequence analyses. Decolorization
experiments were carried out at 120 mg/L concentration of both dyes, at room tempera-
ture, and with an acidic pH of 4.5. Lanaset Brown B was decolorized at a high adsorbance
ratio (96.12%) at the 78th hour. However, Lanaset Navy R was rapidly decolorized in
10 min (46.67%) and showed the highest adsorbance ratio (60.66%) at the third hour.
Freundlich and Langmuir equilibrium isotherm models were used to evaluate the
adsorption of dyes and Freundlich isoterm was more suitable for biosorpsiyon of both
azo dyes. The functional groups on Halobacillus sp. C-22 for decolorization were charac-
terized by FT-IR. This is the first study to reveal potential of Halobacillus sp. for decol-
orization of textile azo-metal complex dyes.
Keywords: Azo-metal complex dyes; Decolorization; Halobacillus sp.
Received: April 22, 2010; revised: August 20, 2010; accepted: August 27, 2010
DOI: 10.1002/clen.201000150

1 Introduction Bioremediation in salty environments inevitably requires the

application of halotolerant and halophilic microorganisms, which
Among industrial wastewaters, dye wastewater from textile dyestuff
are able to grow under harsh conditions. Since externally added
industries is one of the most difficult to treat. This is because dyes
bacteria may have some deleterious effects on the ecosystem, apply-
usually have a synthetic origin and complex aromatic molecular
ing or activating the indigenous microflora is preferred if possible
structures which make them more stable and more difficult to be
[13]. In many cases, the textile effluents contain high salinity,
biodegraded [1]. It is reported that there are over 100 000 commer-
thereby causing problems for conventional biological treatments
cially available dyes with a production of over 7
105 metric tons per
[14]. Halotolerant and halophilic bacteria usually tolerate noticeable
year [2]. Such dyes, when discharged to the environment, are therefore
amounts of toxic metals in their environment. Thus they are utilized
persistent and many are also toxic. In addition, conventional waste-
in bioremediation of oil [15, 16] and oxyanion pollution [17, 18] but
water treatment processes are often inefective in dye removal. Thus,
their effectiveness in decolorization of textile effluents has been
decolorization of textile wastewaters has been a major environmental
reported inadequate.
concern for a long time. Chemical or physico-chemical treatment
Azo compounds are highly colored and they have been used as
methods, such as coagulation/adsorption, complete destruction of
dyes in textile industry for a long time and many microorganisms
dye molecules by electrolysis, or ozonation, etc., are in general ineffi-
are capable of decolorizing the azo dyes, including Gram-positive
cient, costly and of limited applicability, while sometimes producing
and Gram-negative bacteria [19, 20] but there are a few study on the
large amounts of toxic waste which is difficult to dispose of [3–5].
use of halophilic/halotolerant bacteria for decolorization azo dyes
Bioremediation is becoming important, because it is cost-effective and
[21, 22]. Metal-complex azo dyes are an important class of dyes in the
environmentally friendly and produces less sludge [6, 7]. Many micro-
textile industry and the more important dyes are chromium, cobalt,
organisms are capable of decolorizing the azo dyes, including Gram-
and copper complexes with azo ligands. A few studies of the removal
positive and Gram-negative bacteria [8–10] and fungi [11, 12].
of azo-metal complex dyes have been established by white rot fungi
[23] and bacterial cells of Shewanella strain J18143 [24].
Exploitation of the salt tolerant bacteria in the bio-treatment
system would be a great improvement of conventional biological
Correspondence: Professor Dr. K. Güven, Faculty of Science, Department treatment systems and the bio-treatment concept.
of Biology, Anadolu University, TR26470 Eskiş¸ehir, Turkey. Çamaltı Saltern is the biggest coastal solar saltern on the Aegean
E-mail: kguven@anadolu.edu.tr
cost of Turkey. Sea salt is produced by evaporation of seawater (SW)
Abbreviation: SW, seawater. in coastal lagoons [25].

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178 A. Demirci et al. Clean – Soil, Air, Water 2011, 39 (2), 177–184

Table 1. Names and proportion of textile dyes used, CAS names are also given

Dye Chemical Entity CAS No. Proportion (%)

Lanaset Black B C.I. Acid Black 172 57693-14-8 30–40

Disodium [1-[(2-hydroxy-3,5-dinitrophenyl)azo]-2-naphtholato(2)][3-hydroxy-4- 70236-55-4 5–10
[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3)]chromate(2-)
Lanaset Bordeaux B C.I Acid Red 260 52333-30-9 1–5
Chromate(2-)[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3- 83833-37-8 30–40
hpyrazol-3-onato(2-)][4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-
hydroxyl-1-naphthalenesulfonato(3-)]
Lanaset Brown B Disodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4- 52587-68-5 15–20
hydroxybenzenesulphonato(3-)][1-[[2-hydroxy-5-(phenylazo)phenyl]azo]-2-
naphtholato(2)]chromate(2-)
Disodium [2,4-dihydro-4-[(2-hydroxy-5-ni trophenyl)azo]-5-methyl-2-phenyl-3H- 70236-60-1 30–40
pyrazol-3-onato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-
sulphonato(3-)] chromate(2-)
Lanaset Green B Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis(imino-4,1- 70161-19-2 60–70
phenyleneoxy)]bis(benzenesulfonate)
Lanaset Navy R C.I Acid Blue 225 70209-96-0 1–5
C.I Acid Blue 317 68541-71-9 15–24

The purpose of our study was to screen and then investigate the
role of halotolerant/halophilic bacteria isolated from a saline
environment, Çamaltı Saltern, in decolorization of some of textile
azo-metal complex dyes in a saline aqueous solution.
2 Materials and methods
2.1 Chemicals
Five commercially used textile azo and azo-metal comlex dyes
including Lanaset Black B, Lanaset Bordeaux B, lanaset Brown B,
Lanaset Green B, and Lanaset Navy R (Tab. 1) were CIBA Geighy and
they were kindly obtained from a textile factory in Uş¸ak-Turkey.
Concentrated dye solutions were filter sterilized and aseptically
added to the medium.
2.2 Isolation of bacteria and screening for
resistance to dyes
Water and soil samples were taken from Çamaltı Saltern in 2008.
Isolation of strains was performed on SW agar (medium (g/L): 0.65 g/L
NaBr, 0.167 g/L NaHCO3, 5 g/L KCl, 0.723 g/L CaCl2, 49.49 g/L
MgSO4  7 H2O, 34.567 g/L MgCl2  6 H2O, 195 g/L NaCl, 1 g/L yeast
extract (YE), 20 g/L agar) [26]. One milliliter of several dilutions
(from 101 to 105) of the original water or soil samples was used
to inoculate the plates by a ‘‘spread plating’’ technique. After incu-
bation of the plates for 15–20 days at 378C, colonies grown were
restreaked on the same medium for single colony isolation.
Screening for resistance to dyes was carried out by using SW agar
medium supplemented with any of any of dye solution (50 mg/L). A
total of six bacterial isolates were tested for screening of dyes tested.
Bacterial culture growth as a pure culture was streaked out onto
plates of azo dye. After incubation for 15–20 days at 378C, colonies
grown were restreaked onto azo dye medium and chosen for decol-
orization assay.
2.3 Identification of the isolate and phylogenetic
analysis
Extraction of genomic DNA, PCR-mediated amplification of 16S
rDNA and direct sequencing of the purified PCR product were done
as in a previous study [26]. 16S rDNA sequences (>1350 bases) of
closely related taxa of the bacterial isolates were retrieved from the
NCBI database by using basic local aligment search tool (BLAST).
These sequences were aligned using the CLUSTAL X program [27] and
a neighbor-joining phylogenetic tree was constructed using
TREECON program [28].
2.4 Decolorization assay
Bacterial growth occurred only with the culture named as strain C-22
on SW agar plates either containing Lanaset Navy R or Lanaset Brown
B. Lanaset Brown B consists of disodium [3-[(4,5-dihydro-3-methyl-5-
oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzenesulphonato(3-)]
[1-[[2-hydroxy-5-(phenylazo)phenyl]azo]-2-naphtholato(2)]chromate(2-)
(15–20%) and disodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-
5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-hydroxy-4-[(2-hydroxy-1
naphthyl)azo]-7 nitronaphthalene-1-sulphonato(3-)] chromate(2-)
(30–40%) having both azo and sulpho forms. Lanaset Navy R is a
mixture of Acid Blue 225 (1–5%) and Acid Blue 317 (15–24%; see
Tab. 2). Afterwards, decolorization experiments were carried out
with the strain C-22 by using Lanaset Brown B and Lanaset Navy
R in sterile saline water (195 g/L NaCl) instead of bacterial growth
medium to prevent precipitation of dye with the ingredients.
Decolorization experiments were carried out at room temperature
by 125 rpm shaking and at pH 4.5 which is the original pH of the
solution.
Strain C-22 was grown in SW broth medium at 378C for 7 days at
125 rpm. The pellet was obtained after centrifugation at 8000 rpm
for 15 min and washed twice in sterile saline water. The pellet was
suspended in sterile saline water (109 cfu/mL) and then 45 mL of
decolorizing medium (SW broth medium and 0.12 g azo dye in
1 L) was inoculated with 5 mL of bacterial suspension to
give 108 cfu/mL cells. Following incubation aliquots of sample
(1.5 mL) was withdrawn at different time intervals (10, 20, 30, 40,
50, 60, 120, 180, 240, 300, 1440, 2880, and 4320 min). Bacterial cell
mass was separated from dye suspension by centrifugation at
8000 rpm for 15 min. The degree of decolorization of the dye was
measured at its maximum adsorption wavelength (520 nm for
Lanaset Brown B and 570 nm for Lanaset Navy R) using a UV–VIS
spectrophotometer (Schimadzu RF-5301 PC). The decolorization effi-
ciency of isolate was expressed as per the following equation.

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Clean – Soil, Air, Water 2011, 39 (2), 177–184 Decolorization of azo-metal complex dyes by a halophilic bacterium 179

Table 2. Chemical structures of azo dyes used for decolorization All decolorization experiments were performend in triplicates.

Lanaset Navy R Mixture of azo and azo-metal complex dyes

2.5 Fourier transform infrared spectrometer
5% Acid Blue 225
(FT-IR)
O HN In order to investigate the functional groups on strain C-22 in
HN
Cl relation to decolarization of dyes, strain C-22 biomass obtained by
O O centrifugation was dried by vacum evaporator at room temparature.
S
O - Na+
O NH2 O About 1 mg of biomass was encapsulated in 100 mg of KBr in order to
prepare translucent sample disk and was analyzed by FT-IR (Perkin
15–24% Acid Blue 317 Elmer Spektrum 100). The adsorption spectrum of dye-unloaded dry
O 2 Na + bacterial biomass was used as control biomass for comparison with
O O
S dye loaded biomass.

N
N NO 2 2.6 Adsorption isotherms
O
Cr3+ O
Adsorption isotherms are plots of dye concentration in the solution
O
O against the biosorption capacity (q) at a constant temperature. Two
N
O 2N N
biosorption equilibrium isotherm models, proposed by Freundlich
and Langmuir were used to evaluate the adsorption of dyes.
Corresponding correlation was used to draw the plots of the con-
centration of adsorbate against the concentration of biomass.
The Freundlich model is based on the relationship between the
Lanaset Brown B Mixture of azo metal complex dyes dye uptake capacity qe (mg/g) of biomass and the residual (equi-
librium) dye concentration Ce (mg/L). The general Freundlich
5–20% Non-Cl acid
dye (CAS no 52587-68-5) equation is as:

qe ¼ Kf Ce1=n (2)
N
N 1
O
log qe ¼ log Kf þ log Ce (3)
3+ O
n
Cr
O O where intercept log Kf is a measure of adsorption capacity and the
N
N slope 1/n is the intensity of adsorption.
N N
The general Langmuir equation is commonly presented as:
Me +
O S O 2 Na
Ce 1 Ce
O ¼ þ (4)
qe Q b Q

30–40% Non-Cl acid dye where qe is the amount of dye removed (mg/g), Ce the equilibrium
(CAS no 70236-60-1) concentration (mg/L), Q and Qb are the Langmuir constants related to
adsorption capacity and adsorption energy, respectively [29].
N
N
O
O
3 Results and discussion
Cr3+
O O 3.1 Isolation and identification
N
N
N N Out of six isolates tested only C-22 grew on the plates of SW agar
supplemented with azo-metal complex dyes either Lanaset Brown B
Me +
O S O 2 Na
or Lanaset Navy R. Isolate C-22 had been obtained from sediment of a
O
crystalization pool.
Identification of the isolate C-22 was carried out after PCR ampli-
fication and the sequencing of 16S rRNA genes. The strain was
identified as Halobacillus sp. by BLAST analysis. Its similarity percent-
Decolorization (R) by strain C-22 was determined as: age was 99% with the type strains. The phylogenetic tree (Fig. 1) was
constructed by using the neighbor-joining method. This indicated
A0 At that the isolate C-22 was part of the cluster within genus Halobacillus.
R¼
100 (1)
A0 Halobacillus are Gram-positive moderately halophilic bacteria living
in different saline environments [30, 31] but their biotechnological
where R is the percentage of dye biosorption by biomass in percent- possibilities have not been extensively exploited. Therefore, this is
age, A0 the initial concentration of dye in mg/L and At is the final the first study to reveal potential of Halobacillus sp. for decolorization
concentration of dye in mg/L [21]. of textile dyes.

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180 A. Demirci et al.
Figure 1. Phylogenetic relationships of strain Halobacillus sp. C-22 and
selected relatives. Biostrap values >72% are shown. The tree was con-
structed using the neighbor joining method [32] and rooted using
Salimicrobium halophilum as out-group. GenBank accession numbers
are given beside the strains. The scale bar represents one nucleotide
substitution per 1000 sequence positions.
Biostrap values greater than 72% are shown in Fig. 1. The tree was
constructed using the neighbor-joining method [32] and rooted
using Salimicrobium halophilum as out-group.
3.2 Decolorization assays
No pH variable could be tested in this study since the azo-metal
comlex dyes formed a precipitation at different pH values in saline
conditions. Therefore, only effect of contact time at a high concen-
tration of dyes was evaluated.
In this study, Halobacillus sp. C-22 grew in the medium having azo-
metal complex dyes (50 mg/L), however, the decolorization rate was
not checked at this concentration. Decolorization experiments of
both azo-metal complex dyes (Lanaset Brown B and Lanaset Navy R)
were carried out at a concentration of 120 mg/L which is higher than
in the screening medium. Decolorization of Lanaset Brown B with
Halobacillus sp. C-22 showed that absorbance ratio was highest
(96.12%) at the 78th hour but, rapidly decreased after then (Fig. 2).
These results indicate that Halobacillus sp. C-22 has a high capacity for
decolorization of Lanaset Brown B in 3–4 days and Halobacillus sp. was
effective in the azo dye decolorization. Lanast Navy R was rapidly
decolorized in 10 min (46.66%). Decolorization of Lanaset Navy R
Figure 2. Decolorization (%) of Lanaset Brown B by Halobacillus sp. C-22.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Clean – Soil, Air, Water 2011, 39 (2), 177–184
Figure 3. Decolorization (%) of Lanaset Navy R by Halobacillus sp. C-22.
with Halobacillus sp. C-22 was more rapid than lanaset Brown B
and reached the maximum level (60.6%) in 3 h (180 min; see Fig. 3).
Azo dyes generally contain one, or more sulphonic-acid groups on
the aromatic rings, which might act as detergents, thereby inhibit-
ing the growth of the microorganisms. Such dyes may affect DNA
synthesis since it has also been reported that dyes are inhibitors of
the nucleic acid syntheses, or cell growth [7, 33]. The different
decolorization of the tested azo dyes were affected by their molecu-
lar weights, substitution group of the dye molecules, and the intra-
molecular hydrogen bond between the azo and hydroxyl groups at
the same time [22]. Azo-metal complex dyes include metals in com-
mercial terms, the more important metal complex dyes are
chromium, cobalt, and copper with azo ligands. Although copper
and chromium salts and to a lesser degree, cobalt salts are harmful
to humans, animals, and plants, the usage and production of many
of these dyes has ceased. Nevertheless, the share of this class of dye is
estimated to be approximately 30% in wool dyeing and 40% in
polyamide dyeing [24]. Microbial decolorization is a cost-effective
method for removing these pollutants from the environment and a
few studies of the removal of azo-metal complex dyes have been
established [24, 34, 35].
Previous studies [21, 22] carried out by halophilic and halotolerant
bacteria showed that Halomonas sp. isolated from effluents of textile
industries have a remarkable ability in decolorizing the widely
utilized azo dyes. The strains were able to decolorize azo dyes in
a wide range of NaCl concentration (up to 20% w/v), temperature (20–
408C) after 4 days of incubation in static culture. These strains also
readily grew in and decolorized the high concentrations of dye
(5000 ppm) and could tolerate up to 10 000 ppm of the dye [21].
Guo et al. [22] showed that Halomonas sp. isolated from coastal
sediments possess a broad azo dye-decolorization range under
anaerobic conditions and the decolorization rates of azo dyes were
above 90% in 24 h.
Kumar et al. [36] observed that two aerobic bacterial isolates
brought about maximum decolorization (36.5 and 32.5%) under
optimized conditions in 8 days. Ramachandra [37] observed decol-
orization rate of 60% with Pseudomonas stutzeri, Pseudomonas acidovor-
ans, Enterbacter sp., and Alcaligenes eutrophus. However, Guo et al. [22]
obtained more than 90% decolorization with the Halomonas sp. strain
GTW. The maximum decolorization rates obtained in this study were
also relatively high in comparision with above studies.
3.3 FT-IR spectral analysis
The results of FT-IR analysis of control biomass and samples obtained
after decolorization showed various peaks (Figs. 4 and 5). The IR
adsorption frequencies of peaks and the corresponding functional
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Clean – Soil, Air, Water 2011, 39 (2), 177–184 Decolorization of azo-metal complex dyes by a halophilic bacterium 181

Figure 4. FT-IR spectral analysis of Halobacillus sp. C-22 before (control) Figure 5. FT-IR spectral analysis of Halobacillus sp. C-22 before (control)
and after decolorization of Lanaset Brown B. and after decolorization of Lanaset Navy R.

groups of Halobacillus sp. C-22 control biomass and decolorized bio-
mass were listed in Tabs. 3–5.
The functional groups of control biomass appeared between
517 and 3436 cm1 frequency (Tab. 3). The FT-IR spectra of biomass
adsorbed with Lanaset Brown B displayed peaks at 756, 827,
1051, 1088, 1197, 1285, 1463, 1560, 1587, 2923, and 3435 cm1
(see Fig. 4).
As seen from the figures in Tabs. 3–5, the peaks displayed in the
regions 2923 cm1 (corresponding to CH antisym and sym stretch-
ing) and 3435 cm1 (corresponding to OH, NH, and NH2 stretching)

Table 3. Functional groups of the control biomass of Halobacillus sp. C-22 and corresponding adsorption frequencies

FT-IR peak Frequency (cm1) Functional group assignment and remarks

1 517 C—O—C, C—C — — O bending, NO2 rocking, chain deformation in alk

2 670 C—O—H, O—C — — O, C—C—CHO bending, C—S stretching, CH deformation
3 1059 C—N, C—O, S — — O stretching
4 1163 C—O—C antisymmetry stretching, C—O, S — — O, SO2 stretching, C—C—N bending
5 1240 C—O, C—N stretching, C—O—C antisymmetry stretching, S — — O stretching
6 1401 C—N stretching (amide III. Band)
7 1452 CH2 scissors, CH3 antisymmetry deformation
8 1546 NH deformation (amide II. band), NO2 antisymmetry stretching
þ
9 1648 C—
— C, C —
— N, C —
— O stretching (amide I. Band), NH, NH2, NH3 deformation
10 2342 P—H stretching
11 2925 CH antisymmetry and symmetry stretching
12 3436 OH, NH, NH2 stretching

Table 4. Functional groups of the Halobacillus sp. C-22 adsorbed with Lanaset Brown B and corresponding adsorption frequencies

FT-IR peak Frequency (cm1) Functional group assignment and remarks

1 756 CH deformation, CH2 rocking, C—S stretching

2 827
3 1051
4 1088
5 1197
6 1285
7 1463
8 1560
9 1587
10 2923
11 3435
CH deformation, NH2 wagging
C—N, C—O, S —— O, SO2 stretching
C—O—C antisymmetry stretching, C—N, C — — S stretching
C—O—C, C—N, C—O stretching, C—C—N bending, C—O—C antisymmetry stretching
N—O, C—O, C—N, P — — O stretching, C—O—C antisymmetry stretching
CH2 scissors, CH3 antisymmetry deformation
NH deformation (amide II. Band), NO2 antisymmetry stretching
NH2 deformation, (amide II. Band)
CH antisymmetry and symmetry stretching
OH, NH, NH2 stretching

Table 5. Functional groups of the Halobacillus sp. C-22 adsorbed with Lanaset Navy R and corresponding adsorption frequencies

FT-IR peak Frequency (cm1) Functional group assignment and remarks

1 671 C—O—H, O—C — — O, C—C—CHO bending, C—S stretching, CH deformation

2 1167 C—O—C antisymmetry stretching, C—O, S — — O, SO2 stretching, C—C—N bending
3 1500 NH, NHþ 3 deformation NO2 antisymmetry stretching, ring stretching in benzene
þ
4 1631 C—
— C, C —
— N, C —
— O stretching (amide I. Band), NH, NH2, NH3 deformation
5 2924 CH antisymmetry and symmetry stretching
6 3436 OH, NH, NH2 stretching

ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com

182 A. Demirci et al. Clean – Soil, Air, Water 2011, 39 (2), 177–184

Table 6. Langmuir and Freundlich constants and correlation coefficients for biosorption of azo dyes (Lanaset Brown B and Lanaset Navy R) on the
Halobacillus sp. C-22 biomass

Azo dyes Langmuir isotherm parameters Freundlich isotherm parameters

Q b R2 n Kf R2

Lanaset Brown B 3.06
104 2699.002 0.77 0.65 1.27
108 0.94
Lanaset Navy R 6.678
104 3781.01 0.95 1.116 1.54
106 0.98

were also observed in the products C-22 Lanaset Brown B and Lanaset decolorization were analyzed with FT-IR. Tables 4 and 5 summarize
Navy R. But there were some shifts in the peak positions at some the functional groups of the bacterium corresponding adsorption
frequencies: The FT-IR spectrum of Halobacillus sp. C-22 control frequencies.
(Tab. 3) displays a peak at 1648 cm1 (corresponding to C — — C, C —
— N,
þ
C—— O stretching (amide I. band), NH, NH2, and NH3 deformation).
The FT-IR spectrum of Halobacillus sp. C-22 adsorbed with Lanaset 3.4 Adsorption isotherms
Navy R showed the same peak at 1630 cm1. In the case of Halobacillus The adsorption isotherm models were used to characterize the
sp. C-22 adsorbed with Lanaset Brown B, the band disappeared. The interaction of adsorbtion of azo dyes with the Halomonas sp. C-22
peak of Halobacillus sp. C-22 control biomass displayed at 1546 cm1 biomass preparations. The Langmuir and Freundlich constants cal-
(corresponding to NH deformation (amide II. band), NO2 antisym- culated from the isotherms with the correlation coefficients are
metry stretching) (see Tab. 3). The same band of the Halobacillus sp. presented in Tab. 6. The linearized plots of Langmuir and
C-22 adsorbed with Lanaset Brown B (see Tab. 4) shifted to 1560 and Freundlich isotherm models for decolorization of Lanaset Brown
1587 cm1. In the case of Halobacillus sp. C-22 adsorbed with Lanaset B and Lanaset Navy showed that Freundlich isoterm (see Fig. 6)
Navy R (see Tab. 5), the band disappeared. The FT-IR Spectrum of was more suitable for biosorpsiyon (R2 ¼ 0.94 and R2 ¼ 0.98) of both
Halobacillus sp. C-22 control biomass (see Tab. 3) displayed a peak at azo dyes than Langmuir izoterms (see Fig. 7).
1452 cm1 (corresponding to CH2 scissors and CH3 antisymmetry
deformation). While the same band of the Halobacillus sp. C-22
adsorbed with Lanaset Brown B (see Tab. 4) shifted to 1463 cm1, 4 Conclusions
whereas this band was not observed in the spectrum of the Bacterial decolorization under aerobic conditions usually results in
Halobacillus sp. C-22 adsorbed with Lanaset Navy R (see Tab. 5). The adsorption of dyestuffs on bacteria rather than oxidation [38] and
peak displayed at 1401 cm1 (corresponding to C—N stretching this is the simplest mechanism of color removal by bacterial cells
(amide III. band) in the FT-IR spectrum of Halobacillus sp. C-22 control [39]. During adsorption, the dye will be concentrated onto the bio-
biomass (Tab. 3) disappeared in the FT-IR spectrum of the Halobacillus mass, which will become saturated with time.
sp. C-22 adsorbed with Lanaset Navy R and Halobacillus sp. C-22 Tan et al. [40] reported a novel salt-tolerant Exiguobacterium sp. for
adsorbed with Lanaset Brown B (see Tabs. 3–5). The FT-IR spectrum azo dyes decolorization and the decolorization proceeded primarily
of Halobacillus sp. C-22 control biomass (see Tab. 3) displayed a peak at by enzymatic reduction associated with a minor portion of bioad-
1240 cm1 (corresponding to C—O, C—N stretching, C—O—C antisym- sorbtion to inactivated microbial cells. Biocatalytic effects of anthra-
metry stretching, S — — O stretching), whereas the same band of the quinone on the anaerobic reduction of the dye were also observed. In
Halobacillus sp. C-22 adsorbed with Lanaset Brown B (see Tab. 4) this study, no enzymatic activity for decolorization was tested.
shifted to 1197 cm1 and the same band shifted to 1167 cm1 in However, future studies may examine the enzymatic decolorization
the spectrum of Halobacillus sp. C-22 adsorbed with Lanaset Navy R process.
(see Tab. 5). The FT-IR spectrum of Halobacillus sp. C-22 control bio-
mass (see Tab. 3) displayed a peak at 1163 cm1 (corresponding to
C—O—C antisymmetry stretching, C—O, S — — O, SO2 stretching, C—C—N
bending). The same band of the product Halobacillus sp. C-22 adsorbed
with Lanaset Brown B (see Tab. 4) shifted to 1051 and 1088 and
1167 cm1 in Halobacillus sp. C-22 adsorbed with Lanaset Navy R
(see Tab. 5).
Decolorization of dyes by bacteria could be due to adsorption by
microbial cells, or to biodegradation. In the case of adsorption cell
mats become deeply colored because of the adsorbed dyes, whereas
those retaining their original colors occur when biodegradation
takes place [7]. Zhao [23] reported that decolorization of azo-metal
complex dyes was carried out by processes involving both physical
adsorption and degradation by enzymes. However, Li and Guthrie
[24] reported that bacterial reduction of dyes was observed.
In this study, the cells were deeply colored due to the uptake of the
dye. This indicates that the color removal by Halobacillus sp. C-22 may
be largely attributed to adsorption. Therefore, biomass of Halobacillus Figure 6. The linearized Freundlich plot for Lanaset Brown B and Lanaset
sp. C-22 (control) and biomass treated with textile dyes obtained after Navy R biosorption by Halobacillus sp. C-22.

ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com

Clean – Soil, Air, Water 2011, 39 (2), 177–184 Decolorization of azo-metal complex dyes by a halophilic bacterium
Figure 7. The linearized Langmuir plot for Lanaset Brown B and Lanaset
Navy R biosorption by Halobacillus sp. C-22.
In this study, only live bacterial cells were used for decolorization
and functional groups of adsorption shown by FT-IR conclude that it
is a physical adsorption. Future studies may be carried out to further
reveal an enzyme or any other bioproduct might be involved in
decolorization as is the case with Exiguobacterium sp. [40].
It is already known that moderate halophiles accumulate high
cytoplasmatic concentrations of low molecular weight organic com-
pounds to cope with the osmatic stress and to maintain positive
turgor pressure. The ability to accumulate high concentration of
these compounds makes moderate halophiles useful for the biotech-
nological aims [41].
In conclusion, this study suggests that Halobacillus sp. C-22 strain
isolated from Çamaltı Saltern have a potential for decolorization of
colored effluents in high salt environments.
Acknowledgments
This study was supported by a project of the Anadolu University
Research Foundation number 071018.
The authors have declared no conflict of interest.
References
[1] S. Seshadri, P. L. Bishop, A. M. Agha, Anaerobic/Aerobic Treatment of
Selected Azo Dyes in Wastewater, Waste Manage. 1994, 14, 127–137.
[2] H. Zollinger, Colour Chemistry–Chemistry-Synthesis, Properties and
Application of Organic Dyes and Pigments, VCH, Weinheim 1987.
[3] I. M. Banat, P. Nigam, D. Singh, R. Merchant, Microbial
Decolorization of Textile Dye-Containing Effluents — a Review,
Bioresour. Technol. 1996, 58, 217–227.
[4] P. Verma, P. Baldrian, F. Nerud, Decolorization of Structurally
Different Synthetic Dyes Using Cobalt(I)/Ascorbic Acid/Hydrogen
Peroxide System, Chemosphere 2003, 50, 975–979.
[5] F. Zhang, A. Yediler, X. Liang, A. Kettrup, Effects of Dye Additives on
the Ozonation Process and Oxidation by Products: A Comparative
Study Using Hydrolysed CI Reactive Red 120, Dyes Pigm. 2004, 60, 1–7.
[6] I. M. Robinson, G. Mcmullan, P. Nigam, Remediation of Dyes in
Textile Effluent: A Critical Review on Current Treatment
Technologies, Bioresour. Technol. 2001, 77, 247–255.
[7] K. C. Chen, J. Y. Wu, D. J. Liou, S. C. J. Hwang, Decolorization of the
Textile Azo Dyes by Newly Isolated Bacterial Strains, J. Biotechnol.
2003, 101, 57–68.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
183
[8] R. K. Sani, U. C. Banerjee, Decolorization of Triphenylmethane Dyes
and Textile and Dyestuff Effluent by Kurthia sp., Enzyme Microb.
Technol. 1999, 24, 433–437.
[9] K. M. Kodam, I. Soojhawon, P. D. Lohande, K. R. Gawai, Microbial
Decolorization of Reactive Azo Dyes under Aerobic Conditions,
World J. Microbiol. Biotechnol. 2005, 21, 367–370.
[10] S. Moosvi, H. Kehaira, D. Madamwar, Decolorization of Textile Dye
Reactive Violet 5 by a Newly Isolated Bacterial Consortium RVM
11.1, World J. Microbiol. Biotechnol. 2005, 21, 667–672.
[11] D. S. L. Balan, R. T. R. Monteneiro, Decolorization of Textile Indigo
Dye by Ligninolytic Fungi, J. Biotechnol. 2001, 89, 141–145.
[12] P. Verma, D. Madamwar, Decolorization of Azo Dyes Using
Basidiomycete Strain PV 002, World J. Microbiol. Biotechnol. 2005, 21,
481–485.
[13] R. Margesin, F. Schinner, Bioremediation (Natural Attenuation and
Biostimulation) of Diesel–Oil-Contaminated Soil in an Alpine
Glacier Skiing Area, Appl. Environ. Microbiol. 2001, 67, 3127–3133.
[14] E. Mellado, A. Ventosa, Microorganisms for Health Care, Food and Enzyme
Production (Ed: J. L. Barredo), Research Signpost, Kerala, India 2003, p.
233.
[15] D. Delille, A. Basseres, A. A. Dessomemes, Effectiveness of
Bioremediation for Oil-Polluted Antarctic Seawater, Polar Biol.
1998, 19, 237–241.
[16] R. Margesin, F. Schinner, Biological Decontamination of Oil Spills in
Cold Environments, J. Chem. Technol. Biotechnol. 1999, 74, 381–389.
[17] J. O. Nriagu, J. M. Payna, Quantitative Assessment of Worldwide
Contamination of Air, Water and Soils by Trace Metals, Nature 1988,
333, 134–139.
[18] B. K. Kinkle, M. J. Sadowsky, K. Johnstone, W. C. Koskinen, Tellurium
and Selenium Resistance in Rhizobia and Its Potential Use for Direct
Isolation of Rhizobium meliloti from Soil, Appl. Environ. Microbiol. 1994,
60, 1674–1677.
[19] C. M. Craliell, S. J. Barclay, N. Naidoo, C. A. Buckley, D. A. Mulholland,
E. Senior, Microbial Decolorization of a Reactive Azo Dye under
Anaerobic Conditions, Water SA 1995, 21, 61–69.
[20] H. Fang, H. Wenrong, L. Yuezhong, Biodegradation Mechanisms and
Kinetics of Azo Dye 4BS by a Microbial Consortium, Chemosphere
2004, 57, 293–301.
[21] S. Asad, M. A. Amoozegar, A. A. Pourbabaee, M. N. Sarbolouki, S. M.
M. Dastgheib, Decolorization of Textile Azo Dyes by Newly Isolated
Halophilic and Halotolerant Bacteria, Bioresour. Technol. 2007, 98,
2082–2088.
[22] J. Guo, J. Zhou, D. Wang, C. Tian, P. Wang, M. S. Uddin, A Novel
Moderately Halophilic Bacterium for Decolorizing Azo Dye under
High Salt Condition, Biodegradation 2008, 19, 15–19.
[23] C. F. Zhao, The Biodegradation of Metal Complex Dyes by White Rot
Fungi, Huazhong Keji Daxue Xuebao, Ziran Kexueban 2001, 29, 108–110.
[24] T. Li, J. T. Guthrie, Colour Removal from Aqueous Solutions of Metal-
Complex Azo Dyes Using Bacterial Cells of Shewanella Strain J18143,
Bioresour. Technol. 2010, 10, 14291–14295.
[25] M. B. Mutlu, K. Guven, Isolation and Characterization of Halophilic
Bacteria from Çamaltı Saltern Turkey, New Biotechnol. 2009, 25 (1),
S81.
[26] M. B. Mutlu, M. Martı́nez-Garcı́a, F. Santos, A. Peña, K. Guven, J.
Antón, Prokaryotic Diversity inTuz Lake, a Hypersaline
Environment in Inland Turkey, FEMS Microbiol. Ecol. 2008, 65, 474–
483.
[27] J. D. Thomson, T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins,
The CLUSTAL-X Windows Interface: Flexible Strategies for Multiple
Sequence Alignment Aided by Quality Analysis Tools, Nucleic Acids
Res. 1997, 25, 4876–4882.
[28] Y. van de Peer, Y. De Wachter, TREECON for Windows: A Software
Package for the Construction and Drawing of Evolutionary Trees for
the Microsoft Windows Environment, Comput. Appl. Biosci. 1994, 10,
569–570.
[29] E. Y. Küçükgül, S. Kutlu, Single Adsorption of Copper and Zinc from
an Aqueous Solution onto Activated Carbon, DEÜ Müh. Fak., Çevre
Mühendisliği Böl., Derg. 2006, 2, 81–90.
www.clean-journal.com
184 A. Demirci et al.
[30] Y. Jung-Hoon, K. So-Jung, L. Choong-Hwan, W. O. Hyun, O. Tae-
Kwang, Halobacillus yeomjeoni sp. Nov., Isolated from a Marine
Solar Saltern in Korea, Int. J. Syst. Evol. Microbiol. 2005, 55 (6), 2413–
2417.
[31] K. Ripka, et al., Molecular Characterisation of Halobacillus Strains
Isolated from Different Medieval Wall Paintings and Building
Materials in Austria, Int. Biodeterior. Biodegrad. 2006, 58, 124–132.
[32] N. Saitou, M. Nei, The Neighbour-Joining Method: A New Method for
Reconstructing Phylogenetic Trees, Mol. Biol. Evol. 1987, 4, 406–
425.
[33] K. Wuhrmann, K. Mechsner, T. Kappeler, Investigation on Rate
Determining Factors in the Microbial Reduction of Azo Dyes,
Appl. Microbiol. Biotechnol. 1980, 9, 325–338.
[34] A. Stolz, Basic and Applied Aspects in the Microbial Degradation of
Azo Dyes, Appl. Microbiol. Biotechnol. 2001, 56, 69–80.
[35] M. H. Vijaykumar, Y. Veeranagouda, K. Neelakanteshwar, T. B.
Karegoudar, Decolorization of 1:2 Metal Complex Dye Acid Blue
193 by a Newly Isolated Fungus, Cladosporium cladosporioides, World J.
Microbiol. Biotechnol. 2006, 22, 157–162.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
View publication stats
Clean – Soil, Air, Water 2011, 39 (2), 177–184
[36] V. Kumar, L. Wati, P. Nigam, I. M. Banet, G. McMullan, D. Singh,
R. Marchant, Microbial Decolorization and Bioremediation of
Anaerobically Digested Molasses Spentwash Effluent by Aerobic
Bacterial Cultures, MICROBIOS 1997, 89, 81–90.
[37] C. V. Ramachandra, Devolopment of Indigenous Technology for the
Removal of Sulfur Compounds and Colour from Distillery Effluent,
Annual Progress Report, Industrial Toxicology Res. Centre,
Lucjnow 1993.
[38] U. Pagga, D. Brown, The Degredation of Dyestuffs II. Behavior of
Dyestuffs in Aerobic Biodegradation Test, Chemosphere 1986, 15, 479–
491.
[39] R. Bras, I. A. Ferra, H. M. Pinheiro, I. C. Goncalves, Batch Tests for
Assessing Decolorisation of Azo Dyes by Methanogenic and Mixed
Cultures, J. Biotechnol. 2001, 89, 155–162.
[40] L. Tan, Y. Qu, J. Zhou, A. Li, M. Gou, Identification and Characteristics
of a Novel Salt-Tolerant Exiguobacterium sp. for Azo Dyes
Decolorization, Appl. Biochem. Biotechnol. 2009, 159, 728–738.
[41] A. Ventosa, J. J. Nieto, A. Oren, Biology of Moderately Halophlic
Aerobic Bacteria, Microbiol. Mol. Biol. Rev. 1998, 62, 504–544.
www.clean-journal.com