Study on the Decolorization of Commonly Used Reactive Dyes...
Transcript of Study on the Decolorization of Commonly Used Reactive Dyes...
Chapter I
Study on the Decolorization of Commonly Used Reactive Dyes in the Textile Industry
Reactive Dyes
eactive dyes, including many structurally different dyes, are extensively used in
the textile industry because of their wide variety of color shades, high wet
fastness profiles, ease of application, brilliant colors, and minimal energy
consumption. A reactive dye, according to a useful definition by Rys and Zollinger, is a
colored compound which has a suitable group enable of forming a covalent bond between a
carbon atom of a hydroxy, an amino or a mercapto group respectively of the substrate
(Mansoor, 2008). They point out that this definition excludes mordant dyes and 1: 1
chromium azo dye complexes, which are used in dyeing protein fibres, may form covalent
bonds between metal ion and nucleophilic groups of the fibre. The idea that the
establishment of a covalent bond between dye and substrate would result in improved
wash fastness compared with that of ordinary dye-substrate systems where weaker forces
were operative is an old one.
Attempts were made by various dye firms from about 1906 onwards to achieve this
aim but it was not until 1956 that the first successful reactive dyes, the Procions, were
introduced by ICI for the dyeing and printing of cellulose fibres, following the work of
Rattee and Stephen from 1954 onwards (Mansoor, 2008). The invention consisted in the
synthesis of dyes containing a reactive group, the 2,4,6-dichlorotriazinylamino group which
has two labile chlorine atoms activated by the electron-withdrawing action of the three N
atoms, and the Devising of dyebath conditions, which, while bringing about the formation
of a covalent bond, were mild enough to avoid serious damage to the fibre. The dyeings
R
were carried out at ordinary temperatures, ‘fixation’ being brought about by the addition of
sodium bicarbonate, thus raising the pH. The reaction with cellulose may be represented as
nucleophilic substitution by the attaching species RO- or HO
- where R = cellulose moiety
(Fig 1.1). Attach by HO-, derived from the water of the dyebath, occurs simultaneously, but
that of cellulose onion predominates since the dye is absorbed by the cellulose fibres and
dye-substrate reaction is therefore facilitated. It is necessary to remove hydrolysed unfixed
dye by thorough soaping and washing otherwise inferior fastness to wet treatment results.
Among the reactive dyes, three most common groups are azo, anthraquinone and
phthalocyanine dyes (Axelsson et al., 2006), most of which are toxic and carcinogenic
(Acuner and Dilek, 2004). Disposal of these dyes into the environment causes serious
damage, since they may significantly affect the photosynthetic activity of hydrophytes by
reducing light penetration (Aksu et al., 2007) and also they may be toxic to some aquatic
organisms due to their breakdown products (Hao et al., 2000).
Fig 1.1 Dye molecule reacting with cellulose (nucleophilic substitution)
Reactive dyes like other dyes can be removed from wastewater by chemical and
physical methods including adsorption, coagulation–flocculation, oxidation and
electrochemical methods (Lin and Peng, 1994, 1996). However, both the physical and
chemical methods have many disadvantages in application, such as high-energy costs, high-
sludge production and formation of by-products (Sarioglu et al., 2007). Conversely,
bioprocessing can overcome these defects because it is cost saving and environmentally
benign. It is well known that bacteria can degrade and even completely mineralize many
reactive dyes under certain conditions (Asad et al., 2007; Chen et al., 2003; Kapdan and
Erten, 2007; Moosvi et al., 2005). Even better, the products of intermediate metabolism
during the decolorization process, such as aromatic amines, can be degraded by the
hydroxylase and oxygenase produced by bacteria (Pandey et al., 2007).
Some new bacterial strains capable of decolorizing a broad-spectrum of dyes have
also been isolated and characterized (Deng et al., 2008). Bacterial degradation of reactive
dyes is often initiated under anaerobic conditions by an enzymatic biotransformation step
(Carvalho et al., 2008; Park et al., 2007). The resulting products such as aromatic amines
are further degraded by multiple-step bioconversion occurring aerobically or anaerobically
(Barragan et al., 2007; Xu et al., 2006). The present study is focused on the isolation of dye-
decolorizing bacteria from contaminated soil of an industrial estate in Tirupur and its
evaluation on seven reactive dyes commonly employed in the Tirupur textile dyeing
industries.
Among the commonly used reactive dyes In Tirupur textile dyeing units, seven were
chosen for the decolorization study and they were Reactive Black HFGR, Reactive Black,
Reactive Blue, Reactive Red, Colonial Red, Reactive Yellow I and Reactive Yellow II.
Microbes (especially bacteria) were isolated from the contaminated soil within a popular
textile dyeing industry in Tirupur called ‘Emperor Textiles’ which was known to exist for
more than five decades. Individual steps involved in the experimental process are given
below as main objectives:
Collection and study of absorption spectrum of all the seven dyes chosen.
Isolation of bacteria from the contaminated soil in an industrial estate (Emperor
Textiles Pvt. Ltd, Tirupur, Tamil Nadu, India).
Screening of bacterial isolates for their ability to decolorize all the seven chosen
dyes separately and as consortia.
Study on the effect of different carbon and nitrogen sources in decolorization
process.
Study of various inoculum concentration/ size on the decolorization of selected dyes
by bacterial consortia.
Identification of the best decolorizing bacterial isolate by 16S rRNA sequencing
method.
Materials and Methods
Commercial Dyes and Chemicals
The dyes were procured from Emperor Textiles, Tirupur, Tamil Nadu, India (Fig 1.2.
a, b). The common names of all the dyes have been used for convenience and they were
Reactive Black HFGR, Reactive Black, Reactive Blue, Reactive Red, Colonial Red, Reactive
Yellow I and Reactive Yellow II. All other chemicals and reagents were of Analytical grade
(Himedia, Mumbai, India).
Spectrum Study of the Dye
Dyes procured from the industry was initially studied for absorption spectrum in a
UV-Vis Spectrophotometer (Jasco Double Beam Spectrophotometer, UK) from 250nm to
800nm (Safia et al., 2005; Muhammad Asgher et al., 2007).
Lsolation of Dye-Decolorizing Bacteria
Soil samples were collected aseptically from the dumping grounds of the sludge
within the textile industrial complex and carefully transported to the lab (Fig 1.2. c, d, e &
f). Dumping ground of the sludge was chosen for soil collection due to obvious reasons that
the location had been under usage for over 5 decades since the establishment of the dyeing
industry (Fig 1.3 b, c). The soil samples were serially diluted by following the standard
protocol and the dilution series of 10-2 to 10-7 was plated in Nutrient Agar (Himedia,
Mumbai) medium. Each dilution was maintained in triplicates. All the plates were
incubated at 370C for 24 hours (Cappuccino and Sherman. 6th Edn. 2004; Franciscon et al.,
2009). Cultures were identified based on their morphology and color and were transferred
aseptically into sterile agar slants for raising pure cultures to perform further study.
Culture Maintenance and Media
All the isolated bacterial strains (Stock Culture) were maintained routinely on
Nutrient Agar containing (g/l): NaCl 5.0, bacteriological peptone 10.0, Yeast Extract 2.0,
Beef Extract 1.0 and Agar Agar 15.0, stored at 4°C until use (Jadhav et al., 2010). The
organisms from stock culture were used for the decolorization studies after pre-culturing
in Nutrient Broth (g/l): Peptone 10.0, NaCl 5.0, Yeast Extract 2.0 and Beef Extract 1.0 at 37
±2°C for 16 hours under shaking condition (120 rpm) at neutral pH.
Screening of Decolorizing Ability
Use of Nutrient Broth
The Decolorizing ability of the bacterial isolates were tested individually on all the
seven chosen dyes. Decolorization experiments were performed in three sets. A loopful of
log phase pure culture was inoculated into 250 ml Erlenmeyer flask containing 100 ml
nutrient broth with the dye concentration of 100mg/l (Vijaya and Sandhya, 2003; Kalyani
et al., 2008). The flask was incubated at 37°C for 24 hours in a shaker-incubator at 150rpm.
Decolorization Assay
Decolorization was detected by UV-Vis spectrophotometer (Jasco UV-Vis
Spectrophotometer, UK) at respective max using the supernatant from the liquid culture
medium after centrifugation at 10,000 rpm for ten minutes in a refrigerated centrifuge
(Remi C24, Mumbai, India). The removal of the color was reported as % decolorization.
[% = A0-At/A0 x 100]
Where A0 and At were absorbance of the dye solution initially and at cultivation time
(t), respectively. Each decolorization value is a mean for three parallel experiments. Abiotic
controls (without microorganisms) were also included (Mohandas et al., 2007).
Use of Minimal media
The Decolorizing ability of the bacterial isolates were also tested in minimal media
containing the dye at a concentration of 100 mg/l. Minimal medium contained (g/l):
Potassium dihydrogen phosphate 3.0, Disodium hydrogen phosphate 6.0, Ammonium
chloride 5.0, Sodium chloride 5.0, Glucose 8.0 and Magnesium sulphate 0.1. The pH was set
to 7.0 (Cappuccino and Sherman. 2004).
Effect of Different Sources of Carbon and Nitrogen on Dye
Decolorization
As an alternative medium to nutrient broth and minimal media, Bushnell & Haas
medium (Safia et al., 2005) was also used to test the efficiency of the isolates to decolorize
the chosen dyes under different sources of carbon and nitrogen. Solution containing the
following components in (g/l) formed the basal composition for Bushnell and Haas Medium
(BHM): Magnesium sulphate 0.2, Di-potassium hydrogen phosphate 1.0, Calcium chloride
0.02, Ferric Chloride 0.05 and Ammonium Nitrate 1.0. The different combinations of carbon
and nitrogen supplements tested are provided in Table 1.1
Screening of the Developed Bacterial Consortium: Development of
Consortium – SK-C
A mixture of bacterial isolates that demonstrated best decolorizing ability was
prepared as consortium and employed in the decolorizing experiment. For this, equal
volumes of separate pure broth cultures were mixed together as a starter culture and used
in the inoculation of decolorizing experiment performed in Nutrient Broth, Minimal media
and BHM with special supplements of carbon and nitrogen as indicated in Table 1.1.
Strain Identification
The chromosomal DNA of the strains with best decolorization potential was
isolated according to the procedure described by Rainey et al. (1996). A partial DNA
sequence for 16S rRNA gene was amplified by using 5’ - ATG GAT CCG GGG GTT TGA TCC
TGG CTC AGG-3’ (forward primer) and 5’-TAT CTG CAG TGG TGT GAC GGG GGG TGG-3‘
(reverse primer) (Jing et al., 2004). Amplifications were performed in 50 µl reaction
mixtures containing the template DNA, 40ng, 0.2 µM, for each of the primers, dNTPs
200µM, Taq DNA polymerase 2.5U and 10 x Taq buffers 5µl. The mixture was subjected to
the following amplification conditions; 2 min at 940C, followed by 30 cycles of 940C for 1
min, and ended by a final extension step at 720C for 7 min. The PCR products were
electrophoresced on 0.7% agarose gels. The PCR reaction mixture was then sent for
sequencing to Chromous Biotech Pvt. Ltd, Bangalore, India. The nucleotide sequence
analysis of the sequence was done at Blast-n site at NCBI server
(http://www.ncbi.nlm.hin.gov/BLAST). The alignment of the sequence was done by using
CLUSTALW program V1.82 at European bioinformatics site
(http://www.ebi.ac.uk/clustalw). The sequence was refined manually after crosschecking
with the raw data to remove ambiguities and submitted to the NCBI.
Results
Absorption Spectrum
The absorption spectra of seven chosen dyes were studied (250 nm to 800 nm) in a
double beam UV-Vis Spectrophotometer (Fig 1.8 to 1.11). From the optical density at 2 nm
bandwidth, the absorption maximum was determined and presented in Table 1.2.
Isolation of Bacterial Cultures
From the soil sample collected, serial dilution was performed. Based on different
colony morphology, 24 different bacterial strains were raised as pure culture and named
SK1 to SK24 (Fig 1.3 a, d & e).
Decolorization Study in Nutrient Broth
All the 24 isolates obtained, when subjected to decolorization of the seven chosen
dyes individually, demonstrated varying levels of efficiency, as calculated in % of
decolorization from the optical density. Every isolate were capable of decolorizing all the
seven dyes to varying extents from almost ‘No Decolorization’ (ND) to 95 % of
decolorization. However, two isolates namely the SK20 and SK21 were capable of
decolorizing all the dyes equally well showing a decolorization from 40% to 94.8%. The
isolate SK03 was also equally capable but could decolorize only three of the seven dyes
namely Reactive Blue, Reactive Black and Black HFGR.
Of all the seven dyes, Colonial Red was effectively decolorized by eleven bacterial
isolates namely SK09 (90.90 ± 0.45 %), SK11 (92.85±0.14 %), SK12 (92.20 ± 0.74 %), SK13
(92.20 ± 0.64 %), SK14 (92.85 ±0.74 %), SK15 (94.15 ±1.0 %), SK17 (93.50 ± 0.24 %), SK20
(94.80± 0.21 %), SK21 (94.15 ±0.33 %) (Fig. 1.6, 1.8), SK23 (94.15 ± 0.84 %), SK24 (93.50
± 0.14 %). In contrast Reactive Red, was decolorized only to a maximum of 57.85 ± 0.04 %
by SK21 (Fig 1.5), followed by SK20 with 44.45 ± 1.22 % where as other bacterial isolates
were insignificant with decolorization abilities (Table 1.3).
In case of reactive yellow I, SK21 was the only isolate among the 24, to decolorize to
about 91 ± 0.06 % (Fig 1.10), followed by SK 20 (40.00 ± 0.88%), whereas, none of the
other isolates could decolorize the reactive yellow I efficiently. Reactive yellow II also
followed similar pattern of decolorization where SK21 decolorized the dye to about 91.66 ±
0.41 % (Fig. 1.11), followed by SK20 54.62 ± 0.98 % while rest of the isolates could not
decolorize to any significant level (Table 1.3).
SK20 demonstrated decolorization of reactive blue to a maximum of 85.88 ± 0.99 %
followed by SK21 (Fig. 1.7) and SK3 with 72.94 ± 0.14 % and 58.82 ± 0.32 % respectively.
Few other isolates such as SK02, SK04, SK05, SK06, SK07, SK09, SK11, SK12, SK14, SK23
and SK24 could only exhibit a maximum of 25% of decolorization, while the rest of the
isolates could not decolorize the dye at all. From the seven dyes chosen for the study, Black
HFGR and Reactive Black demonstrated the least amount of decolorization of 53.48 ± 0.44,
54.65 ± 1.25 %, and 59.30 ± 0.65 % by SK03, SK20 and SK 21 (Fig. 1.4, 1.9) respectively
(Table 1.3).
Decolorization of the Dyes in Minimal Media
Among seven reactive dyes tested, as colonial red was decolorized by almost all the
24 isolates, it was chosen for decolorization study under minimal media. Isolates that
produced more than 90% of decolorization was considered best and therefore chosen for
the decolorization of colonial red in minimal media. The best ten isolates were SK11, SK12,
SK13, SK14, SK15, SK17, SK20, SK21, SK23 and SK24. Unfortunately, there was no
significant variation in the Optical Density demonstrating the inability of all the isolates to
decolorize the dye within 24 to 48 hours. However, after 72 hours of incubation, almost all
the ten best isolates exhibited an average of 50% of decolorization (Table 1.4). Among
these, SK21 demonstrated the highest percentage of decolorization (62.57±0.40%). There
was no further increase in decolorization by any of these isolates when incubated beyond
72 hours. Therefore, further investigation was carried out in minimal media with different
sources of Carbon and Nitrogen.
Effect of Different Sources of Carbon and Nitrogen
Decolorization study in the Minimal media did not exhibit significant result and
therefore, the experiment was repeated with supplements of different sources of carbon
and nitrogen for which Bushnell and Haas medium was selected as the basal mineral
composition. Colonial Red dye was again used as the model dye in this decolorization
experiment due to the observation of best results in the initial screening process. Best ten
isolates employed in minimal media were tested again in this experiment. Among the seven
different combinations of nutrient supplements in BHM basal media, the composition of
BHM supplemented with Glucose and Yeast extract was found to be the optimal
composition for maximum decolorization. In this, SK21, SK20 and SK23 demonstrated a
remarkable decolorization to 88.31±0.26%, 85.06±0.31% and 84.41±0.22% respectively.
Even though decolorization was comparable to that in nutrient broth, the process required
48 hours of incubation at shaking condition (Table 1.5).
Decolorization by the Bacterial Consortium: SK-C
The efficiency of the best ten bacterial isolates that performed well in earlier
experiments was used in this consortium as well with Colonial Red as the model dye. The
decolorization efficiency was tested in three different media composition such as Nutrient
broth, Minimal media and BHM (supplemented with Glucose and Yeast Extract). In nutrient
broth, decolorization was achieved to 90.27±0.20 % in 24 hours while that of BHM (with
glucose and Yeast Extract) to about 80.31±0.32 % in 72 hours. In minimal media, the same
consortium reported only 60.21±0.41 % of decolorization even after incubating for 72
hours (Table 1.6). From the performance of the consortium it is clearly evident that there is
not much difference in the % of decolorization by the individual strains (where SK20 alone
produced 94.80±0.21 % of decolorization).
Strain Identification
The bacterial isolates SK20 and SK21 demonstrated maximum/efficient
decolorization in five dyes namely Colonial Red, Reactive Red, Reactive Yellow I, Reactive
Yellow II and Reactive Blue among the seven dyes tested. However, Reactive Black HFGR
and Reactive Black was decolorized by only SK03, SK20 and SK21 to over 60%
approximately. This observation suggested for the potent use of these three bacterial
strains for further investigation and field application thereof. Therefore, SK03, SK20 and
SK21 were subjected to 16SrDNA sequencing method of identification and found to be
Bacillus sp. (FJ966212), Bacillus firmus (FJ974057) and Paenibacillus lautus (FJ974058)
respectively.
Table 1.1 Showing different sources of carbon and nitrogen used with BH basal medium (BHM – Bushnell & Haas Medium; YE – Yeast Extract).
Dye λ max (nm)
Black HFGR 598
Reactive Black 595
Reactive Red 522
Colonial Red 512
Reactive Blue 604
Reactive Yellow I 411
Reactive Yellow II 411
Table 1.2 Showing absorption maxima (λ max) of the chosen reactive dyes
S. No. Source of Carbon & Nitrogen 1. BHM 2. BHM + glucose 3. BHM + YE 4. BHM + Glucose + YE 5. BHM + Sucrose + YE 6. BHM + Starch + YE 7. BHM + Lactose + YE
S. No Isolate Black HFGR R. Red R. Yellow 1 R. Black R. Blue R. Yellow 2 C. Red
1 SK1 4.65 ± 0.23 4.13 ±0.33 6.00 ±.77 1.16 ± 0.87 ND 10.10 ± 0.45 77.27 ± 0.56
2 SK2 10.46 ± 0.53 6.61 ±0.43 8.00 ±.67 1.16 ± 0.75 7.00 ± 0.64 16.66 ± 0.66 49.35 ± 0.42
3 SK3 53.48 ± 0.44 23.96 ± 0.88 26.00 ± 0.45 53.48 ±
0.64 58.82 ± 0.32 2.40 ± 1.77 37.66 ± 1.23
4 SK4 6.97 ± 1.03 6.61 ± 1.45 8.00 ± 0.84 4.65 ± 0.45 25.88 ± 1.01 18.51 ± 0.11 65.58 ± 0.44
5 SK5 4.65 ± 0.63 4.13 ± 0.33 7.00 ± 0.78 1.16 ± 0.15 16.47 ± 0.83 13.88 ± 0.15 45.45 ± 0.18
6 SK6 6.97 ± 0.45 7.43 ± 0.52 12.00 ± 0.34 2.32 ± 0.54 16.47 ± 0.45 16.66 ± 0.66 1.94 ± 0.45
7 SK7 10.46 ± 0.75 4.13 ± 0.64 7.00 ± 0.78 1.16 ± 0.55 8.23 ± 0.65 13.88 ± 0.77 69.48 ± 0.85
8 SK8 6.97 ± 0.84 4.95 ± 0.82 4.00 ± 0.78 ND 15.29 ± 0.77 17.59 ± 0.48 62.33 ± 0.66
9 SK9 23.25 ± 0.48 11.57 ± 0.37 9.00 ± 0.65 5.81 ± 0.45 1.17 ± 0.47 19.44 ± 0.68 90.90 ± 0.45
10 SK10 6.97 ± 0.75 9.09 ± 0.48 10.00 ± 0.14 ND ND 16.66 ± 0.48 7.79 ± 0.68
11 SK11 9.30 ± 0.47 4.13 ± 0.48 5.00 ± 0.14 ND 10.58 ± 0.14 13.88 ± 0.74 92.85 ± 0.14
12 SK12 9.30 ± 0.47 4.95 ± 1.24 10.00 ± 1.5 0.00 ± 0.78 3.52 ± 0.44 10.18 ± 0.21 92.20 ± 0.74
13 SK13 3.48 ± 0.47 9.91 ± 0.45 10.00 ± 0.75 10.46 ±
0.45 ND 13.88 ± 0.15 92.20 ± 0.64
14 SK14 15.11 ± 0.46 9.91 ± 0.75 7.00 ± 0.51 8.13 ±0.18 1.17 ± 0.65 18.51 ± 0.41 92.85 ± 0.74
15 SK15 4.65 ± 0.42 10.74 ± 0.18 6.00 ± 0.75 8.13 ± 0.45 ND 18.51 ± 0.94 94.15 ± 1.0
16 SK16 ND 8.26 ± 0.33 12.00 ± 0.35 ND ND 16.66 ± 0.47 6.49 ± 0.38
17 SK17 ND 2.47 ± 0.55 16.00 ± 0.17 17.44 ±
0.14 ND 31.48 ± 0.25 93.50 ± 0.24
18 SK18 ND ND 3.00 ± 0.39 3.48 ± 0.16 1.17 ± 0.32 13.88 ± 0.41 ND
19 SK19 ND ND 4.00 ± 0.21 ND ND 12.03 ± 0.19 77.92 ± 0.21
20 SK20 54.65 ± 1.25 44.45 ± 1.22 40.00 ± 0.88 58.13 ±
0.39 85.88 ± 0.99 54.62 ± 0.98 94.80 ± 0.21
21 SK21 51.16 ± 0.14 57.85 ± 0.04 91.00 ± 0.06 59.30 ±
0.65 72.94 ± 0.14 91.66 ± 0.41 94.15 ± 0.33
22 SK22 ND 3.30 ± 0.43 11.00 ± 0.25 1.16 ± 0.84 0.00 ± 0.45 13.88 ± 0.84 83.76 ± 0.21
23 SK23 1.16 ± 0.42 1.62 ± 0.64 13.00 ± 0.72 1.16 ± 0.04 3.52 ± 0.88 12.03 ± 0.16 94.15 ± 0.84
24 SK24 ND ND 5.00 ± 0.46 ND 3.52 ± 0.79 14.81 ± 0.64 93.50 ± 0.14
Table 1.3 Decolorization of seven reactive dyes in nutrient broth by bacterial isolates (SK 21- SK 24). All the experiments were performed in triplicates and the average was calculated to represent the decolorization activity in percentage (%).
ND: No Decolorization
S. No. Isolate % of decolorization
@ 72 hrs
1 SK 11 38.92 ± 0.72
2 SK 12 41.47 ± 0.62
3 SK 13 39.42 ± 0.47
4 SK 14 40.33 ± 0.73
5 SK 15 45.24 ± 0.52
6 SK 17 44.12 ± 0.19
7 SK 20 59.74 ± 0.23
8 SK 21 62.57 ± 0.40
9 SK 23 57.11 ± 0.36
10 SK 24 50.28 ± 0.25
Table 1.4 Decolorization of colonial red in minimal media by the best 10 bacterial isolates. All the experiments were performed in triplicates and the average was calculated to represent the decolorization activity in percentage (%).
S. No.
Isolate BHM BHM+GLU BHM+YE BHM+G+YE BHM+SU+YE BHM+ST+YE BHM+LAC+
YE
1 SK 11 5.62 ± 0.17 36.17 ± 0.17 37.17 ± 0.41 35.71 ± 0.62 43.01 ± 0.61 52.68 ± 0.24 54.54 ± 0.71
2 SK 12 6.97 ± 0.31 40.28 ± 0.58 38.46 ± 0.51 29.32 ± 0.42 43.56 ± 0.23 51.35 ± 0.48 56.62 ± 0.41
3 SK 13 8.21 ± 0.40 36.27 ±0.32 38.41 ± 0.31 32.46 ± 0.27 45.16 ± 0.51 50.16 ± 0.84 49.27 ± 0.34
4 SK 14 12.18 ± 0.37 35.71 ± 0.50 34.61 ±0.29 38.96 ± 0.19 43.08 ± 0.10 54.36 ± 0.41 46.61 ± 0.19
5 SK 15 7.27 ± 0.51 41.10 ± 0.34 35.89 ± 0.61 62.33 ± 0.27 39.78 ± 0.58 57.19 ± 0.26 46.27 ± 0.24
6 SK 17 12.19 ± 0.43 42.18 ± 0.09 51.17 ± 0.26 55.84 ± 0.59 56.36 ± 0.38 54.27 ± 0.21 51.29 ± 0.70
7 SK 20 26.72 ± 0.21 57.52 ± 0.23 56.89 ± 0.49 85.06 ± 0.31 68.51 ± 0.61 61.09 ± 0.21 71.62 ± 0.28
8 SK 21 38.11 ± 0.76 63.52 ± 0.17 58.62 ± 0.23 88.31 ± 0.26 70.27 ± 0.51 70.71 ± 0.42 68 ± 0.20
9 SK 23 20.14 ± 0.31 51 ± 0.37 56.57 ± 0.41 84.41 ± 0.22 54.48 ± 0.41 54.48 ± 0.21 50.18 ± 0.26
10 SK 24 18.27 ± 0.52 41.62 ± 0.45 51.72 ± 0.39 44.80 ± 0.52 60.41 ± 0.72 49 ± 0.28 58.23 ± 0.35
Table 1.5 Decolorization of colonial red by best 10 bacterial isolates in Bushnell & Hass medium with
different carbon and nitrogen sources. All the experiments were performed in triplicates and
the average was calculated to represent the decolorization activity in percentage (%).
Media/duration 24 hrs 48 hrs 72 hrs
BHM+G+YE 56.29 ± 0.39 79.62 ± 0.24 80.31 ± 0.32
Minimal Media 31.37 ± 0.41 48.33 ± 0.19 60.21 ± 0.41
Nutrient Broth 90.27 ± 0.20 92.56 ± 0.41 90.17 ± 0.33
Table 1.6 Decolorization of colonial red by the developed bacterial consortium SK-C in different nutrient
composition. All the experiments were performed in triplicates and the average was
calculated to represent the decolorization activity in percentage (%).
Figure 1.8: Spectrum of Colonial Red before and after decolorization. (Solid line –
Native dye; dashed line – after decolorization).
Figure 1.9: Spectrum of Reactive Black HFGR dye before and after decolorization. (Solid
line – Native dye; dashed line – after decolorization).
Figure 1.10: Spectrum of Reactive Yellow I dye before and after decolorization. (Solid
line – Native dye; dashed line – after decolorization).
Figure 1.11: Spectrum of Reactive Yellow II dye before and after decolorization. (Solid
line – Native dye; dashed line – after decolorization).
Discussion
o develop an efficient dye degradation biotechnology, the key step is to obtain
broad-spectrum and highly efficient dye-decolorizing bacteria (Suizhou et al.,
2006). In the present study, isolation of potent decolorizing bacteria were
targeted from the contaminated soil sample taken within a Textile Industry (Emperor
Textiles (P) Ltd., Tirupur, Tamil Nadu, India) that have adapted to the presence of
commonly used reactive dyes. From this, a total of 24 bacterial isolates were obtained and
screened for decolorizing abilities. Among the seven dyes that were used as decolorizing
indicators, Colonial Red was found to be the easiest dye to degrade with reactive black
HFGR and reactive black being the most difficult. This is possibly due to their structural
differences and complexities (Hu, 2001). Zimmermann et al., (1982), reported similar
observation while investigating the degradability of different structures of azo dyes. It
should be noted that although the percentages did not reach 100%, the liquid appeared
colorless indicating efficient decolorization process to have involved and also the potent
strain that could possibly be investigated to apply in this regard (Khadijah et al., 2009).
Studies with nutrient broth demonstrated excellent decolorization activities over 90
% (in Colonia Red by most of the isolates) within 24 hours. This consequently lead to study
on a cheaper source of nourishment for the growth of the bacteria and decolorization.
Therefore, decolorization experiment was repeated with minimal media, which resulted in
an average/moderate decolorization of 62% after 72 hours of incubation. Further
extension of incubation did not reveal any further decolorization.
T
The eventual cessation of decolorization is likely to be due to nutrient depletion
(Saratale et al., 2006). Dye being deficient in carbon source, the biodegradation of dyes
without any extra carbon source is very difficult (Nigam et al., 1995; Senan and Abraham,
2004) and therefore, supplementation of defined sources of carbon and nitrogen was
provided with Bushnell and Haas medium as the basal composition (Safia et al., 2005, Bella
et al,. 2009).Decolorization occurred efficiently only when a carbon and energy source
were available in the growth medium. (Coughlin et al., 1997). Among different sources of
carbon and nitrogen, BHM supplemented with glucose and Yeast extract demonstrated a
suitable medium for the growth of the isolates and decolorization of Colonial Red dye.
Glucose and Yeast extract employed as metabolizable co-substrates seems to be obligatory
for the functioning of dye-decolorizing bacteria (Nigam et al., 1996b).
Many researchers have mentioned that the higher degree of biodegradation and
mineralization can be expected when co-metabolic activities within a microbial community
complement each other. In such a consortium, the organisms can act synergistically on a
variety of dyes and dye mixtures. One organism may be able to cause biotransformation of
the dye which consequently renders it more accessible to another organisms that
otherwise is unable to attack the dye (Nigam et al., 1996a, b). Knapp and Newby (1995)
reported an example of this approach using a mixed culture containing atleast four distinct
microbial strains for the degradation of dye azo linked chromophore in an industrial
effluent.
In the present study, a total of ten bacterial isolates which performed well in
screening for decolorization of Colonial red dye were mixed together as consortium and
applied for the decolorization study with colonial red as the model dye. In contrast to the
earlier reports, the consortium studied here did not decolorize to any better level than
when tested as individual strains in terms of % of decolorization and duration of
incubation as well. This is probably due to the absence of any complementary metabolic
activities of the respective bacterial isolates that could otherwise degrade the dye or
achievement of the conditions for the same (Haug et al., 1991).
Tracking back the sources of the selected isolates revealed that all these isolates
were obtained from a site where the industry had been depositing the sludge for over 5
decades since its establishment. These isolates probably have acquired natural adaptation
to survive in the presence of the dyes used in the industry and in the study thereof. This is
evident from the decolorization study in the minimal medium. Chen et al. (2003) and Senan
and Abraham (2004) reported isolation and screening of microorganisms capable of
decolorizing various azo dyes from sludge samples collected from waste water treatment
sites contaminated with dyes. Several bacterial strains that can aerobically decolorize
azodyes have been isolated during the past few years. Many of these strains require organic
carbon sources as they cannot utilize dye as the growth substrate (Stolz, 2001; Anjali et al.,
2007). From various studies carried out, SK03, SK20 and SK21 were the most efficient in
decolorizing more than one dye with color removal ranging 50% to 96% under aerobic
condition and identified to be Bacillus sp., Bacillus firmus and Paenibacillus lautus
depending on the dyes.
Many investigations in last few years have revealed the potent use and identification
of Bacillus species with respect to reactive dye decolorization (Sugiura et al., 1999; Suzuki
et al., 2001, Maier et al., 2004). To conclude, the isolation of efficient dye decolorizing
bacteria from the samples collected from dye contaminated soil indicates the natural
adaptation of these microorganisms to survive in the presence of the toxic dyes. The
advantages of these three bacterial strains are apparent and further exploitation these
selected isolates will be beneficial in textile wastewater/ effluent treatment. The results of
this study will form the basis for development of a cost effective and robust indigenous
process for bioremediation of textile dyes – based effluent.