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.