Chapter IV

Treatment of Textile Effluent using Novel Bacterial Isolates

Search for a Solution

ur biosphere is under constant threat from continuing environmental

pollution. Impact on its atmosphere, hydrosphere and lithosphere by

anthropogenic activities cannot be ignored. Man made activities on water by

domestic, industrial, agriculture, shipping, radio-active, aquaculture wastes; on air by

industrial pollutants, mobile combustion, burning of fuels, agricultural activities, ionization

radiation, cosmic radiation, suspended particulate matter; and on land by domestic wastes,

industrial waste, agricultural chemicals and fertilizers, acid rain, animal waste have

negative influence over biotic and abiotic components on different natural ecosystems.

Some of the recent environmental issues include green house effect, loss in bio-diversity,

rising of sea level, abnormal climatic change and ozone layer depletion etc. In recent years,

different approaches have been discussed to tackle man made environmental hazards.

Clean technology, eco-mark and green chemistry are some of the most highlighted

practices in preventing and or reducing the adverse effect on our surroundings.

Among many engineering disciplines–Civil Engineering, Mechanical Engineering,

Electrical Engineering etc., Textile Engineering has a direct connection with environmental

aspects to be explicitly and abundantly considered. The main reason is that the textile

industry plays an important role in the economy of the country like India and it accounts

for around one third of total export. Out of various activities in textile industry, chemical

processing contributes about 70% of pollution. It is well known that cotton mills consume

large volume of water for various processes such as sizing, desizing, scouring, bleaching,

O

mercerization, dyeing, printing, finishing and ultimately washing. Due to the nature of

various chemical processing of textiles, large volumes of waste water with numerous

pollutants are discharged. Since these streams of water affect the aquatic eco-system in

number of ways such as depleting the dissolved oxygen content or settlement of suspended

substances in anaerobic condition, a special attention needs to be paid. Thus a study on

different measures which can be adopted to treat the waste water discharged from textile

chemical processing industries to protect and safeguard our surroundings from possible

pollution problem has been the focus point of many recent investigations. This

communication highlights such studies carried out in the area of textile effluent treatment.

Effluent treatment plants are the most widely accepted approaches towards

achieving environmental safety. But, unfortunately, no single treatment methodology is

suitable or universally adoptable for any kind of effluent treatment. For instance, in the

past, biological treatment systems had been used extensively but they are not efficient for

the color removal of the more resistant dyes (Mckay, 1979). Therefore, the treatment of

waste stream is done by various methods, which include physical, chemical and

occasionally biological treatment depending on pollution load. Decolorization of industrial

textile wastewater can be obtained by ozonization (50–60% of color reduction);

floculation–filtration (up to 80% of color removal) and by alkalinization with calcium

hydrossulfite. These methods can be pre-treatments or can be employed after biological

depuration (Ginocchio et al., 1985). The treatment of wastewater containing dyes and its

decolorization involves serious problems. A wide range of several pH intervals, salt

concentrations and chemical structures very often add to the complications.

Among low cost viable alternatives available for effluent treatment and

decolorization, the biological systems seem to be the best ones. In biological treatment, the

effluent of secondary sludge is partially decolorized, however dyes are not degraded and

stay adsorbed on the biomass and in the sludge from the treatment center (Conchom et al.,

1997; Morais et al., 1999). The sludge turns out to be the colored pollutant agent generated

in wet base about 1–10 ton day−1 in a medium size industry (consumption of 50 m3 water

per h). However, biological systems are recognized by their capacity to reduce biochemical

oxygen demand and chemical oxygen demand by conventional aerobic biodegradation. But

there is a problem with its inability to remove color (O’Neill et al., 2000). Although the

decolorization is a challenge for textile industry as well for wastewater treatment systems

the literature suggests that there is a great potential for developing microbiological

decolorization systems with total color removal, in some cases within few hours (Balan,

1999; Balan and Monteiro, 2000).

In the present study, an effort has been made to investigate the potential use of 46

bacterial isolates for the decolorization of the effluent. These bacterial isolates were

obtained from the soil sample taken from the effluent disposal site of United Bleacher’s Pvt.

Ltd, Mettuppalayam, Tamil Nadu, India and have already been tested against the individual

dye stuff that is commonly used in the respective textile industry (as described in chapter

II).

Materials and Methods

Effluent Collection

The raw-discharge (Raw effluent) of the dying unit of United Bleacher’s Pvt. Ltd,

Mettuppalayam, Tamil Nadu, India, was collected in barrels and transported the laboratory

within 24 hours.

Physico-Chemical Properties of the Raw Effluent

Physic-chemical properties of the raw effluent such as the pH, absorption maxima,

TDS, TSS and total solids were analyzed.

Screening of Effluent Decolorizing Isolates

All the 46 isolates obtained through serial dilution were maintained as pure culture

(as explained in Chapter II) were used for the degradation studies after pre-culturing in

nutrient agar. To screen for the ability to decolorize the effluent, an experiment was

conducted in an Erlenmeyer’s flask containing the effluent and nutrient medium to which

pure strains were inoculated and incubated for about 24 hours at 370C under shaking (150

rpm in a shaker-incubator) and static conditions. The flasks were further incubated to

observe the time required for the decolorization. Aliquots (3ml) of the culture media were

withdrawn at different time intervals, centrifuged at 10,000 rpm for 15 minutes to separate

the bacterial cell mass. Decolorization of the textile effluent was analyzed using UV-Vis

spectrophotometer (UV-Vis 1800, Schimadzu, Japan) at max. All decolorization

experiments were performed in three sets and the decolorization activity was expressed in

terms of the percentage of decolorization as described in Chapter I.

Optimization of the Decolorization Conditions

The textile dye effluent was subjected to decolorization study by the best isolate

among the JMC-UBL strains. Decolorization was performed at different pH, temperature,

media composition, shaking and static conditions as described in Chapter III.

Molecular Characterization of the Effluent Decolorizing Bacteria

The chromosomal DNA of the strain with best decolorization potential was isolated

and identified through 16S rRNA gene sequencing and subsequent blast method, as

described in Chapter II.

Results

he physico-chemical properties were analyzed. The total suspended solid in

the raw effluent was 0.30g/dl. Total dissolved solids were around 0.17 g/dl

and the total solids were 0.47 g/dl. The pH was highly alkaline (10.2) in the

raw effluent. Total dissolved solids in the chemically treated effluent were 0.24 g/dl and

the total dissolved solids of biologically treated effluent were around 0.88 g/dl (Table 4.1).

Among the 46 isolates tested for decolorizing the effluent in the nutrient medium, six

bacterial cultures alone demonstrated promising decolorizing activity with over 45% on an

average in shaking condition within 72 hours. Those six isolates were JMC-UBL02, 03, 04,

23, 24 and 27, therefore, were subjected to 16S rDNA sequencing method of identification

and found to be Enterococcus faecalis (HM451428), Bacillus thurunginesis (HM451439),

Bacillus sp. (HM45431), Bacillus megaterium (HM451443), Bacillus flexus (HM451429) and

Comamonas sp.(HM451426) respectively.

Slight change in pH was noted in all the flasks (Table 4.3). All the 46 isolates were

also tested in static condition (microaerophilic) in nutrient broth. In this experiment, JMC-

UBL 27 decolorized the effluent to a maximum of 57.67 ± 0.61 % (Table 4.2). This isolate

was one among the six isolates that performed well in shaking condition. The other five

isolates JMC-UBL02, 03, 04, 23 and 24 produced decolorization to similar levels that of the

shaking condition.

T

Based on these experiments, JMC-UBL-27 was chosen for optimization experiments.

Temperature involved the incubation of triplicate inoculated flasks, (pH 7, static condition,

nutrient broth) at 20, 30, 40 and 500C. The maximum decolorization (57.38 %) was

attained after 72 hours of incubation at 300C (Table 4.5). Further, pH optimization was

carried out at 6 different pH, such as in 4, 5, 6, 7, 8, and 9 at static condition incubated at

370C, in nutrient broth. In this JMC-UBL27 showed a maximum decolorization to about

54.27% at pH 8 (Table 4.4; Fig. 4.1, 4.2). Different media composition was also

experimented and none of the composition showed significant decolorization (Table 4.6).

All these optimization experiments revealed that JMC-UBL27 was the best decolorizer for

the effluent and worked best at pH 8, 30oC in static condition.

Table 4.1 Physico-chemical properties of the textile effluent

S. no Parameters

Treated effluent (Biologically)

Untreated effluent

1 Colour Black Light brown Dark brownish

2 pH 8.0 10.2

3 COD (mg/l) 133 183

4 BOD (mg/l) 62 89

5 TSS (g/dl) 0.36 0.30

6 TDS (g/dl) 0.88 0.17

Table 4.2. Decolorization of textile effluent in nutrient broth under static condition by

46 isolates for 72 hrs. All the experiments were performed in triplicates and

the average was calculated to represent the decolorization activity in

percentage (%). ND- No Decolorization

S. No Isolates % of Dec S. No Isolates % of Dec

01 UBL 01 30.23 ± 0.353 24 UBL 24 34.44 ± 0.33

02 UBL 02 36.28 ± 0.67 25 UBL 25 20.62 ± 0.59

03 UBL 03 40.43 ± 0.18 26 UBL 26 ND

04 UBL 04 42.44 ± .012 27 UBL 27 57.67 ± 0.61

05 UBL 05 14.51 ± 0.41 28 UBL 28 ND

06 UBL 06 36.27 ± 0.35 29 UBL 29 23.59 ± 0.22

07 UBL 07 ND 30 UBL 30 18.72 ± 0.43

08 UBL 08 18.67 ± 0.27 31 UBL 31 30.57 ± 0.47

09 UBL 09 29.73 ± 0.38 32 UBL 32 28.37 ± 0.24

10 UBL 10 ND 33 UBL 33 ND

11 UBL 11 ND 34 UBL 34 ND

12 UBL 12 10.47 ± 0.54 35 UBL 35 17.82 ± 0.26

13 UBL 13 30.53 ± 0.27 36 UBL 36 16.43 ± 0.21

14 UBL 14 20.19 ± 0.31 37 UBL 37 16.72 ± 0.25

15 UBL 15 27.54 ± 0.21 38 UBL 38 ND

16 UBL 16 28.37 ± 0.26 39 UBL 39 15.47 ± 0.27

17 UBL 17 30.33 ± 0.37 40 UBL 40 20.26 ± 0.26

18 UBL 18 18.42 ± 0.52 41 UBL 41 11.49 ± 0.28

19 UBL 19 27.61 ± 0.59 42 UBL 42 22.38 ± 0.55

20 UBL 20 30.43 ± 0.50 43 UBL 43 33.64 ± 0.71

21 UBL 21 16.23 ± 0.41 44 UBL 44 ND

22 UBL 22 20.63 ± 0.19 45 UBL 45 21.12 ± 0.66

23 UBL 23 40.27 ± 0.18 46 UBL 46 18.28 ± 0.41

Table 4.3. Decolorization of textile effluent in nutrient broth under shaking condition by 46 isolates for 72 hrs. All the experiments were performed in triplicates and the average was calculated to represent the decolorization activity in

percentage (%). ND- No Decolorization

No. Isolates % of Dec S. No. Isolates % of Dec

1 UBL 01 31.88 ± 0.56 24 UBL 24 42.85 ± 0.63

2 UBL 02 40.57 ± 0.43 25 UBL 25 25.17 ± 0.29

3 UBL 03 46.37 ± 0.28 26 UBL 26 6.15 ± 0.79

4 UBL 04 47.82 ± 0.19 27 UBL 27 46.37 ± 0.52

5 UBL 05 12.30 ± 0.27 28 UBL 28 1.53 ± 0.42

6 UBL 06 39.13 ± 0.34 29 UBL 29 30.27 ± 0.63

7 UBL 07 7.69 ± 0.38 30 UBL 30 27.28 ± 0.41

8 UBL 08 23.52 ± 0.63 31 UBL 31 36.23 ± 0.18

9 UBL 09 34.28 ± 0.53 32 UBL 32 31.42 ± 0.26

10 UBL 10 10.76 ± 0.49 33 UBL 33 4.83 ± 0.37

11 UBL 11 3.07 ± 0.54 34 UBL 34 18.46 ± 0.42

12 UBL 12 12.30 ± 0.62 35 UBL 35 27.67 ± 0.42

13 UBL 13 38.57 ± 0.17 36 UBL 36 24.53 ± 0.29

14 UBL 14 29.75 ± 0.28 37 UBL 37 16.53 ± 0.53

15 UBL 15 28.57 ± 0.34 38 UBL 38 16.25 ± 0.57

16 UBL 16 30.72 ± 0.18 39 UBL 39 16.92 ± 0.26

17 UBL 17 30.43 ± 0.54 40 UBL 40 25.71 ± 0.79

18 UBL 18 27.65 ±0.31 41 UBL 41 13.84 ± 0.47

19 UBL 19 31.42 ± 0.27 42 UBL 42 30.43 ± 0.58

20 UBL 20 18.46 ± 0.24 43 UBL 43 28.57 ± 0.63

21 UBL 21 19.57 ± 0.39 44 UBL 44 15.38 ± 0.74

22 UBL 22 28.98 ± 0.27 45 UBL 45 27.69 ± 0.49

23 UBL 23 46.37 ± 0.53 46 UBL 46 23.33 ± 0.44

Sl. No. pH % of Dec

1 4 ND

2 5 ND

3 6 ND

4 7 50.35 ± 0.47

5 8 54.27 ± 0.33

6 9 39.32 ± 0.41

Table 4.4. Effect of various pH on the decolourization of textile effluent by UBL-27.

S. No. Temperature % of Dec

1 20 10.67 ± 0.27

2 30 57.38 ± 0.67

3 40 50.31 ± 0.29

4 50 32.63 ± 0.19

Table 4.5. Effect of various temperature on the decolourization of textile effluent by

UBL-27.

S. No. Media % of Dec

1 E + P 2.85 ± 0.26

2 E + BE 8.82 ± 0.53

3 E + YE ND

4 E + P + BE ND

5 E + P + YE ND

6 E + G 3.84 ± 0.32

7 E + MM 4.34 ± 0.50

Table 4.6. Effect of various media composition on the decolourization of textile effluent

by UBL-27

E=effluent; P=peptone; BE=beef extract; YE= yeast extract; G= glucose; MM= minimal

media. All the experiments were performed in triplicates and the average was

calculated to represent the decolorization activity in percentage (%) at 72 hrs. ND- No

Decolorization

Figure 4.1: Flasks showing the decolorization of raw effluent in nutrient broth by the

isolate JMC – UBL 27 under shaking condition

.

Figure 4.2: Flasks showing the decolorization of raw effluent in nutrient broth by the

isolate JMC – UBL 27 under static condition.

Discussion

eal textile dye effluents contain not only dyes but also salts, sometimes at very

high ionic strength and extreme pH values, chelating agents, precursors,

byproducts, surfactants, etc. (Wesenberg et al., 2003). Dyes of different

structures are often used in the textile processing industry, and therefore, the effluents

from the industry are markedly variable in composition (Kalyani et al., 2009). The

difficulties encountered in the wastewater treatment resulting from dyeing operations lies

in the wide variability of the dyes used and in the excessive color of the effluents (Machado

et al., 2006). Thus, in spite of the high decolorization efficiency of some strains,

decolorizing a real industrial effluent is quite troublesome (Wesenberg et al., 2003). This is

evident in the present study. Isolates such as JMC-UBL01, 02, 03, 43 and 45 that

demonstrated significantly high capacity to decolorize the disperse group of dyes, could not

show similar efficiency in decolorizing the real-time textile effluent (from United Bleachers

Pvt. Ltd, Mettuppalayam, Tamil Nadu, India) in this chapter.

For opting biodegradation as the probable route for treatment of wastewater, fungal

strains capable of growing in wide range of pH and temperature conditions and capable of

resisting the toxicity of the dyes even at higher concentrations should be chosen (Kaushik

and Malik 2009). Studies using real dye wastewaters in addition to pure and individual dye

solutions and simulated dye wastewaters should be conducted while evaluating the

biodegradation capabilities of various microorganisms. Such studies will be greatly helpful

in the feasibility and designing of industrial-scale bioreactors for treating dye wastewaters.

R

Thus, bioremediation should not rely only on the Water, Air and Soil Pollution,

biodegradation studies of simulated dye wastewaters but should also be extended to real

dye wastewaters (textile dye effluent) in a realistic approach. However, decolorization

using fungal culture have greater disadvantages due to ‘blanket of biomass’ and in further

downstream processing of the effluent water. For the convenience and effective

management of the treatment plant, use of bacteria is greatly admired.

Physicochemical status of the effluent samples of United Bleachers Pvt. Ltd revealed

a reasonably high load of pollution indicators compared to the prescribed standards of

Pollution board. Color is imparted to a water body by dissolved constitutes (dyes and

pigments) that absorb white light and emit specific wavelength. There was a gradual

change in the color from dark brown to light brown of the effluent from source to the sink

(temporary storage tank for effluent transport and usage in the laboratory) indicating sign

of decolorizaiotn. The decreasing color intensity of the effluent has been related to

absorption / chemical transformation of dyes (including metal complex by biotic or abiotic

components of the effluent) (Adams et al., 1995; Wang, J. Yu , 1998; Blanquez et al., 2004).

The increasing bacterial count at sink might have been responsible for such color change in

the present study.

Initially the temperature of the effluent generated from UBL was considerably high,

however, declined to mesophilic status (300C) at sink, which ultimately have favored

biologically mediated remediation of effluent. This was supported by the finding through

optimization where 30ºC was found to be the optimum temperature for maximum

decolorization of the isolates. This is in consistent with the findings of Swamy and Ramsay,

(2007) Asgher et al., (2008) and Muhammad et al., (2009). The trend in decolorization

decreased above and below 300C.

Incubation temperature is a very critical process parameter which varies from

organism to organism and slight changes in temperature may affect its growth and

ultimately its enzyme production. Higher temperatures may inhibit the growth of organism

and enzyme formation which is responsible decolorization (Babu and Satyanarayana,

1995; Bhatti et al., 2007). Bioremediation at higher temperature (400C) reduces solubility

of gases in water that ultimately express as high BOD/COD. This increase in temperature

reduced the biodecolorization by almost 10% at 400C than that at 300C. High values of

BOD/COD as observed in present case demands significant amount of dissolved oxygen for

enhanced intrinsic remediation of wastewater. Generally alkaline pH of textile effluents is

associated with the process of bleaching (AEPA, 1998; Buckley, 1992; Banat et al., 1996)

and it is extremely undesirable in water ecology (Baker et al., 1994). Both chemically and

biologically mediated adsorption/reduction of dyes are initiated with decreasing pH level

under redox-mediating compounds (Shaul et al., 1991; Youssef, 1993; Van der Zee et al.,

2003). Decrease in pH i.e., from 10.2 to 8.0 of the effluent significantly improved bacterial

count and thereby associated remediation. This is consistent with the findings of Naeem et

al., (2009). TSS and TDS in effluents correspond to filterable and non filterable residues,

respectively. Reduction in pH for bioremediated favored microbial growth and the latter

eventually resulted in increased in flocculation contributing to the rise as TSS. Microbial

community (both aerobic and anaerobic) establishes itself in granulated floc as activated

sludge plays a vital role in biodecolorization/bioremediation of wastewater (Lin and Liu,

1994; Lin and Peng, 1995).

In the present study, among the 46 bacterial isolates screened for the

bioremediation process, only a few isolates show potential decolorizing abilities though of

varying degrees under shaking and non shaking conditions. However, biodecolorization

abilities of the bacterial isolates confirmed through experiments in liquid broth under

anoxic (static) conditions remained below 60%. It clearly indicated need of improvements

in culture conditions (aeration and agitation) to further augment the decolorization

processes. The extremely poor results of the densely colored effluent may be accounted for

higher unused dye concentration that may inhibit the growth of the bacterium (Muhammad

et al., 2009). Therefore, consideration of multiple aspects in degradation studies of such

chemicals cannot be ignored. But, somehow, the phenomenon of natural remediation

seemed to be occurring on-site and it was further more confirmed through laboratory

studies where few bacterial isolates indicated bioremediating abilities. A detailed

physiological understanding of such microbes is much needed for bioremediation

technology in future.