Publications - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/4801/19/19_publications… ·...

Publications

Publications

1. Senthil Kumar S, Mohamed Jaabir MS, Krishna Moorthy S, Manikandan R, Ravikumar R. (2007). Decolorization of Textile Dyes by soil isolates from a textile industry. JARJ 4 (2); 20-24. [ISSN 0973 – 0303].

2. Senthil Kumar S, Veeramani A, Mohamed Jaabir MS, Ravikumar R. (2010). Decolorization of Textile Dye Reactive Black HFGR Using a Novel Isolate Paenibacillus lautus SK21. Journal of Theoretical and Experimental Biology 6 (3): 195-202.

3. Veeramani A, Senthil Kumar S, Mohamed Jaabir MS, Sivagandhi C, Marimuthu R, Ravikumar R. (2010). Eudrilus euginiae as a putative candidate for Textile industry effluent polluted soil bioremediation. Current World Environment 5(1): 131-136. [ISSN 0973-4929].

Papers Accepted

1. Senthil Kumar S, Mohamed Jaabir MS, Ravikumar R. (2010). Biodegradation Perspective of the Textile Dye Disperse Red F3B by an Indigenous Bacteria. International Journal of Environmental Science and Technology.

International Presentation

1. Study on the Decolorization and Degradation of Textile Dyes commonly used in the Textile Industry District of Tamil Nadu, India. The proceedings of 6th International symposium on Ecosystem Behavior (BIOGEOMON 2009) held at Helsinki, Finland.

Other Citations

1. S. Senthil Kumar and M.S. Mohamed Jaabir; College faculty evolves eco-friendly process for treating effluents – The Hindu, July 17, 2009, pp-2.

2. S. Senthil Kumar and M.S. Mohamed Jaabir; College researchers say bacteria helpful

in treating toxic effluents – The Times of India, August 6, 2009.

3. S. Senthil Kumar and M.S. Mohamed Jaabir; ‘Saayakazhivai Suthigarikkum Bacteria’ – An article in Tamil, Kalai Kathir.

Sequence submissions at NCBI

S. No. Accession No. Nucleotide Source

1. FJ966212 16s rRNA partial Bacillus sp. SK03

2. FJ974057 16s rRNA partial Bacillus firmus SK20

3. FJ974058 16s rRNA partial Paenibacillus lautus SK21

4. FJ974059 16s rRNA partial Pseudomonas stutzeri JMC 01

5. GQ120620 16s rRNA partial Bacillus subtilis JMC02

6. HM451426 16s rRNA partial Comamonas species strain JMC-UBL-27

7. HM451427 16s rRNA partial Enterococcus fecalis strain JMC-UBL-31

8. HM451428 16s rRNA partial Enterococcus Fecalis strain JMC-UBL-02

9. HM451429 16s rRNA partial Bacillus flexus strain JMC-UBL-24

10. HM451430 16s rRNA partial Acinetobacter species strain JMC-UBL 15

11. HM451431 16s rRNA partial Bacillus species strain JMC-UBL 15

12. HM451432 16s rRNA partial Acinetobacter species strain JMC-UBL 17

13. HM451433 16s rRNA partial Comamonas species strain JMC-UBL 19

14. HM451434 16s rRNA partial Bacillus species strain JMC-UBL 10

15. HM451435 16s rRNA partial Paenibacillus species strain JMC-UBL 34

16. HM451436 16s rRNA partial Bacillus endophyticus strain JMC-UBL 13

17. HM451437 16s rRNA partial Bacillus anthracis strain JMC-UBL 06

18. HM451438 16s rRNA partial Pseudomonas species strain JMC-UBL 26

19. HM451439 16s rRNA partial Bacillus thuringiensis strain JMC-UBL 03

20. HM451440 16s rRNA partial Bacillus cereus strain JMC-UBL 09

21. HM451441 16s rRNA partial Bacillus pumilus strain JMC-UBL 20

22. HM451442 16s rRNA partial Lysinibacillus species strain JMC-UBL 45

23. HM451443 16s rRNA partial Bacillus megaterium strain JMC-UBL 23

24. HM451444 16s rRNA partial Pseudomonas species strain JMC-UBL 01

25. HM451445 16s rRNA partial Azoarcus species strain JMC-UBL 43

*Corresponding author; Email address: [email protected]

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Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 6 (3): 195-202, 2010

© 2010 Elias Academic Publishers

www.ejteb.org

Decolorization of Textile Dye Reactive Black HFGR Using a

Novel Isolate Paenibacillus lautus SK21

S. Senthil Kumar

1*, M. S. Mohamed Jaabir

1, A. Veeramani

2 and R. Ravikumar

3

1Department of Biotechnology, Jamal Mohamed College (Autonomous),

Tiruchirappalli-620 020, Tamil Nadu, India. 2Department of Botany, Aringar Anna Govt. Arts College, Namakkal-637 002, Tamil Nadu, India. 3Research Department of Botany, Jamal Mohamed College (Autonomous),

Tiruchirappalli-620 020, Tamil Nadu, India.

Received: 27 February, 2010; revised recieved: 6 June, 2010.

Abstract Paenibacillus lautus strain SK21 was isolated from the textile effluent polluted soil in

Tirupur, Tamil Nadu and identified based on Biochemical and 16S RNA Sequence. The

present study was carried out in an attempt to decolorize a commonly used yet tougher dye

to decolorize, Reactive Black HFGR. The decolorization percentage was calculated from

UV-Vis spectrophotometric analysis. Dye decolorization was probably due to the

biotransformation and depended upon the biomass. Replacement of nutrient broth with

minimal media did not show any decolorization property. Decolorization was optimized

and found to be up to 95% at 7pH, 40°C under static and non-aerated condition.

Key words: Reactive dyes, Azo dyes, Bacterial isolates, dye decolorization,

Paenibacillus lautus

Introduction Synthetic dyes find use in a wide range of industries such as textile dyeing, paper printing,

cosmetics and pharmaceuticals (Erdal and Taskin, 2010). Approximately 10,000 different dyes

and pigments are used in industries and over 7 × 105 tons of these dyes are annualy produced

world-wide. Due to inefficiencies of the industrial dyeing process, 10 - 15% of the dyes are lost

in the effluents of textile units, rendering them highly coloured. Among the various classes of

dyes, reactive dyes are more difficult to remove. They contain chromophoric groups such as

azo, anthraquinone, triarylmethane, etc. and reactive groups e.g. vinylsulphone, chlorotriazine,

trichloropyrimidine etc. that form covalent bonds with the fiber. Azo reactive dyes are the

largest class of water soluble synthetic dyes with the greatest variety of colors and structure and

are generally resistant to biodegradation processes (Lin and Peng, 1994; Sanghi et al., 2006;

Daneshvar et al., 2007).

Water pollution control is at present one of the major areas of scientific activity. While

colored organic compounds generally impart only a minor fraction of the organic load to

wastewater, their colour renders them aesthetically unacceptable. Colour is one of the most

obvious indicators of water pollution and discharge of highly coloured synthetic dye effluents

can be damaging to the receiving water bodies (Nigam et. al., 1966). Two percent of dyes that

are produced are discharged directly in aqueous effluent and 10% are subsequently lost during

Senthil Kumar et al / Decolorization of Textile Dye Reactive Black HFGR


196

the textile colouration process (Pearce et al., 2003). Some of the azo dyes, xanthene dyes and

anthroquinone dyes are known to be very toxic and mutagenic to living organisms.

Microbial decolorization has been proposed as a less expensive and less

environmentally intrusive alternative. In the present study, we focused our attention on the

isolation of dye decolorizing microorganisms from contaminated soil of an industrial estate and

analyzed the ability of these isolates to degrade Reactive Black HFGR.

Materials and Methods Chemicals

All chemicals and reagents used in this investigation were of Analytical grade. The common

name of the dye (Reactive Black HFGR) has been used for convenience and was procured from

A.K. Chemi Dyes Enterprises, Mumbai, India. The stock solutions of the dyes were filter

sterilized and added to the growth medium in the concentration of 100ppm (mg/litre).

Spectrum Study of the Dye

The dye procured from the industry was initially studied for absorption maxima in a Double

Beam UV-Vis Spectrophotometer from 350nm to 800nm (Schimadzu, UV-Vis 1800).

Isolation of Bacterial Cultures

Soil sample taken from the dumping grounds of the sludge was used for the isolation of dye –

decolorizing microorganisms owing to long – term usage of the location for over 5 decades

since the establishment. Bacteria from the soil sample were isolated by pour plate method and

serial dilution technique using nutrient agar medium. All the plates were incubated at 37°C for

24 hours.

Study of Decolorization Activity

All decolorization experiments were performed in triplicates. A loopful of each isolated

bacterial culture was inoculated into a separate 250 ml Erlenmeyer flask containing the Reactive

Black HFGR (100 mgl-1

) in Nutrient broth and incubated for 24 h at 37°C for initial screening

of the isolates for the ability to decolorize the dye. Aliquots of the culture (3 ml) was withdrawn

at different time intervals, centrifuged at 5000rpm for 15 min to separate the bacterial cell mass.

Decolorization was determined by measuring the absorbance of the supernatant at 520 nm (

max) and percentage of decolorization was calculated (Saratale et al., 2006) as follows:

(%) Decolorization = (Initial absorbance – Observed absorbance) / Initial absorbance X 100

A loopful of culture was inoculated in 250 ml Erlenmeyer flask containing 100 ml

nutrient broth. Separate study was carried out for different temperatures (20, 30, 40 and 50) and

pH (3-10). Decolorization was also studied under shaking (150 rpm/min) and static (non-

aerated) conditions. Decolorization was also studied in minimal media (mgl-1

): Glucose 1800;

MgSO4.7H2O 250; KH2PO4 2,310; K2HPO4 5,550; (NH4)2SO4 1,980. Effect of the source of

nitrogen in the medium for decolorization of the dye was studied using Yeast extract and

Peptone.

Identification of the Isolate SK20 by 16SrRNA Gene Amplification and Sequencing

DNA was extracted from pure culture of the isolate SK20 that showed prospective application.

A partial DNA sequence for 16SrRNA gene was amplified by using ATG GAT CCG GGG

GTT TGA TCC TGG CTC AGG(forward primer) and TAT CTG CAG TGG TGT GAC GGG

GGG TGG (reverse primer) (Jing et al., 2004). Amplifications performed in 50µl reactions

mixtures containing the template DNA, 40ng, 0.2µM, for each of the primers, dNTPs 200µM,

Taq DNA polymerase 2.5U and 10X buffers 5µl. The mixture was subjected to the following

amplification conditions; 2min at 94 C for 1min, and ended by a final extension step at 72 C for

7min. The PCR products mixture was purified and sequenced at Chromous Biotech, Bangalore,



197

India. The identity of the bacterium determined by sequencing method was verified and

confirmed through biochemical tests.

Results and Discussion

From the isolated soil sample, twenty four bacterial isolates were obtained and named as SK 1

to SK24 based on the Author’s name. The absorption maxima for Black HFGR were found to

be at 600 nm (Table 1). Decolorization occurred only when nutrient broth was available in the

medium. This was confirmed when the minimal media was replaced by the Nutrient Broth.

Among the 24 isolates, SK03, SK20 and SK 21 demonstrated decolorization beyond 50% while

the other isolates did not show any decolorization activity beyond 23.25% (Table 2).

Table 1: Absorption Maxima (λ max) of Reactive Black HFGR dye.

Dye λ max (nm)

Reactive Blue 604

Table 2: Showing % of decolorization of Reactive Black HFGR dye by 24 bacterial isolates.

S. No Isolate Black HFGR

1 SK1 4.65 %

2 SK2 10.46 %

3 SK3 53.48 %

4 SK4 6.97 %

5 SK5 4.65 %

6 SK6 6.97 %

7 SK7 10.46 %

8 SK8 6.97 %

9 SK9 23.25 %

10 SK10 6.97 %

11 SK11 9.30 %

12 SK12 930 %

13 SK13 3.48 %

14 SK14 15.11 %

15 SK15 4.65 %

16 SK16 0.00 %

17 SK17 0.00 %

18 SK18 0.00 %

19 SK19 0.00 %

20 SK20 54.65 %

21 SK21 51.16 %

22 SK22 0.00 %

23 SK23 1.16 %

24 SK24 0.00 %

The optimum temperature for the selective SK03, SK20 and SK21 demonstrated better

decolorization at a temperature of 40°C but the magnitude of decolorization differed with

57.8%, 63% and 97.9% respectively (Figure 1). The optimum pH among the three isolates

SK03, SK20 and SK21 were 6, 7 and 8 respectively. However, highest decolorization was

obtained in SK21 (Figure 2). For all the three isolates, static condition demonstrated the highest



198

percentage of decolorization and SK21 was found to be the best isolate for decolorizing reactive

black HFGR upto 95.5% (Figure 3). When choice of nitrogen source was tested, yeast extract

was suitable for SK03 and SK20 showing 45.8% and 50.8% of decolorization respectively.

Peptone was seen to be suitable for SK21 producing 25% of decolorization.

Figure 1: Effect of Temperature on the Decolorization of reactive black HFGR by SK21.

UV-Vis Spectral Analysis of Biodecolorization

The UV-Vis spectra of the media containing the dye before decolorization showed a maximum

absorption at 600 nm (0 hour). In the final stage after decolorization, the absorption maxima

totally disappeared from 600 nm (12 hours). Disappearance of the peak from 600 nm is a clear

evidence of molecular rearrangements in the dye structure and degradation thereof (Figure 4).

As reported by Asad et al. (2007) decolorization of dyes by bacteria is due to adsorption

by microbial cell as a surface phenomenon or to biodegradation. In case of adsorption, the

UV-Vis absorption peak would tend to decrease approximately in proportion to each other,

whereas, in biodegradation either the major visible light absorbance peak disappears completely

or a new peak appears. The observation of Paenibacillus lautus strain SK21cells mass retained

their natural colour after decolorization of reactive black HFGR. Based on this, it is confirmed

that the reactive black HFGR has undergone biotransformation and not due to simple adsorption

over the surface.

Physiological differences among the bacterial isolates may account for differences in

the decolorization abilities (Reddy, 1995; Asgher et al., 2006). The complex enzymatic system

responsible for the dye degradation and pattern of its expression may also vary among the

isolates (Nagai et al., 2002; Boer et al., 2004); however, the relative rates of decolorization for

the reactive blue dye cannot be easily explained. Degradation of dye involves aromatic ring

cleavage which is dependent on the identity of the ring substituents with the presence of

phenolic, amino, acetamido, 2-methoxy phenol or other easily biodegradable functional groups

resulting in a greater extent of degradation (Spadaro et al., 1992; Mazmanci and Unyayar,

2005). In the present study, reactive black HFGR was found to be decolorized to different

extents by the individual bacterial isolates. Different isolates have degraded the dye to different

levels following a different pattern during the incubation period as is commonly observed in



199

studies elsewhere (Knapp et al., 1997; Toh et al., 2003). However, overall complexity of

structure alone is not an indicator of the difficulty of decolorization of a particular dye (Maas

and Choudary 2005).

Nucleotide Sequence Accession Number

The bacterial isolate SK21 was observed to be gram-positive, rod-shaped bacteria and identified

to belong to Paenibacillus genus based on the biochemical tests (Albert and Anciet, 1999)

(Table 3). This was verified and confirmed with the sequence analysis of the amplified 16S

ribosomal DNA and therefore the isolate SK21 was determined (using the BLAST tool on

http://www.ncbi.nlm.hih.gov) to be Paenibacillus lautus SK21. The sequence data has been

deposited in the GenBank nucleotide sequence databases under accession number FJ974057.

Figure 2: Effect of different pH on the Decolorization of reactive black HFGR by SK21.



200

Figure 3: Effect of shaking and static (non-aerated) condition on the Decolorization of reactive black

HFGR by SK21.

Table 3: Biochemical profile of Paenibacillus genus.

S.No. Biochemical Test Result

1. Glycerol +

2. Esculin +

3. Maltose +

4. D-Arabinose +

5. L-Arabinose +

6. Ribose +

7. D-Xylose +

8. a-Methylxyloside +

9. Rhamnose -

10. Dulcitol -

11. Inositol -

12. Sorbitol -

13. 1-Methyl-Dmannoside -

14. Arbutin +

15. Salicin +

16. Lactose +

17. Starch +

18. Glycogen +

19. D-Tagatose -

20. D-Arabitol -

21. 5-Ketogluconate -

22. 2-Ketogluconate -

23. Gentiobiose +

24. Raffinose +

25. Trehalose +

26. Sucrose +

27. Melibiose +

28. Cellobiose +

29. Amygdalin +

30. N-Acetylglucosamine +

31. Mannose +

32. Glucose +

33. Galactose +

34. Adonitol +

35. D-Fucose +

36. Glycerol +

37. Esculin +

38. Maltose -

39. D-Arabinose -



201

40. L-Arabinose +

+ Positive: - Negative

Figure 4: UV-Vis spectra of Reactive Black HFGR biodegradation at 0 hr (a) and 12 hrs (b).

Reactive black HFGR was completely biotransformed by the novel Paenibacillus lautus

SK21 bacterial strain isolated from textile industry waste land. UV-Vis spectroscopic studies has

revealed the molecular rearrangement of the dye and therefore confirmed to have undergone

biodecolorization and biodegradation. Our study indicates the use of our novel isolate for

environmentally safe disposal of the textile dyes as compared to the existing physico chemical

methods.

References

Albert Manero and Anciet Blanch, R., 1999. Identification of Enterococcus spp. with a biochemical key.

Applied and Environmental Microbiology, 65-10: 4425-4430.

Asad, S., Amoozegar, M.A., Pourbabaee, A.A., Sarbolouki, M.N., and Dastgheib, S.M.M. 2007.

Decolorization of textile Azo dyes by newly isolated halophilic and halotolerant bacteria. Bioresour.

Technol., 98: 2082–2088.

Asgher, M., Shah, S.A.H., Ali, M., and Legge., R.L. 2006. Decolorization of reactive textile dyes by

white rot fungi isolated in Pakistan. World Journal of Microbiology and Biotechnology, 22: 89-93.

Boer, C.G., Obici, L., Souza, C.G.M.de., and Piralta, R.M. 2004. Decolourization of synthetic dyes by

solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main

lignolytic enzyme. Bioresource Technology, 94: 107–112.

Daneshvar, N., Ayazloo, M., Khataee, A.R., and Pourhassan, M. 2007. Biological decolorization of dye

solution containing Malachite Green by microalgae Cosmarium sp. Bioresour. Technol., 98:

1176-1182.

Erdal, S., and Taskin, M. 2010. Uptake of textile dye Reactive Black-5 by Penicillium chrysogenum

MT-6 isolated from cement-contaminated soil. African Journal of Microbiology Research,

4: 618-625.

Jing, X., Shaorong, G., Yong, T., Ping, G., Kun, L., and Shigui, L. 2004. Antagonism and molecular

identification of an antiobiotic bacterium BS04 against phytopathogenic fungi and bacteria. High

Technol. Let., 10 (3): 47-51.

Knapp, J.S., Zhang, F., and Tapley, N.K. 1997. Decolourization of Orange II by a wood-rotting fungus.

Journal of Chemical Technology and Biotechnology, 69: 289–296.



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Lin, S.H., and Peng, F.C. 1994. Treatment of textile wastewater by electrochemical methods. Water Res.,

28: 277-282.

Maas, R., and Chaudhari, S. 2005. Adsorption and biological decolorization of Azo dye Reactive Red 2 in

semi continuous anaerobic reactors. Process Biochemistry, 40: 699–705.

Mazmanci, M.A., and Unyayar, A. 2005. Decolourization of Reactive Black 5 by Funalia trogii

immobilized on Luffa cylindrica sponge. Process Biochemistry, 40: 337-342.

Nagai, M., Sato, T., Watanabe, H., Saito, K., Kawata, M., and Enei, H. 2002. Purification and

characterization of an extracellular laccase from the edible mushroom Lentinula edodes, and

decolorization of chemically different dyes. Applied Microbiology and Biotechnology, 60: 327–335.

Nigam, P., Banat, I.M., Singh, D., and Marchant, R. 1996. Microbial process for the decolourization of

textile effluent containing azo, diazo and reactive dyes. Process Biochemistry, 31: 435–442.

Pearce, C.I., Lloyd, J.R., and Guthrie, J.T. 2003. The removal of colour from textile wastewater using

whole bacterial cells: a review. Dyes and Pigments, 58: 179–196.

Reddy, A. 1995. The potential for white rot fungi in the treatment of pollutants. Current opinion in

Biotechnology, 6: 320-328.

Sanghi, R., Dixit, A., and Guha, S. 2006. Sequential batch culture studies for the decolorisation of

reactive dye by Coriolus versicolor. Bioresour. Technol., 97: 396-400.

Saratale, G.D., Kalme, S.D., and Govindwar, S.P. 2006. Decolorization of textile dyes by Aspergillus

ochraceus. Ind. J. Biotechnol., 5: 407–410.

Spadaro, J.T., Gold, M.H., and Ranganathan, V. 1992. Degradation of Azo dyes by the lignin degrading

fungus Phanerochaete chrysosporium. Applied and Environmental Microbiology, 58: 2397–2401.

Toh, Y.C., Yen, J.J.L., Obbard, J.P., and Ting, Y.P. 2003. Decolourization of Azo dyes by white-rot fungi

(white-rot fungi) isolated in Singapore. Enzyme and Microbial Technology, 33: 569–575.

http://www.sciencedirect.com/science/journal/13595113

Cu rrent World Environment Vo l . 5 (1 ) , 131 -136 (2010 )

Eudritus eugeniae as a putative candidate for textileindustry effluent polluted soil bioremediation

A. VEERAMANI1, S. SENTHIL KUMAR2, M.S. MOHAMED JAABIR2*'

C. SIVAGANDHI2, R. MARIMUTHUz and R' RAVIKUMAR3

lDepartment of Botany, Arignar Anna Government Arts College, Namakkal - 637 002 (lndia).2Department of Biotechnology, Jamal Mohamed college (Autonomous)'

Tiruchirappalli - 620 020 (lndia).3Department of Botany, Jama| Mohamed Co||ege (Autonomous)'

Tiruchirappalli ' 620 020 (lndia).

(Received: April 12, 2010; Accepted: June 17, 2010)

ABSTRACT

Efforts were taken to understand the impacf of textile effluent on the population of earthwormsmimicking the condition prevalent in Textile belt of Tamil Nadu where effluents are drained into the

cultivable land through the water stream. Categories of earthworms were maintained and drained with

calculated amountJof raw, chemically and biologically treated effluents for a period of 8 weeks in

which fecundity and growth *ere recoided. There was significantly high fecundity in the soil treated

with raw effluent than that of control and the treated effluents suggesting us to use of these worms for

bioremediation of textile effluent polluted soil'

Key words: Eudrilus eugeniae, Textile effluent pollution, Bioremediation.

INTRODUCTION

Textile industries consume large amount

of water (60'4001/kg of fabric) and chemicals for

wet processingl. Tlle chemical reagents used in

textile sector are diverse in chemical composition

ranging f rom inorganic to organic' The inputs of wide

range of chemicals, which, if not incorporated in

the final products (fabric), become waste and turn

out to be part of water ecology' Generally, textile

effluent is colored, varying in hydraulic flow rate'

having high; pH, temperature, biological oxygen

demand (BOD), chemical oxygen demand (COD)'

total dissolved solids (TDS) and total suspended

solids (TSS)'z{.

Color is imparted to text i le eff luents

because ol various dyes and pigments used' Many

dyes are visible in water at concentrations as low

as 1 mg/L. textile wastewaters, typically with dye

content in the range of 10'200m9/L are therefore

highly colored. In addition to this, various salts and

chemicals are major sources of heavy metals in

wastewaters. Sediments, suspended and dissolvedsolids are important repositiores for toxic heavy

metals and dyes6'7 causing rapid depletion of

dissolved oxygen leading to oxygen sag in the

receiving waters.

The key environmental issues associatedwith textile manufacture are; water use, treatment

and disposal of aqueous effluent. Textile effluents

are most ly d ischarged a f te r min ima l o r no

pretreatment into the adjoining water channels,

streams and estuariess'1o. There is a growing

emphasis on biological remediation associated with

their cost effective and long lasting nature. Presently'

textile belt of Tamil Nadu as popularly called had

been draining their effluents into the streams and

waste lands that were once cultivable until strict

rules and regulations were passed to confine iheir

effluents. Since then, the industries have been

132 veeramani et ar., curr. worrd Environ, Vor. 5(1), 131-136 (2010)

treating their etfluents either by chemical treatmentor using biological agents such as bacterium orfungi. However, the large quantities of waterultimately reach the ecosystem even after carefulrecycling and purification processes as being donerecently. Since earthworms are major componentsin the cultivable lands where the textile industrypolluted waters drain into, a study has beenundertaken to record the impact of these textile dyeindustry effluents (raw and chemical or biologicallytreated) on the population of these worms. Eudriluseugeniae was employed for the purpose.

MATERIAL AND METHODS

Collection of Raq chemically and biologicallytreated textile dye effluent

Textile dye effluents were collected fromthe United Bleachers Limited at Mettupalayamseparately as raw effluent, chemically treatedeffluent and biologically treated effluent.These werebrought to the lab and used for the restoration ofmoisture content in the earthworm medium as perthe treatment regime.

Collection of earthworms, treatment and theirmaintenance

Earthworm, Eudrilus eugeniae werecollected from the Periyar Research Organizationfor Bio-Technique & Eco-System (PROBE), PeriyarManiyammai University, Vallam, Tanjore Dist, TamilNadu, South India. Organic waste served as amedium of growth for the worms in the sievedgarden soil and cow dung mixed in the ratio of 2:1.The mixture was allowed to dry under sun-light for10 hours. After mixing subsequent amount of water,the worms were allowed to be contained in suitableperforated plastic containers with the soil (Fig.1a,1b). The study was carried out in four groups suchas the Control (Group l) receiving only tap water,Group ll: receiving raw effluent, Group lll receivingthe chemical ly treated eff luent and Group lVreceiving the biologically treated effluent(Fig.1c,1d)..The effluents were given for the first ten daysfollowed by the tap water for all the groups in orderto accommodate study in a small volume of soiltaken in the culture-basket.The study was prolongedfor a period of 8 weeks with a changeover of only30% of medium (vvt/wt) at the end of 4 weeks. Carewas taken to rear the worms with 82o/o of moisture

in the immediate surroundings. The opt imumtempera ture was main ta ined cons tan t ly byspreading some banana leaves over the culturebasket. Viability of the worms, cocoon production,hatchlings and weight of the worms were recordedfor every seven days from day 1. At the time ofrecording, the worms were taken out gently in acotton mesh and washed with a gentle flow of water.For this, the soil from each basket was carefullyplaced on a clean white surface and the worms,cocoons and hatchlings were collected carefully andweighed or counted as appropriate.

RESULTS AND DISCUSSION

Number and weight of the worms, numberof hatchlings and cocoons were recorded at aninterval of seven days for 8 weeks in all the treatmentgroups. Viability of the worms were recorded for aperiod of 8 weeks where, worms in Group ll andthe Control (Group l) were allviable. Group lll andGroup lV worms demonstrated mortality to 33%and 100 % respectively at sixth week (Fig. 2).Absolule mortality observed in Group lV worms ispossibly due to the odour, heavy microbial load and/ or the metabolites present in the biologically treatedeffluent.

The mean weight of the worms havereduced significantly (p<0.001) from 3.48t0.92 toabout 1.65t0.65 in the control group L A similarobservation was made in the treatment groups lland lll also where there is a reduction in size ofworms by 2-3 folds. However, in the biologicallytrealed group (lV), there was high mortality after 5weeks (Fig.3). In Group lV in week 5, the weight ofthe worms reduced to nearly 3 folds when comparedto the control which took 8 weeks for s imi larobservation. All the three worms in the biologicallytreated effluent-soil died in the sixth week itself. Asa measure of the populat ion growth, cocoonproduction was recorded among all the threetreatments in comparison with the control. Anaverage of 7 cocoons were seen in the control groupwhereas, in the group-ll (raw effluent) it was raisedto a maximum of 22 in the fifth week and finallywere 17 at the end of eight weeks. This is asignificant increase when compared to the controlgroup-l (Fig. a). In the chemically treated efftuent-soil, the number of cocoons was comparable to that

in the control without any significant change. Incontrast to this, there were no cocoon productionsseen in the biologically treated-soil group (lV).Hatchlings were counted every seven days andthere were two hatchlings at the end of four weeks

133

in the contro l group- l which was s igni f icant ly(p<0.001) different from the (Group ll) raw effluenttreated-soil which produced 7 cocoons. Chemicallytreated and biologically treated-soil groups (lll andlV) did not produce hatchlings through cocoons

Veeramani et al., Curr.World Environ, Vol.5(1), 131-136 (2010)

Fig. 1: Soil and cowdung mixed in the ratio of 1 :1 (1.a); Basket wilh many perforations to

allow aeration of the soil (1.b); Experiments conducted in triplicates (1.c); Earthwormsmaintained in baskets receiving tap water (control Group l), raw effluent (Group ll)'

chemically treated (Group lll) and biologically treated effluent (Group lV) (1.d)

-{-Control(Group"1}

+ Raw effluent(Group-2)

*** Chernically Treatedtffluent{Group-3)

*Biologically TreatedEfflucn t(Group-4 )

L 2 3 4 5 6 7 8Weeks

Fig. 2: Weight of the worms in all the Groups for a period of eight weeks

4.5

4

3 .5tro -3t u L . )

o L

;.9p 1.5o=

I

n q

0

134 Veeramani et al., Curr. World Environ., Vol. 5(1), 131-136 (2010)

(Fig. 5). There was significant loss in weight of theworms throughout the experimental period which isprobably due to the limited space and soil availablein the basket containing the soil. Chemically treatedeffluent-soil in group-lll demonslrated stress andtoxicity due to chemicals to certain levels that causedmorbidity and mortality though not to the level of thebiologically treated effluent-soil group-lV.

A greater proport ion (>80%) of thebiomass of terrestrial invertebrates is representedby earthworms which play an important role instructuring and increasing the nutrient content of

the soi l . Therefore, they can be suitable bio-indicators of chemical contamination of the soil interrestrial ecosystems providing an early warningof deterioration in soil gualilytt-to. This is importantfor protecting the health of the natural environmentand is of increasing interest in the context ofprotecting human healthl4 as well as other terrestrialvertebrates which prey upon earthwormsls. Thesuitability of earthworms as bioindicators in soiltoxicity is largely due to the fact that they ingestlarge quantity of decomposed litter, manure andother organic matter deposited on soil, helping toconvert it into rich topsoill&17. Moreover, studies have

I Control(Grorip-1)

r Raw ef f luerr t (Group-2 )

Weeks

Flg. 3: Viability of worms in all the Groups for a period of eight weeks

25

t n -I

E

3-g

.g

ei

.i

z

2 3

: Che.micallV TrcatedEffluent(Group-3!

I Biologically TreatedEffluent(Group-4)

+Control(Group-l!

-*-Raweflluent(Group-2 I

**r - Chemically TreatedEffluent(Group-3)

+giolqgisxlly TreatedEfflue nt(Group-41

o 1 5

uq 1 0

A

z5

l a 3 a 9 6 1 CWpeks

Fig. 4: Number of Cocoons in all the Groups for a period of eight weeks

Veeramani et al., Curr. World Environ, Vol. 5(1), 131-136 (2010) 135

shown that the earthworm skin is a significant routeof contaminant uptakels and thus investigation ofearthworm biomarkers in the ecological r iskassessment (ERA) can be helpful 1e. They have alsobeen observed to be valuable as index organismsin eva lua t ing the impact o f so i l -bornecontaminants2o-22. With the increasing rate ofindustrialization and urban development, there isno doubt that the environment we live in today isexposed to greater pollution. There is a needtherefore to evaluate an off-side test for assessingthe impact of textile industry effluent on the activitiesof the earthworm species Eudrilus eugeniae.

From the present investigations, it is clearthat the earthworms are known not to suffersignificant morbidity from exposure to raw textileindustry effluent. Moreover, they seem to flourishwith high fecundity in the presence of raw textileeffluent than that of the control water and treatedeffluents.This is a unique and striking finding whichis different from the expected outcome indicatingus to conduct further studies on testing the putativeemployment of earthworms in the bioremediationof such soils and their uses thereof to promotegrowth, development and yield of a crop plant.

--.""- Co n t rol (G ro u p- 1 )

-*- Raw effluent(Group-2)

-r-Cheilrically TreatedEmuent(Group-3)

-Biologicnlly TreatedEfflucnt{6roup-4)

Weeks

Fig.5: Number of hatchlings in allthe Groups for a period of eight weeks

REFERENCES

1 . AEPA (Australian Environmental ProtectionAuthority), Environmental guidelines for thetextile Dyeing and Finishing /ndustry StateGovernment of Victoria, Melbourne, Victoria,Australia (1998).Buckley, C. {. Membrane technology tor thetreatment of dyehouse effluents, Water Sc[Technol. 25(1 0): 203'209 (1 992).Banat , l .M, N igam, P , S ingh. D ' andMarchant. R. Microbial decolorization oftextile dye containing effluents, a review,Bioresour. Technol. 58: 217 -227 (1 996).Ghoreishi, S. M. and Haghighi, R. Chemicalcatalytic reaction and biological oxidation for

treatment of non-biodegradable text i leeffluent, Chem. Eng. J. 95: 163-169 (2003).Wagner, S. lmprovements in products andprocessing to diminish environmenlal impact,in: COTTECH Conference, Raleigh, NC,November 11-12 (1993).Tamburlini, G, Ehrenstein, O.V. and Bertollini,R. Chi ldren's health and environment: areview of evidence, Environmental lssueReport No. 129, WHO/EuropeanEnvironment Agency,WHO Geneva. 223(2002).Chapman, P.M, Romberg, G.P. and Vigers,G. A. Design of monitoring studies for priority

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poffutants, J.Water Pollut. Control Fed. 54(3):292-297 (1982).Ademoroti-C, M. A, Ukponmwan, D.O. andOmode, A. A. Studies of textile effluentdischarges in Nigeria, Environ. Stud. 39(4):291-296 (1es2).Yusuff, R. O, Sonibare, J. A. Characterizationof textile industry'effluents in Kaduna, Nigeriaand pollution implications, Global Nest Int.6(3):212-221 (2004).Robinson, T. McMullan, G. and Marchant,R. Remediation of dyes in textile effluent: ac r i t i ca l rev iew on cur ren t t rea tmenttechnologies with a proposed alternative,Bioresour. Tech noL 77 : 247 -255 (2001 ).Culy, M.D. and Berry, E.C. Toxicity of soil-applied granular insecticides to earthwormpopufations in cornfields. Down to Earth.50:'20-25 (199s).Sorour, J, and Larink, O. Toxic effects ofbenomyl on the u l t ras t ruc tu re dur ingspermatogenesis of the earthworm Eiseniafoetida. Ecotoxicol Environ Saf. 50: 1 80-1 88(2001).Bustos-Obregon, E. and Goicochea, R.l.Pes t ic ide so i l con taminat ion main lyaffectsearthworm male reproduct iveparameters. Asian J Androl.4(3): 195-199(2001).Beeby, A. What do sent inels stand for?Environ Pollut. 112: 285-298 (2001 ).Dell'Omo, G.Turk, A. and Shore, R.F. Reviewof Biological Proceedings. Environ ToxicolChem. 18: 237 -240 (1 999).

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Reinecke, S.A. and Reinecke, A.J. Lysosomalresponse of earthworms coelomocytesinduced by long-term experimental exposureto heavy metals. Pedobiologia 43: 585-593(1 eee).Sandoval, M.C, Veiga, M, Hinton, J. andKlein, B. Review of Biological indicators formetal mining effluents: A proposed protocolusing earthworms. Proceeding of the 2ghAnnual British Columbia ReclamationSymposium. 67-79 (2001 ).Lord, K.A, Briggs, G.G, Neale, M.C. andManlove, R. Uptake of pesticides from waterand soil by earthworms. Pestic Scr. 11 : 40.1-408 (1980).Sanchez-Hernandez, J .C. Ear thwormbiomarkers in ecological risk assessment.Rev Environ Contam Toxicol. 188: 85-126(2006).l re land, M. P . Meta l accumula t ion byearthworms Lumbricus rubellus,Dendrobaena veneta and Eisenia tetraedraliving in heavy metal polluted sites. Environ.Pollut., 13: 66-69 (1979).Callahan, C.A, Menzie, C.A, Burmaster, D.E,Wiborn, D.C. and Erns, T. On site methodfor assessing chemical impacts on the soilenvironment using earthworms. A case studyat the Baird and McGuire superfund site.Envi ron. Toxicol. Chem., 10:, 817 -826 (1 991 ).Schaefer . M. Ear thworms in c rude o i lconlaminated soils: Toxcity tests and effectson crude oil degradation. Environ. Asses.Remed., 8: 35-37 (2001 ).

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