45 Int.J.Curr.Biotechnol. Volume 2; Issue 4; Apr, 2014

Rhishikesh Dhanve, Jyoti Jadhav, Sanjay Govindwar, A study of textile effluent ecotoxicity and its biodegradation by anExiguobacterium sp. RD3, Int.J.Curr.Biotechnol., 2014, 2(4):45-50.

A study of textile effluent ecotoxicity and its biodegradation by an Exiguobacterium sp. RD3

Rhishikesh Dhanve1*, Jyoti Jadhav2, Sanjay Govindwar2

1 Department of Biotechnology, Shriram Pratishthan Mandal’s Lokmangal Institute of Versatile Education (Affiliatedto Solapur University), Wadala, Solapur – 413 222, India.

2 Department of Biochemistry, Shivaji University, Vidyanagar, Kolhapur- 416 004, India.

A R T I C L E I N F O A B S T R A C T

Article History:Received 12 April 2014Received in revised form 18 April 2014Accepted 24 April 2014Available online 30 April 2014

Key words:Exiguobacterium, Chromosomal aberra-tion, Micronuclei, Mortality rate.

Biodegradation of textile effluent by an isolate from effluent contaminatedsite up to a threshold level was monitored. Along with biodegradation byExiguobacterium sp. RD3 the concentration of fluoride, nitrate, chloride,iron, and calcium were found to be lowered. Exposure to control effluentexerted occurrence of different chromosomal aberrations in root tip cells ofAllium cepa were also studied. Aquatic fish under_study (Acantopsischoirorhyncus) showed anxious movements in presence of effluent. Hema-tological studies of the blood cells showed abnormalities in the shapes oferythrocytes and formation micronuclei. Mortality rate of fish under_studywas observed to drop off in the environment with presence of the biode-graded metabolites of textile effluent.

IntroductionDuring textile processing, inefficiencies in dyeing resultin large amounts of the dyestuff being directly lost to thewastewater, which ultimately finds its way into the naturalreservoirs (McMullan et al., 2001). Direct discharge ofthese effluents causes formation of toxic aromatic aminesunder anaerobic conditions in receiving media (Asgheret al., 2006). Textile wastewater is diverse in chemicalcomposition and is considered as most polluted amongall industrial sectors (Ali et al., 2008). The aquaticenvironment is the ultimate destination for almost allindustrial wastes and water quality gets seriouslyimpacted by these waste products (Talapatra et al., 2006).It has been shown that fish are suitable sentinelorganisms for monitoring genotoxic pollutants in theaquatic environment because they play an important rolein the food web, they are bio-concentrators and areresponsive to mutagens at low concentrations such asenvironmental pollutants. The latent affects of themutations caused by such pollutants may take aconsiderable length of time to become apparent and mayhave adverse consequences on the ecosystem (Ngan etal., 2007). Textile dying effluents have reported to inducecytogenetic and geotoxic affects on aquatic animal cells

(Lee and Steinert, 2003). Along with adverse affect onroot elongation, chromosomal aberrations likefragmentation, bridge formation, lagging chromosome,multipolarity, lack of cytokinesis etc. have been observedin plants like Allium cepa (Dane et al., 2006). Due toreported mutagenicity of some synthetic dyes, disposalof untreated textile dyeing effluent water has raisedconcern about the safety of the aquatic environment(Sumathi et al., 2001). Studies have reported that theecotoxicity of individual xenobiotic dyes as well as itstextile effluent can be lowered in presence of potentialmicroflora (Prasad et al., 2013; Govindwar et al., 2014). Inthis study we have tried to focus on the comparativestudy of toxic effects of textile effluent and the sameeffluent after biodegradation.

Materials and methodsEffluent and chemicalsTextile effluent used for biodegradation studies wasobtained from textile industry, Solapur, Maharashtra. Thechemicals Tris, Di-sodium ethylene dichloro tetraamide(Na

2 EDTA), Sodium hydroxide (NaOH), Triton, Dimethyl

sulfoxide (DMSO) were obtained from Hi-mediaLaboratories Pvt. Ltd., Mumbai, India., ferrous ammoniumsulfate, potassium dichromate, Leishman’s stain, acetoorcein stain, were obtained from Sisco ResearchLaboratories, Mumbai. Ethyl alcohol, acetic acid,methanol was obtained from Qualigens Fine Chemicals,Mumbai, India.

*Corresponding author.Email address: rhishodh@gmail.com

International Journal of CurrentBiotechnology

Journal Homepage : http://ijcb.mainspringer.comISSN: 2321 - 8371

Volume 2; Issue 4; Apr, 2014 Int.J.Curr.Biotechnol. 46

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

400

430

460

490

520

550

580

610

640

670

700

730

760

790

Wavelength (nm)

Abs

orba

nce

Control EffluentDegraded effluent

Figure – 1: UV-Visible spectrophotometric analysis of textile effluent (f&); and its degradation product ( %).

Sr. No. Test for Effluent

(ppm)

Metabolite

(ppm)

1 Fluoride 3 0.5

2 Nitrate 25 15

3 Chloride 10 -

4 Iron 0.6 0.3

5 Calcium 110 70

6 Sulphide - -

7 Hardness 200 (ppm CaCO3) -

8 Alkalinity 200 (ppm CaCO3) -

9 BOD 63 mg l -1 36 mg l -1

10 COD 181 mg l-1 84 mg l -1

Table - 1: Characterization of textile effluent

Sample Root elongation (cm)

Effluent 0.4 ± 0.018

Water 5.2 ± 0.014* Metabolite 4.3 ± 0.012*

Table – 2: Effect of the effluent on root elongation of Allium cepa

The values are mean of three experiments andSEM (±) is significantly different from the Alliumcepa root elongations in control water at * P <0.01 by one-way ANOVA Test with Tukey KramerMultiple Comparison Test.

47 Int.J.Curr.Biotechnol. Volume 2; Issue 4; Apr, 2014

a b c d

e f g h

i j k l

n o p m

Figure 2: Mitotic aberrations in Allium cepa (a) Anaphase, (b, c, and d) Anaphase with bridge, (e and f) Anaphase withlaggard formation, (g) Anaphase with chromosome loss, (h and i) Breaks showing chromosomal fragments, (j) Stickchromosome, (k) Micronucleus formation, (l, m, n, and o) Binucleated telophases, (p) Telophase with bridge formation.

Triticum aestivum Phaseolus mungo

Water Textile effluent Extracted metabolite Water Textile effluent

Extracted metabolite

Germination (%) 100 50 80 100 90 90 Plumule (cm)

9.5 ± 1.27

5.6 ± 1.33

8.5 ± 1.21

16.32 ± 1.26

8.14* ± 1.23

15.44$ ± 1.37

Radical (cm)

6.83 ± 1.45

2.95 ± 1.10

5.33 ± 1.17

11.03 ± 1.34

1.42* ± 0.18

7.25$ ± 1.41

Table–3: Phytotoxicity of textile effluent on Triticum aestivum and Phaseolus mungo.

The values are mean of ten germinated seeds of three sets SEM (±). Significantly different from the seeds germinatedin plain water * P < 0.001 and significantly different from the seeds germinated in Textile effluent at $ P < 0.001 by One-way Analysis of Variance (ANOVA) Test with Tukey Kramer Multiple Comparison Test.

Volume 2; Issue 4; Apr, 2014 Int.J.Curr.Biotechnol. 48

Biodegradation analysesThe textile effluent was subjected to biodegradationanalysis by growing an efficient biodegradationmicroorganism in presence of 40% diluted sterilized textileeffluent. This organism which was isolated from the soilcontaminated with textile effluent and was identified asExiguobacterium sp. RD3. Routinely it was maintainedon selective medium having composition (g/l) nutrientbroth 25, glucose 5 and yeast extract 3. After incubationof 60 hours, the inoculated effluent was made cell freeand was analyzed for its absorption maxima using UV-Visible spectrophotometer (Hitachi U 2800).Characteristics of effluent like fluoride content, nitratecontent, chloride content, iron content, calcium contentsulphide content, hardness, and alkalinity were analyzedusing water analysis kit from Bangalore genie. COD andBOD analyses were carried out using spectralab CODand BOD analyzers.

Cytotoxicity study in Allium cepaAfter 1 week exposure to dilute effluent (1:2), the roottips of Allium cepa were fixed in Carnoy fixative (3 alcohol:1 acetic acid) and hydrolyzed in 1N HCL at 50 oC for 5 minfollowed by squashing in a 2% aceto orcein stain. Slideswere kept in a freezer and examined within a month. TheAllium test was conducted according to Rank and Nielson(1994) with slight modification. For the mutagenicityassessment dividing cells with the irregular anaphases(e.g. disorganized structure, lag chromosomes ormultipolar anaphases), cells with stick chromosomes,micronuclei, and binuclear and/or multinucleate cells wereobserved.

The roots of Allium cepa were allowed to sprout indifferent dilutions of effluent and the elongation in rootswas observed after an incubation period of one week.During the incubation period the sprouted roots wereregularly supplied with respective effluent dilutions.From the number of divided and undivided cells thepercentage cell division was determined as mitotic index.

Phytotoxicity studyPhytotoxicity studies were carried out by using plantsTriticum aestivum and Phaseolus mungo. The plants weresupplemented with equal volumes of effluent dilution(1:2), water, and degradation metabolites, incubated for 7days. Observations were done in terms of root, shootgrowth and percentage germination.

Fish toxicity studyThe fish (Acantopsis choirorhyncus) were brought fromlocal fish market, Kolhapur. They were kept in regularwater before experiment and supplied with fish foodgranules. To study the effect of textile effluent on fishmortality, different dilutions of textile effluent were maderanging from 10% to 60%. Regular water was used fordilutions. Changes in behavior of fish and their mortalityrate in different effluent dilutions were observed for thetime span of 48 hours. To study the abnormalities inerythrocytes of fish understudy in effluent environment,about 10 µl of blood from the fish showing anxiousmovement was drawn by puncturing the caudal vein. Athin smear of blood was prepared on a clean grease freeslide. The blood cells were then stained by Leishman’sstaining and observed under oil immersion lens.

Statistical analysisFor the analysis of data, one-way analysis of variance(ANOVA) with Tukey-Kramer multiple comparisons testwas used.

ResultsWhen characterization of effluent was performed resultsas shown in Table 1 were observed. Along withbiodegradation by Exiguobacterium sp. RD3 theconcentration of fluoride, nitrate, chloride, iron, andcalcium were found to be lowered. Also there wasdepletion in BOD and COD values. When degradation oftextile effluent was monitored spectrophotometrically, asteep decrease in absorbance was observed afterbiodegradation of textile effluent by the Exiguobacteriumsp. RD3. Decrease in absorbance confirmed thebiodegradation of compounds in textile effluent byExiguobacterium sp. RD3. Biodecolorization (83.57%)was also observed due to cleavage of chromophoricgroups in the compounds (Fig. 1). Textile dye content ofany effluent is variable and is the result of particularprocessing being applied. When the textile dyes used inpresent effluent generation were studied in pure form forindividual biodegradation; different degradationproducts were formed. In our previous studies, based onGCMS analysis they were proposed to beenvironmentally safe concentrations of compounds like8 nitrosonaphthol, naphthalene, 5 aminonaphthalene,1,3,4-triol, Naphthalene, Nitrophenylamine, N,N benzene1,4 diamine, N, N-dimethyl benzene- 1,4 diamine, 2 Aminobenzoic acid (Dhanve et al., 2008; Dhanve et al., 2009).

In presence of control effluent, the decrease in rootgrowth was observed as compared to that in presence ofits degradation product. Besides the root tip squashpreparations showed the occurrence of differentchromosomal aberrations like bridge formation, laageringof chromosome, binuclear cells, micronucleus formationetc. This confirmed the interference of the recalcitrantcomponents of effluent in the cell division of the Alliumcepa root tip cells. As a result of which there wasdiminished root growth in presence of control effluent.The aberrations observed, are as shown in Fig. 2.

In presence of control effluent, there was very muchinhibition of root elongation of Allium cepa. Inhibitionof elongation of the root length was observed to bedirectly proportional with effluent concentrations. Thebiodegradation product did not show any inhibition effecton root elongation. Indeed the roots grew well in presenceof degradation product.. Inhibition effect was due to theaberrations in mitosis of growing cells. The dye inhibitedcell division and there was decline in the mitotic indexwith the increase in dye concentration and duration oftreatment. Number of undivided cells was more inpresence of textile effluent. The effluent, water anddegradation metabolite of effluent showed the mitoticindex of 0.25, 0.83, 0.71 respectively (Table 2). In presentcase, suppression of mitotic activity and aberrations inmitosis are mainly responsible for occurrence of stuntedroot length of Allium cepa in presence of textile effluent.

Phytotoxicity studies with Triticum aestivum andPhaseolus mungo revealed that there was reduction inroot, shoot development as well as the germination ofseeds understudy in presence of control effluent.Whereas the degradation product of the same effluentdid not show such reduction in root, shoot developmentand seed germination. Statistical results of phytotoxicityare as shown in Table 3. This confirmed the abatement oftoxic recalcitrant compounds by their bioconversion tonontoxic product. The nontoxic nature of the degradationproduct rendered the biodegradation byExiguobacterium sp. RD3 as a significant ecofriendlyprocess.

49 Int.J.Curr.Biotechnol. Volume 2; Issue 4; Apr, 2014

When the fish Acantopsis choirorhyncus was exposedto the effluent containing environment, deliberate affectsof effluent toxicants were observed on movements andmortality of the fish. Within 48 hours of incubation withregular food supplements, in 40% dilution of the effluent,more than half of the population of the fish understudydied. Uneasy motility of the fish was observed to beincreased along with the increasing concentrations ofeffluent dilutions. This cleared that the textile effluentused in present study exerted hazardous health affectsin Acantopsis choirorhyncus . In contrast, thebiodegradation product of the effluent did not induceany observable hazardous health effects on the fishunderstudy.

In this study the textile effluent was found to induceerythrocyte nuclear abnormalities in fish Acantopsischoirorhyncus. DNA damage was confirmed by Cometassay and was observed in form of decreased tail lengthand increased fragmentation in case of damaged DNA(Table 4). When blood cells of Acantopsis choirorhyncuswere observed for any abnormalities in erythrocytes byLeishman’s staining method, it was observed that therewas considerable change in shape of erythrocytes in fishunder influence of effluent environment (Fig. 3). Theabnormality of shape was inferred by comparison withthe shape of erythrocytes from normal healthy fish.

DiscussionBesides their visual effect and adverse impact in terms ofchemical oxygen demand, many synthetic dyes are toxic,mutagenic and carcinogenic (Jin et al., 2007). Due toreported mutagenicity of some synthetic dyes, disposalof untreated textile dyeing effluent water has raisedconcern about the safety of the aquatic environment(Sumathi et al., 2001). Characterization of the effluentunderstudy was done to monitor the extent ofbiodegradation process. Different physicochemicalmethods have been reported earlier to elicit thebiodegradation of wastewater ingredients.Physicochemical methods have their own merits anddemerits. However biological treatment methods are morefeasible due to their cost effectiveness, reproducibilityand reusability of microbes. Enzymatic metabolic systemsof different microbes make them capable of utilizingcomplex organic pollutants. The metal ion compositionof textile effluents may vary qualitatively andquantitatively, and metals can be used for monitoringwater pollution (Grinevicius et al., 2008). Since most ofthe textile dye effluents are discharged into an aquaticenvironment, evaluation of their toxicity is important inhelping to reduce potential hazardous affects on aquaticorganisms (Birhani and Ozmen, 2005). Differentpollutants have been reported earlier as causes ofappearance of erythrocyte nuclear abnormalities in fish(Bolognesi et al., 2006). Appearance of micronucleus is awell-known indicator of genotoxic pollution, but there isno consistent data on the origin of erythrocyte nuclearabnormalities. Fish are ectothermic organisms, anddifferent abiotic parameters can influence theirphysiological status and sensitivity to otherenvironmental demands (Perovic et al., 2009). Cytogeneticand DNA damage studies in peripheral erythrocytes dueto pollutants have been reported earlier (Avasx andKonen, 2007). Genotoxicity - DNA damage in the fishblood cells was also confirmed by the comet assay whichhas been mostly stated as a sensitive and efficient assayfor studying DNA damage (Rivero et al., 2008; Dhawanet al., 2009). Main reason for the deaths may beunavailability of dissolved oxygen in the effluentenvironment. Besides this, reddish rashes were also

observed on the skin of many fish in effluentenvironment, which supported the toxic nature of theeffluent constituent. Similar to that the effluent was alsoobserved to exert cytotoxic and phytotoxic effects.Biodegradation potential of the present organism madeit possible to reduce its toxicity upto a certain extentunder controlled conditions.

ConclusionIn conclusion the textile effluent used in present studywas ecotoxic since it exerted hazardous affects on plantsources Allium cepa, Triticum aestivum and Phaseolusmungo. Monitoring of biodegradation of textile effluentby Exiguobacterium sp. RD3, rendered it as ecologicallysafe phenomenon in the toxicity point of view.

AcknowledgementThe authors want to thank Mr. Vikas Jadhav, Departmentof Environmental Science and Mr. Yogesh Gaikwad,Department of Zoology, Shivaji University, Kolhapur fortheir technical assistance during the study.

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Table – 4: DNA damage studies in blood cells of Acantopsis choirorhyncus

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Con trol cells C ells exposed to effluen t

Olive tail moment 1.75 ± 0.040 1.05 ± 0.008*

Extent tail mome nt 2.14 ± 0.038 1.27 ± 0.014*

Tail DNA% 7.64 ± 0.028 7.21 ± 0.007*

Tail length (µM) 28.02 ± 0.042 17.72 ± 0.057*