RESEARCH ARTICLE

Decolorization does not always mean detoxification: casestudy of a newly isolated Pseudomonas peli for decolorizationof textile wastewater

Afef Dellai & Dorra Dridi & Valerie Lemorvan &

Jacques Robert & Ameur Cherif & Ridha Mosrati &Hedi Ben Mansour

Received: 14 January 2013 /Accepted: 25 February 2013 /Published online: 8 March 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The textile industry is a favor to the Tunisian econ-omy by offering several job positions. However, it’s notenvironmentally friendly. In fact, textile industries dischargehigh volumes of wastewater which contain several toxic pol-lutants such as dyes, fixator, and whiteness. In our study,Pseudomonas peli, isolated and characterized from OuedHamdoun (center of Tunisia), was found able to decolorizetextile effluent about 81 % after 24 h shaking incubation. Onthe other hand, the in vitro antiproliferative effects of theuntreated and treated effluent was evaluated by their potentialcytotoxic activity using the MTT colorimetric method against

three human cancer cell lines (A549, lung cell carcinoma;HT29, colon adenocarcinoma; and MCF7, breast adenocarci-noma). Results showed that intact textile effluent and itscontent azo dyes didn’t inhibit the proliferation of all testedcell lines. However, the cytotoxic effect was remarkable whenwe tested effluent obtained after treatment by P. peli in a dose-dependent manner. This activity was attributed to the pres-ence, in our treated effluent, of some azo products of dyeswhich are responsible for inhibition of human cell lines pro-liferation. Thus, the use of this strain for testing on the indus-trial scale seems impossible and disadvantageous.

Keywords Textile effluent . Biotreatment . Cytotoxicity .

Pseudomonas peli

Introduction

The textile industry is one of the oldest and largest orga-nized sectors in Tunisia. There are over 2,086 large-scaletextile industries concentrated mainly (80 %) in the coast ofTunisia. States of Monastir, Sousse, and Mahdia, are themost important centers of ethnic textile prints having greaterdemand in the international market. Indeed, the ennoble-ment branch consumes over 300 tons/year of dyes and pro-duces more than 116 millionm3 (Mm3) of effluent.Generally, textile industries consume large amounts of water(60–400 l/kg of fabric) and chemicals for wet processing(Ali et al. 2009). The chemical reagents used in textile sectorare diverse in chemical composition ranging from inorganicto organic. The input of a wide range of chemicals, which, ifnot incorporated in the final products (fabric), becomeswaste and turns out to be part of the water ecology. Gener-ally, textile effluent is colored, varying in hydraulic flow

Environ Sci Pollut Res (2013) 20:5790–5796DOI 10.1007/s11356-013-1603-3

Responsible editor: Philippe Garrigues

Afef Dellai and Dorra Dridi contributed equally in this work

A. Dellai :D. Dridi :A. Cherif :H. B. MansourLaboratoire de Biotechnologie et Valorisation de Bio GéoRessources Institut Supérieur de Biotechnologie (LR11-ES31),Université de la Manouba BioTechPole Sidi Thabet, 2020Sidi Thabet, Ariana, Tunisia

A. Dellai :V. Lemorvan : J. RobertLaboratoire de Pharmacologie des Médicaments Anticancéreux,Université Victor Segalen Bordeaux 2, Institut Bergonie,229 cours de l’Argonne,33076 Bordeaux Cedex, France

R. Mosrati :H. B. MansourLaboratoire des Aliments Bioprocédés, Toxicologie,Environnements (ABTE-EA 4651) IUT de Caen, Universitéde Caen Basse-Normandie, Campus 2 Boulevard Maréchal Juin,14032 Caen Cedex, France

H. B. Mansour (*)Laboratoire de biotechnologie et Valorisation de Bio GéoRessources (LBVBGR) Institut Supérieur de Biotechnologie,ISBST BioTechPole Sidi Thabet Université Manouba, Sidi Thabet,Ariana 2020, Tunisiae-mail: hdbenmansour@gmail.com

rate, having high pH, temperature, biological oxygendemand, chemical oxygen demand, total dissolvedsolids, and total suspended solids (Buckley 1992;Ghoreishi and Haghighi 2003).

The potential hazards of textile effluent to ecosystemsand human health have aroused great concern since textileeffluents usually contain some toxic substances, such asadditives, detergents, surfactants, and dyes, which arecarcinogenic, mutagenic, or teratogenic to various organisms(Vanhulle et al. 2008; Ben Mansour et al. 2012a). In addition,the presence of dyes or their degradation products in water canalso cause human health disorders such as nausea, hemor-rhage, and ulceration of skin and mucous membranes, and cancause severe damage to the kidney, reproductive system, liver,brain, and central nervous system (Puvaneswari et al. 2006).These concerns have led to strict regulations on the dischargeof textile wastewater, compelling dye manufacturers and treat-ment plants to adopt more effective approaches.

Therefore, treatment of textile dye effluents prior to theirrelease in nearby water streams is necessary. The physicochem-icalmethods, viz., ozonation, photooxidation, electrocoagulation,adsorption, froth flotation, reverse osmosis, ion exchange, mem-brane filtration, flocculation, etc. (Robinson et al. 2001;Daneshwar et al. 2007; Mittal et al. 2005), employed for dyedecolorization are less efficient, costly, and generate secondarysolid waste. Traditional biological treatment methods alone or incombination with physical and/or chemical methods have alsobeen attempted. However, the advanced biological processeshave received increasing attention due to low cost, effectivity,less sludge generation, and eco-friendly nature (Chen et al. 2003). It is well known that a variety of bacteria are not only capable ofdecolorizing, but also able to completely mineralize many reac-tive dyes under certain specific optimum cultural conditions. Ithas also been reported that bacterial process is much faster thanfungal decolorization/degradation of dyestuffs (Karapinar et al.2000; Pourbabaee et al. 2006).

This concern leads us to focus our studies to search forbacteria gifted with the ability to decolorize a textile effluentin Tunisia. The performance of our treatment was evaluatedby the cytotoxic effect of textile effluent before and afterbiotreatment. Cytotoxicity was evaluated towards threehuman cell lines A549 (lung cell carcinoma), HT29 (colonadenocarcinoma), and MCF7 (breast adenocarcinoma).

Materials and methods

Characteristics of the textile wastewater

Tested wastewater was obtained from polyester and polyamidedyeing industry (Medyl ennoblement industry) located in KsarHellal City (center of Tunisia) in May 2012. The samplingpoint in the wastewater treatment plant was the homogenization

tank. Effluent was composed of three acid dyes: Brown YellowNylosane ERLN, Blue Nylosane EBGL, and Bemesside RedE-3BS, and two disperses dyes: Ecarlate Foron RD-FRS andBrown Yellow Foron RD2RS. The latter were used in order tocolor the polyamide fabric. The tested effluent was alsocontained by an acid generator MEROPANT EF (pH=4.5)and the dispersing agent Univadine MC.

Bacterial

Bacterium used in this study was isolated from the OuedHamdoun River (area from discharge pollutant of textileindustries). The 16 s region of the isolated bacteria strainwas amplified using the primers S-D-Bact-0008-a-S-20 andS-D-Bact-1495-a-S20 (Biomatik, USA). The amplifiedproduct was purified using the QIAquick gel extraction kit(QIAGEN) and the sequencing was performed in the ABIPRISM 377–3.0 automated sequencer using the Taq dyedeoxyterminator cycle sequencing kit (Perkin-ElmerApplied Biosystems). The obtained sequence was identifiedby comparison with available sequences in the GenBankdatabase using BLAST Software (Altschul et al. 1990). Thisstrain was identified as Pseudomonas peli, a nonpathogenicand aerobic bacterium.

Agar disk diffusion method

The agar disk diffusion method was employed to exhibit thetoxicity of the tested pharmaceutical effluent against P. peli.The procedure was used as described previously (BenMansour et al. 2012b). A suspension of P. peli diluted priorto 10−1, 10−2, and 10−3 (1 ml of 108cells/ml) was spread ona Mueller-Hinton solid agar medium in petri dishes. Filterpaper disks (6 mm in diameter) were soaked in 10 μl of thetextile wastewater and placed on the inoculated plates andallowed to dry for 15 min, and then incubated at 37 °C for24 h. The diameters of the inhibition zones were measuredin millimeters.

Determination of cell dry weight

Cell dry weight was determined according to the methoddescribed by Meyer et al. (2004) and modified according toour conditions. Firstly, a suspension of P. peli was used andanalyzed by spectrophotometer in order to determine themaximum of absorbance (λmax) which was identified asλmax=650 nm. This suspension of P. peli was diluted 10−1

to 10−10 and the DO630nm was determined for each suspen-sion. Then, 10 ml of the diluted suspensions werecentrifuged (6,000 rpm for 10 min). The pellet was washedthree times with 10 ml of 0.9 % NaCl solution and wasincubated 1 h at 105 °C. After 10 min cooling at roomtemperature, the pellet was weighed. Then, we can calculate

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the cell dry weight expressed by gram of dry cell per liter ofculture medium. Finally, we plot the curve of variation ofDO630nm according to the obtained weights. The cell dryweight concentration can be determined according to thefollowing relation (Eq. 1):

Dry cell weight concentration g=lð Þ¼ A630nm � 1:189 ð1Þ

Acclimatization and biodegradation

P. peli was replicated in Mueller-Hinton solid agar mediumsupplemented with 20 % of tested textile effluent in petridishes. P. peli was grown at 37 °C in 250-ml flasks,containing 50 ml of medium, under rotary shaking incuba-tion at 200 rpm. The growth medium contains yeast extract(15 g/l), peptone (4.5 g/l), and glucose (5 g/l). Textilewastewater (5 ml) was then added to the culture mediumin order to induce the bacterium metabolization system.After cultivation of P. peli on an enzymatic inductive medi-um (nutrient broth supplemented with 10 % of textile waste-water), the exponential phase culture (≈1 g/l of dry cells)was centrifuged (5,000 rpm for 10 min at 37 °C), and thencells were harvested and transferred into a second flask(100 ml in a 500-ml flask) containing only effluent. Biodeg-radation of textile effluent was conducted in flasks at 37 °Cunder oxygenated conditions assured by agitation (200 rpm)and continuous air injection.

Decolorization percentage was calculated as follows(Eq. 2).

Decolorization %ð Þ ¼ DOi � DOf=DOið Þ � 100 ð2Þ

DOi Absorbance of textile effluent before incubation withP. peli

DOf Absorbance of textile effluent after incubation withP. peli

All decolorization experiments were performed in threeindependent replicate experiments. Abiotic (without micro-organism) controls were always included. After incubationof P. peli during 24 h under aerobic condition, the“decolorization medium” was centrifuged (5,000 rpm,10 min, 4 °C). The supernatants were filtered through0.22 μm filters and were directly used for the cytotoxic assay.

Cytotoxicity

Cell culture

The human tumor cell lines A549, HT29, and MCF7 wereobtained from the American Type Culture Collection(ATCC, Manassas, VA). Cells were routinely grown with

DMEM supplemented with 10 % fetal calf serum and 1 %penicillin/streptomycin, all obtained from Biochrom AG(Berlin, Germany). They were grown on flasks (Nunc,Denmark) at 37 °C in a humidified atmosphere containing 5%CO2. Cells were replicated every 4–5 days and the mediumchanged once in between.

Viability assay

The potential effects on cell viability were investigatedaccording to previously reported conditions (Hu and Robert1995), using the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich Chimie,Saint-Quentin-Fallavier, France)] as an indicator of meta-bolically active cells (Martine et al. 2008).

The known number of A549, HT29, or MCF7 cells (103)were transferred into 96-well plates (Nunc, Denmark) in avolume of 200 μl of culture medium and incubated for 24 hbefore addition of test compounds. Cells were then exposed for24 h at 37 °C to known concentrations of the fractions to betested. After drug exposure, the cells were washed withphosphate-buffered saline and then reincubated in fresh culturemedium for a further 48 h, then the culture medium wasremoved and 200 μl of MTT reagent (diluted in culture medi-um, 0.5 mg/ml) was added. Following incubation for 4 h, theMTT/mediumwas removed and DMSO (200 μl) was added todissolve the formazan crystals. Absorbance of the coloredsolution was measured on a microplate photometer (BioTekInstruments) using a test wavelength of 570 nm and a referencewavelength of 630 nm. Results were evaluated by comparingthe absorbance of the treated cells with the absorbance of wellscontaining the solvent control. Conventionally, cell viabilitywas estimated to be 100 % in the solvent control. All experi-ments were performed at least twice in triplicate.

Results

Biodecolorization of textile wastewater

The tested textile wastewater exhibited no toxicity againstbacterium as determined by the agar disk diffusion method.This strain used was isolated from the Oued HamdounRiver, and identified by BLAST as P. Peli. The curvebiomass versus time (Fig. 1) reached a plateau after 6 hshaking incubation, with a final biomass of 2.09 g/L. On theother hand, P. peli was able to decolorize textile wastewaterand the kinetics of biodegradation plotted as increasingpercentage of decolorization versus time is shown inFig. 1. The rate of decolorization was significantly lowerin the first 6 h (% of decolorization=12 %) and thereafteraccelerates to reach 81 % after 24 h of oxygenatedincubation.

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Cytotoxicity

The MTT reduction assay predicts the response of tumors tocytotoxic drugs. The acid dyes, Brown Yellow NylosaneERLN, Blue Nylosane EBGL, and Bemesside Red E-3BS,and the disperse dyes, Ecarlate Foron RD-FRS and BrownYellow Foron RD2RS, were tested for their effects on cel-lular viability against three human tumor cell lines A549,HT29, and MCF7 over a concentration range (50–1000 μg/ml) to determine their potency (IC50-50 % inhibi-tion of cell growth). Assay was performed in vitro onexponentially growing cells. The activity was evaluated bymeasuring the levels of surviving cell after incubation for24 h with the test samples, using the MTT colorimetric assay(Martine et al. 2008; Suganumak et al. 2003) based on theability of metabolically active cells to convert the paleyellow MTT to a blue formazan product, which is quantifi-able spectrophotometrically. The results of this primaryscreening are reported in Figs. 2, 3, 4, 5, 6. The BrownYellow Nylosane ERLN, Blue Nylosane EBGL, BemessideRed E-3BS, and Ecarlate Foron RD-FRS dyes were inactive(Figs. 2 to 5). However, within the series studied, BrownYellow Foron RD2RS revealed a significant activity against

the three human tumor cell lines tested at concentration-related manner; the results are shown in Fig. 6. Inhibitionof cell growth (50 %) was obtained at concentrations of 770,970, and 500 μg/ml respectively against human tumor celllines tested A549, HT29, and MCF7.

Untreated and biological-treated effluents were also test-ed for their effects on cellular viability against the samehuman tumor cell lines (Figs. 7, 8). Dilutions used were 5,12.5, 25, 50 and 75 %. Effluent exhibited a rather moderateinhibition at 75 % against HT29 cells, whereas the P. peli-treated effluent revealed a significant activity against thethree human tumor cell lines tested at concentration-relatedmanner, the results are shown in Fig. 8. Inhibition of cellgrowth (50 %) was obtained at dilutions of 66, 56, and53.1 % respectively against human tumor cell lines testedA549, HT29, and MCF7. In term of cell line sensitivity,comparable responses were observed against the A549, theHT29, and the MCF7 cells.

Discussion

In Tunisia, water is a strategic social and economic devel-opment because of its rarity. The most recent assessment of

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Fig. 1 Decolorization of textilewastewater by P. peli underaerobic conditions. Data areexpressed as mean ± standarddeviation from three replicates

Fig. 2 Effect of the Brown Yellow Nylosane ERLN on cellular growthagainst three human tumor cell lines (A549, lung cell carcinoma;HT29, colon adenocarcinoma; and MCF7, breast adenocarcinoma).Data are expressed as mean ± standard deviation

Fig. 3 Effect of the Blue Nylosane EBGL on cellular growth againstthree human tumor cell lines (A549, lung cell carcinoma; HT29, colonadenocarcinoma; and MCF7, breast adenocarcinoma). Data areexpressed as mean ± standard deviation

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water resources was reported to 4,503 Mm3 annual re-sources; including 2,700 Mm3 surface water and1,800 Mm3 groundwater (Ben Mammou 2007). However,textile industry alone consumes more than 200 Mm3 annu-ally (Ben Mansour et al. 2011) and generates huge amountsof polluted wastewater. At first sight, P. peli seems to behighly effective at reducing pollution in the tested effluent.In fact, P. peli exhibited a high ability to decolorize thetextile wastewater collected from a Tunisian ennoblementindustry. This result is very important as far as the conven-tional physical–chemical treatment of the tested effluentshowed a weak efficiency (Robinson et al. 2001).

At this level, the question asked was if this discolorationmeans detoxification. To answer this question, the cytotoxiceffect of samples was tested against three human tumor celllines A549, HT29, and MCF7. Results showed a normalproliferation of all cell lines treated with various dilutions ofintact textile effluent. This could be explained by the firstpresence of the four dyes, Brown Yellow Nylosane ERLN,Blue Nylosane EBGL, Bemesside Red E-3BS, and EcarlateForon RD-FRS, which showed a weak cytotoxic effect

against the three human tumor cell lines. These dyesdisplayed the greatest level of protection towards the cyto-toxicity of Brown Yellow Foron RD2RS which could beexplained by antagonism phenomena.

Curiously, it appears from this study that biological-treated effluent strongly inhibited all cell lines proliferation,although discoloration was very extensive (% of decolori-zation=81 %). We can explain this observation by thepresence of some metabolites in the biodegradation mediumwhich exhibited a high cytotoxic effect. We can also suggestthe presence of the synergic effect of metabolite compounds.In fact, it has been reported that a number of bacteria areknown to catalyze the reductive cleavage of the azo bond bythe action of azoreductase using reduced coenzyme aselectrodonor (Zimmermann et al. 1982), which leads to theformation of aromatic amines. Reduction of azo dyes maythen produce compounds that are either more or less toxicthan the parent molecules so that azo-reduction may de-crease or increase any indirect toxic effects of the dyes

Fig. 4 Effect of the Bemesside Red E-3BS on cellular growth againstthree human tumor cell lines (A549, lung cell carcinoma; HT29, colonadenocarcinoma; and MCF7, breast adenocarcinoma). Data areexpressed as mean ± standard deviation

Fig. 5 Effect of the Ecarlate Foron RD-FRS on cellular growth againstthree human tumor cell lines (A549, lung cell carcinoma; HT29, colonadenocarcinoma; and MCF7, breast adenocarcinoma). Data areexpressed as mean ± standard deviation

Fig. 6 Effect of the Brown Yellow Foron RD2RS on cellular growthagainst three human tumor cell lines (A549, lung cell carcinoma;HT29, colon adenocarcinoma; and MCF7, breast adenocarcinoma).Data are expressed as mean ± standard deviation

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Fig. 7 Effect of the textile effluent on cellular growth against threehuman tumor cell lines (A549, lung cell carcinoma; HT29, colonadenocarcinoma; and MCF7, breast adenocarcinoma). Data areexpressed as mean ± standard deviation

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(Fang et al. 2004). This result is in agreement with thatdescribed in previous works (Ben Mansour et al. 2009a, b),which revealed that azo products of dyes exhibits a highgenotoxic effect, as evidenced by SOS chromotest (BenMansour et al. 2007, 2009a), and a high mutagenic effect asevidenced by Ames Assay (Ben Mansour et al. 2009b). Thisresult is very important as far as our study demonstrates thatalthough the decolorization of textile effluent is remarkable,however, the toxicity could be increased significantly. Forthis, the removal of coloration is not a reliable parameter forthe contamination control of textile wastewater. Thesefindings are in discordance with our previous observations(Ben Mansour et al. 2011, 2012a). It was reported thatPseudomonas putida mt-2 biodegradation process of textileeffluent should receive increasing interest owing to its costeffectiveness, its ability to produce less sludge, and due toits potential to significantly reduce dye concentrations andassociated toxicities. In fact P. putida was found to beable to totally oxidize azo dyes and their aromatic aminederivatives, which undergo metabolization pathways involvingoxygenases.

Conclusion

It was reported that the biological wastewater treatmenttechnique is considered as one of the most promising treat-ment approaches for removal of contaminants from waste-water because of its affordable cost and high efficiency.However, in our case, within the series of acids dyes stud-ied, Brown Yellow Foron RD2RS revealed a significantcytotoxic activity. Effluent exhibited a rather moderate in-hibition too. But the surprise was that biological-treatedeffluent inhibited strongly all cell lines proliferation, al-though discoloration was very extensive. For this, we canconclude that the removal of coloration is not a reliable

parameter for the contamination control of textilewastewater.

Acknowledgments The authors gratefully acknowledge the financialsupport of the “Agence Universitaire de la Francophonie (AUF),”Paris, France.

Conflict of interest The authors report no financial conflicts ofinterest. The authors alone are responsible for the content and writingof this paper.

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