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International Biodeterioration & Biodegradation 90 (2014) 145e151

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

In vitro toxicity evaluation of organic extract of landfill soil and itsdetoxification by indigenous pyrene-degrading Bacillus sp. ISTPY1

Swati, Pooja Ghosh, Mihir Tanay Das, Indu Shekhar Thakur*

School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110 067, India

a r t i c l e i n f o

Article history:Received 29 January 2014Received in revised form1 March 2014Accepted 3 March 2014Available online 25 March 2014

Keywords:BacillusBiodegradationOkhla landfillComet assayMTT assay

* Corresponding author. Tel.: þ91 11 2670 4321.E-mail addresses: swati.wanwari@gmail.com ( Swa

(P. Ghosh), remember.mihir@gmail.com (M.T. Dindushekhart@gmail.com (I.S. Thakur).

http://dx.doi.org/10.1016/j.ibiod.2014.03.0010964-8305/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Organic contaminants present in okhla landfill soil belong to the group polyaromatic hydrocarbons,pharmaceutical compounds, steroidal compounds, personal care products and their derivatives. Anindigenous pyrene-degrading Bacillus sp. ISTPY1 was used to treat the Okhla landfill soil. GCeMS analysisof the organic extract before and after biodegradation with Bacillus sp. ISTPY1 showed the elimination ofvarious polyaromatic hydrocarbons and other persistent aromatic compounds. Toxicity study was doneon human hepato-carcinoma cell line HepG2 before and after treatment. The bacterium treated sampleinitially showed reduction in toxicity till 48 h. This was increased after 120 h due to formation of qui-nones intermediate and further decreased after 360 h. The LC50 value (MTT assay) also showed the samepattern. The reduction in Olive Tail Moment was observed after 360 h treatment. Result of the studyindicated biodegradation and detoxification of major contaminants of Okhla landfill by Bacillus sp.ISTPY1.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Solid waste is an important and emerging environmentalproblem. It was estimated that 0.5e4.5 kg per person per day ofsolid waste is produced in different regions of the world (Bakareet al., 2005). The most common ways to manage such wastedisposal are landfills and incinerators. Actually up to 95% total MSWcollected is disposed off in landfills worldwide (El-Fadel et al., 1997)and landfills are considered as most widely practiced methods ofMSW disposal. Landfills were thought to be the safe disposalmethod of MSW but it is true only for properly engineered landfillsites. An engineered landfill site allows final disposal of solid wastein a secure manner by minimizing the impacts on the environmentasmodern landfills are often linedwith layers of absorbentmaterialand sheets of plastic to keep pollutants from leaking into the soiland water. Delhi’s landfill sites are not properly engineered and allthe chemicals and contaminants infiltrates in the lower layers ofsoil which ultimately lead to land pollution. It is a cause of concernas workers and rag pickers are continuously under the exposure ofcontaminants.

ti), pooja.ghosh9@gmail.comas), isthakur@hotmail.com,

The improper management of waste disposal raises publicconcern over potential harmful effects to local communities andthe environment. These concerns probably becomemore pragmaticwhen recent intensive studies demonstrated increased humanhealth risk caused by exposure to toxic chemicals, such as dioxinsand related compounds, and heavy metals in these dumping sites(Agusa et al., 2003; Minh et al., 2003). Landfills containing haz-ardous materials are under critical observation today for potentialhazards, resulting in the need for thorough risk analyses along withthe soil and groundwater that have been contaminated withchemicals leaching from landfills. Several reports have been pub-lished which are documented on the leachate characterisation andits effect on groundwater pollution but little information is avail-able on the effect of landfills on the soil contamination and itstoxicological effects.

During the last three decades bioremediation has gainedacceptance as an alternative for pollutant removal. But for suc-cessful bioremediation, detailed study of biodegradation process bythe potent microbial strain is required. Microbial biodegradation isa useful tool for removing pollutants in a cost-effective manner.Several micro-organisms including fungi (Singhal and Thakur,2009), algae (Dilek et al., 1999) and bacteria (Mishra and Thakur,2010) have been explored for their potency to detoxify contami-nated effluent. Some of the studies have reported microbialdegradation of persistent toxic compounds such as dibenzofuran(Jaiswal et al., 2011), chrysene (Hadibarata et al., 2009),

Swati et al. / International Biodeterioration & Biodegradation 90 (2014) 145e151146

pentachlorophenol (Chandra et al., 2009); however, to be practi-cally useful for the bioremediation of the contaminated landfillsites, the degradation and detoxification studies should be done ina complex carbonmixture and at concentrations that are present inlandfill soil.

Many reports were on the use of phytotoxicity bioassays fordetoxification study like seed germination tests and root elongationtests (Jadhav et al., 2010). But due to some limitations like theirtime-consuming, non-specific and more qualitative nature they donot find regular use in studying environmental samples. Somestudies used alkaline single cell gel electrophoresis (comet assay)with yeast cells to demonstrate bioremediation and detoxificationefficiency (Singhal and Thakur, 2009; Mishra and Thakur, 2010), butthey alone cannot find out much about the different aspects oftoxicity such as, overall cytotoxicity, endocrine disruption potentialand moreover, it does not give an idea about the chemical class ofthe pollutant.

In vitro systems are popularly used now days for screeningpurposes and for generating more comprehensive toxicologicalprofiles. They have become well-established tools for rapid evalu-ation of toxicity at chronic, sub chronic and acute levels with fairreproducibility (Tai et al., 1994; Chang et al., 2007). The expressionof many nuclear receptor proteins in human hepato-carcinoma celllines (HepG2) that regulate the expression of xenobiotic metabo-lizing enzymes make these cells ideal in vitro models fortoxicological studies (Tai et al., 1994). In this regard, use of humanhepato-carcinoma HepG2 cell line is very promising for in vitrodetoxification studies. In the present investigation, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),7-ethoxyresorufin-O-deethylase (EROD) and alkaline comet assayswere carried out using HepG2 cell line for comparative toxicityevaluation of landfill soil extract before and after bacterial treat-ment. The cytotoxicity was analysed by MTT assay which is basedon the reduction of dissolvedMTT (yellow) into insoluble formazan(blue) by living cells in the presence of mitochondrial succinatedehydrogenase enzyme (Mosmann, 1983). The dioxin-like behav-iours of environmental contaminants have long been investigatedby EROD assay (Laville et al., 2004; Louiz et al., 2008; Kinani et al.,2010). This assay measures CYP1A1-dependent catalytic activitiessuch as ethoxyresorufin-O-deethylase (EROD) and is sensitive tothe presence of chemicals like furans, dioxins, poly aromatichydrocarbons (PAHs) and poly chlorinated biphenyls (PCBs) (Tillittet al., 1991). The present research was aimed at assessing the soilpollution in the vicinity of Okhla landfill site in Delhi and itsdetoxification by indigenous Pyrene-degrading Bacillus sp. ISTPY1.The study also provides a comprehensive picture of the varioustoxicological endpoints of the complex organic mixture extractedfrom landfill soil.

2. Material and methods

All chemicals were purchased from SigmaeAldrich (St. Louis,MO, USA) or Merck (Darmstadt, Germany) and were used withoutfurther purification unless stated otherwise.

2.1. Soil sample collection

Soil samples were collected from Okhla landfill site, Delhi in tenreplicates at depth from 0 to 10 cm, combined together andconsidered as a representative sample. They were collected insterilized plastic containers. Freeze dried and grounded soil sam-ples were homogenized by sieving through a stainless steel 0.2-mmsieve, and stored in sealed containers at �4 �C until chemicalanalysis.

2.2. Enrichment and isolation of pyrene-degrading bacteria

Isolation was performed using continual enrichment method(Churchill et al., 1999). The enrichment was done in minimal saltmedia (MSM) containing (g l�1) Na2HPO4$2H2O, 7.8; KH2PO4, 6.8;MgSO4, 0.2; NaNO3, 0.085; NH4 (CH3COO)3 Fe, 0.01; Ca(N-O3)2$4H2O, 0.05; trace element solution, 1 ml l�1 with pyrene(50 mg l�1) as carbon source. The trace element solution (g l�1) wasprepared according to Pfennig and Lippert (1966) with slightmodifications (ZnSO4, 0.10; MnCl2, 0.03; H3BO3, 0.30; CoCl2 0.6 H2O,0.20; CuCl2, 0.01; NiCl2.2H2O, 0.02; Na2MoO4 .2H2O, 0.02). TheMSM pHwas adjusted to 7.2 for bacteria. Contaminated landfill soilsample (5.0 g) was added to 45 ml MSM containing 50 mg l�1

of pyrene. Enrichment was carried out with shaking (125 rpm) for4e5 weeks in the dark until there was turbidity. After 4 consecutivetransfers, pyrene degraders were detected on pyrene-coated,mineral media agar plates (Kiyohara et al., 1982). The biodegrada-tion experiment was set up with the isolated strain to confirm thedegradation potential of the bacterium (Obayori et al., 2008).Pure isolates were maintained in glycerol: LB broth medium(50:50) at e 20 �C.

2.3. 16S rDNA analysis

DNA was extracted from cells grown on LB medium with theGenomic DNA isolation Kit (Qiagen Inc., USA) as described by themanufacturer. 16S rDNA was amplified using the universal primer50-GAG AGT TTG ATC CTG GCT-30(forward) and 50-CTA CGG CTA CCTTGT TAC-30(reverse) (Cello et al., 1997). The polymerase chain re-action (PCR) product was separated by agarose gel electrophoresisand the expected size was purified using the Gel Extraction Kit(QIAGEN), according to the manufacturer’s instruction and sent tothe Eurofins Genomics India Pvt Ltd for sequencing. The 16S rDNAsequence was compared against the GenBank database using theNCBI Blast program. The 16S rDNA sequences of strains ISTPY1 havebeen deposited in GenBank under accession no. KC924957.

2.4. Substrate specificity

The bacterial isolate was grown on pure hydrocarbon substratesto evaluate substrate specificity in liquid media containing50 mg l�1 of respective hydrocarbons as a sole carbon and energysource. Visual monitoring was done for turbidity and degradation.The hydrocarbons tested include naphthalene, phenanthrene,anthracene, benzene, phenol, dibenzothiophene and biphenyl.Liquid hydrocarbons were filter sterilised and separately added tosterile MSM at 0.1% (v/v). Incubation was carried out for 10 days.

2.5. Extraction of compounds from soil

Organic compounds were extracted from soil sample accordingto US EPA method 3500C. 200 g dry weight of soil sample wasSoxhlet extracted for 18 h with 300 ml of dichloromethane (DCM)/Acetone in 1:1 v/v ratio. The organic extract was evaporated todryness using a vacuum rotator evaporator at room temperatureand dissolved in dimethyl sulphoxide (DMSO) (10 ml) for bacterialbiodegradation and detoxification experiments (Das et al., 2012).

2.6. Microorganism and culture condition

One indigenous Bacillus sp. strain ISTPY1 isolated by enrichmentwas found to degrade pyrene in minimal salt medium (MSM) andshowed broad substrate specificity when grown in the presence ofdifferent substrates. The Bacillus sp. strain ISTPY1maintained in theMSM containing pyrene as carbon source was used to inoculate

Table 1Comparative growth properties on different carbon sources by Bacillussp. strain ISTPY1.

Substrate Qualitative growth

Naphthalene þAnthracene þþPhenanthrene þþPyrene þþþBenzene þPhenol e

Biphenyl þþDibenzothiophene þþ

e: No growth;þ: Poor growth;þþ: Growth; þþþ: Luxuriant Growth.

Swati et al. / International Biodeterioration & Biodegradation 90 (2014) 145e151 147

MSM agar plates containing (g l�1), Na2HPO4$2H2O,7.8; KH2PO4,6.8; MgSO4, 0.2; NaNO3, 0.085; NH4(CH3COO)3Fe, 0.01; Ca(N-O3)2$4H2O, 0.05; trace element solution, 1 ml l�1 with pyrene(1 mg ml�1) as carbon source. After growth on the selective solidmedium, the bacterial colonies were inoculated into 100 ml MSMbroth (pH ¼ 7.2) containing 2% v/v crude sediment extract (Daset al., 2012) and incubated up to 360 h in different shaking flasks(125 rpm) under aerobic conditions at 30 �C. The inoculum size at0 h was 3 � 104 CFUml�1 and growth was monitored every 24 h upto 360 h by measuring turbidity (A600).

2.7. Sample extraction and GCeMS analysis

Samples for GCeMS analysis were prepared according to US EPAmethod 3500C. Both control (0 h) and treated samples (50 ml each)werecentrifugedto removebacterial biomassat5000 rpmfor10min.The supernatant was equally divided in two parts and pHwas set upat 2 and 12 respectively. The extraction of acidic and basic fractionwas done separately with three volumes of DCM. The acid and baseextracts were combined and dehydrated by anhydrous Na2SO4. Theextracts were divided into two parts and both parts were evaporatedto dryness by a vacuum rotary evaporator. One part was dilutedwith1ml of DMSO for toxicological analysiswhile the otherwas dissolvedin 1ml DCM for GCeMS analysis. The analysis was done using a GCeMS instrument (Shimadzu QP2010 Plus) as described by Das et al.(2012). Data were compared with the inbuilt standard mass spectralibrary system (NIST-05 andWiley-8) of GCeMS.

2.8. Toxicological analysis

2.8.1. Cell cultureToxicological analysis was done on HepG2 which was main-

tained in Dulbecco’s Modified Eagle’s Medium (DMEM) supple-mented with 1% antibiotic antimycotic solution (finalconcentrations: streptomycin, 0.1 mg ml�1; amphotericin B,2.5 ng ml�1; penicillin, 100 U ml�1) and 10% foetal bovine serum ina humidified atmosphere of 5% CO2 at 37 �C (Das et al., 2012). ForEROD and MTT assays, seeding of HepG2 cells was done in 96-wellpolystyrene tissue culture plates (Corning 3596) whereas foralkaline comet assay cells were seeded in 12-well plates (Costar,Corning 3513) both at a density of approximately2.5 � 105 cells ml�1. All positive control chemicals and test sampleswere dissolved in DMSO and were added to the cell cultures indifferent dilutions to work out the dose response relationships. Thefinal concentration of DMSO in the medium was 0.5% and all ex-periments were carried out in triplicates.

2.8.2. EROD assayThe EROD assay was carried out as described by Laville et al.

(2004) with some modification. Medium removed from theHepG2 cell line culture plates after 6 h was replaced by 100 mL offresh culture medium containing 5 mM 7-ethoxyresorufin and10 mM dicumarol. Culture plates were incubated for 30 min at 37 �Cand then methanol (130 ml) was added to stop the reaction.Resorufin-associated fluorescence was measured at 530 nm exci-tation and 590 nm emission by using a multi-well fluorescenceplate reader (SpectraMax M2, Molecular Devices). The results wereexpressed as percentage of EROD activity induced by the positivecontrol (BaP, 1 mM). BaP-equivalents (BaP Eq) for each sample weredetermined as the ratio of the EC50 of BaP expressed as g l�1 to thatof the sample expressed as g Sed Eq l�1 (Louiz et al., 2008; Kinaniet al., 2010). Sigmoid doseeresponse curves for BaP and differentsamples along with their EC50 values were derived from the globalcurve fitting analysis with four parameter logistic curve equation(Das et al., 2012).

2.8.3. Cell viabilityThe effect of samples or test compounds on cellular viability of

the cell lines was evaluated using the MTT assay (Mosmann, 1983;Laville et al., 2004). It was expressed as percentage of the corre-sponding control (DMSO 0.5%). The LC50 values and sigmoid doseeresponse curves were plotted as described in Section 2.8.2.

2.8.4. Comet assayAlkaline comet assay was performed as described by Tice et al.

(2000). HepG2 cells were treated with test samples(10 g Sed Eq ml�1), positive control (BaP, 1 mM) or negative control(DMSO 0.5%) for 24 h. The slides were stained with ethidium bro-mide (2 mg ml�1,100 ml per slide) and visualised with a 40� lensfitted to a fluorescence microscope with an excitation and emissionsetting of 518/605 nm. The percentage of DNA in tail, tail momentand olive tail moment (OTM) of 50 randomly selected cells wereanalyzed from each slide by using CometScore Freeware software(www.tritekcorp.com). The comets were divided into five classeson the basis of amount of DNA in the tail; Class I, less than 1% DNAin tail (intact nucleus); Class II, 1e20% DNA in tail; Class III, 20e50%DNA in tail; Class IV, 50e75% DNA in tail and Class V, more than 75%DNA in tail (Miyamae et al., 1998).

2.9. Statistical analysis

All experimental data were expressed as means � standarddeviation of three replicates. All statistical analyses including globalcurve fitting was performed with sigma plot 11 statistical package(Systat Software, San Jose, CA). The differences between the controland treated cells were examinedwith the aid of ANOVA followed bymultiple comparisons (Dunnett’s Method). A value of P < 0.05 wasused to determine significance in statistical analyses.

3. Results

3.1. Enrichment and identification of pyrene-degrading bacterium

pt?>One pyrene-degrading strain ISTPY1 was isolated fromokhla landfill soil sample by enrichment. The colonies surroundedby clearing zones on pyrene-coated agar plates were considered aspositive and were further analysed for pyrene degradation by GCeMS (Fig. S1, S2). The formation of phthalic acid and decrease in theGCeMS peak area of pyrene sufficiently suggest the utilisation ofpyrene as a carbon source by bacterium. The bacterium alsoshowed broad substrate specificity as shown in Table 1. Usinguniversal primers, 1.4 kb of 16S rDNA sequence of strain ISTPY1 wasamplified, partially sequenced and submitted to GenBank andaccession number KC924957 was obtained. The BLAST searches ofthe obtained sequence showed 99% sequence similarity with Ba-cillus sp. The phylogenetic tree of the isolated bacterium is shown inFig. 1.

Fig. 1. Phylogenetic tree of Pyrene degrading bacterial strain, Bacillus sp. ISTPY1, isolated from Okhla landfill soil. Bootstrap consensus tree (1000 replicates) was drawn by multiplesequence alignment with neighbour joining method using software MEGA, version 5.1.

Time (in hrs.)

0 24 48 120 360

mn595taecnabrosb

A

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Fig. 2. Growth of Bacillus sp. ISTPY1 in organic extract from Okhla landfill soil ascarbon source in MSM at 30 �C. The value represents mean � standard deviation.

Swati et al. / International Biodeterioration & Biodegradation 90 (2014) 145e151148

3.2. Microbial degradation of contaminants of landfill soil

Degradation of 2% v/v crude landfill soil organic extract in MSMwas done by an indigenous bacterium, Bacillus sp. strain ISTPY1(Fig. 2). The samples were analyzed before and after treatment withbacterium by GCeMS to obtain the biodegradation pattern ofcompounds present in the landfill soil organic extract. The chro-matograph corresponding to compound extracted from UT andT360 samples are shown in Fig. S3 and S4 respectively and theidentified compounds are summarised in Table 2. The degradationproducts mentioned in the table were identified from the availablestandards of the authentic compounds documented in NIST-05 andWiley-8 libraries. The chromatograph of samples analyzed quali-tatively shows the degradation of organic compounds as totalnumber of peaks decreased from 150 to 105 of the UT and T360sample respectively. Most of the major peaks of persistent organicpollutants disappeared in the T360 sample along with theappearance of more peaks of the simpler compounds as shown inTable 2.

3.3. EROD assay

The samples (treated and untreated) were tested for their abilityto induce EROD activity. This activity was induced by varioussamples in dose dependent manner (10�3 to 103 g Sed Eq l�1) asshown in Fig. 3a.The four parameter logistic equation was consid-ered for analysis of EC50 values and calculation of biological BaP-Eqs(Das et al., 2012). The significant EROD activity was found in un-treated sample which was shown in terms of BaP Eq and EC50values. The highest EROD activity was induced in 120 h treatedsample. However, Bacillus sp. was able to reduce the toxicity after360 h of treatment which has been indicated by the BaP-Eq valuesin Table 3.

3.4. Cell viability

The dose response curves (Fig. 3b) and MTT assay derived LC50values of the untreated and treated samples showed that the levelof toxicity of landfill organic extract decreased with increasingduration of bacterial treatment. The lowest value of LC50 was forthe UT sample (7.3361) which increased by about 3 times after 15days (22.0921) of bacterial treatment. The 120 h sample showed anincreased cytotoxic effect which was further reduced in the 360 hsample. The present study indicated that the EROD EC50 valueincreased nearly 13 times after 360 h bacterial treatment whereas

Table 2Identification of metabolites formed at different stages of bacterial degradation.

Retentiontime (min.)

Present in sample Name of the compound

UT T24 T48 T120 T360

7.480 þ þ e e e 4-Allylphenol8.633 e e þ þ þ 3-acetyl-4-methyl- 5-Hexen-2-one9.000 þ þ þ e e Benzamide9.990 þ þ e e e N-Hexyl benzoate11.300 e e e þ e 2-ethoxy-Naphthalene11.992 e e þ þ þ Nonadecane12.075 e þ þ þ e Phthalic acid13.842 e þ þ þ e 3,4-Benzocinnoline14.317 þ e e e e Phenanthrene15.942 e e e þ e 2,6-ditert-butylbenzo-1,4-quinone16.150 þ e e e e 1A,9B-Dihydro-1H-

cyclopropa[a]anthracene

Table 3EROD EC50 and MTT LC50 value, of untreated and treated samples along with thecorresponding Bap-Eq values.

Treatments EROD EC50 R2 (EROD EC50) BaP-Eq MTT LC50 R2 (LC50)

BaP 1.9358e�6 0.9961 e e e

UT 0.2021 0.9943 9.578e�6 7.3361 0.9846T24 0.2449 0.9942 7.904e�6 11.5408 0.9877T48 0.3451 0.9889 5.609e�6 17.2487 0.9987T120 0.1736 0.9864 11.15e�6 10.5854 0.9898T360 2.7006 0.9974 7.16e�7 22.0921 0.9987

Swati et al. / International Biodeterioration & Biodegradation 90 (2014) 145e151 149

the MTT assay-derived LC50 values showed only about a 3-timesincrease under the same conditions.

3.5. Comet assay

The results of the single cell gel electrophoresis (Comet assay)with biodegraded (24 h, 48 h, 120 h and 360 h) and untreated

Fig. 3. Toxicity evaluation of treated and untreated samples by Bacillus sp. strainISTPY1. Acronyms correspond to the different samples to which cells were exposed;BaP: Benzo (a) Pyrene (Positive control), UT: Un-treated crude extract, T24: 24 htreated extract, T48: 48 h treated extract, T120: 120 h treated extract, T360: 360 htreated extract. Values represent the mean � SD, n ¼ 3. (a) EROD induction measuredafter 6 h exposure period; 100% EROD induction was considered for 1 mM BaPtreatment. (b) Cell viability measured after 24 h exposure period; 100% cell viabilitywas considered for 0.5% DMSO treatment.

sample is shown in Fig. 4a and b. Tail moments of 50 randomlyselected comets are presented as quantile box plots. The plot showsthat distribution of comets became more homogenous with lowertail moment (2.2022 � 2.1675) in the 360 h treated sample incomparison to untreated sample tail moment (13.3366 � 8.2914).The olive tail moment data showed a decreasing trend withincreasing duration of bacterial treatment. 360 h treated sampleresulted in a 3.6 fold decrease in DNA migration(OTM ¼ 3.6078 � 3.2487) in comparison to that of the untreatedsample (OTM ¼ 11.7596 � 5.3055).

4. Discussion

Landfills are the major depositories for a wide range of solidwastes which are a source of emerging contaminants such aspharmaceuticals, personal care products, plasticizers and steroidalcompounds. Several studies in the past have reported the prevalenceof toxic organic contaminants in landfill soil (Zakaria et al., 2005;Minh et al., 2006; Nduka et al., 2013) and health risk associated

Fig. 4. Genotoxicity of the contaminants before and after treatment with Bacillus sp.strain ISTPY1 (a) The tail moment and the olive tail moment plotted against differentsamples. Tail moments of 50 randomly selected comets are presented as quantile boxplots. The edges of the box represent the 25th and the 75th percentiles; a solid line inthe box presents the median value. Error bars indicate 90th and 10th percentiles andthe black circles indicate outlying points beyond 5th and 95th percentiles. Olive tailmoments of same 50 comets are shown as the mean � standard deviation and Dun-nett’s test was performed for data validation (b) Images of different classes of cometsseen under fluorescent microscope after stained with ethidium bromide. Roman nu-merical indicates class of comet.

Swati et al. / International Biodeterioration & Biodegradation 90 (2014) 145e151150

with it (Vrijheid, 2000). Ray et al. (2005) reported high incidence ofimpairment of lung function among the landfill workers. Respira-tory, dermatological and neurobehavioural problems have also beenreported in landfill workers by Poulsen et al. (1995) and Gelberg(1997). The major classes of organic compounds detected in thepresent study included polycyclic aromatic hydrocarbons (PAH),steroidal compounds, pharmaceutical compounds and their de-rivatives. The improper disposal of medical waste may be thepossible source of pharmaceutical compounds in the landfill. Largenumber of other contaminants may also be present at low concen-trations below the detection limit whose toxicity cannot be ignored.

The presence of toxic organic contaminants in landfill soil helpsin the evolution of potent microorganisms that can utilize theseorganic contaminants as a carbon source. Bacillus sp. strain ISTPY1isolated from the landfill soil was able to use 50 mg l�1 pyrene assource of carbon and energy. It also showed broad substrate spec-ificity and ability to grow on mixture of organic contaminantspresent in Okhla landfill. Several bacterial strains are known fordegradation of HMW PAHs like Mycobacterium (Schneider et al.,1996; Churchill et al., 1999; Vila et al., 2001), Pseudomonas sac-charophila strain P15, Pseudomonas stutzeri strain P16, Sphingomo-nas yanoikuyae strain R1 and Bacillus cereus strain P21 which playan active role in pyrene metabolismwhen grown in minimum saltsbuffer (Kazunga and Aitken, 2000). As most of the persistentcompounds present in okhla landfill have an aromatic structure,the degradation and detoxification potential of pyrene-degradingBacillus sp. strain ISTPY1 was studied. The degradation studyshowed the efficient removal of highly toxic polycyclic aromaticcompounds like phenanthrene and 1A,9B-Dihydro-1H-cyclopropa[a]anthracene in the mixture of contaminants and formation ofsimple compounds like phthalic acid and quinone as intermediatewhich are further seems to be degraded to aliphatic compoundslike Nonadecane and 3-acetyl-4-methyl- 5-Hexen-2-one. Previousstudies have also reported the formation of phthalic acid duringphenanthrene degradation by Mycobacterium sp. strain AP1 (Ariaset al., 2008). The formation of oxidised intermediate like quinonesuggests the involvement of an oxidative pathway by Bacillus sp.ISTPY1 for degradation of mixture of contaminants present in thelandfill soil. Formation of quinone has also been reported previ-ously in the degradation of pyrene by Mycobacterium PYR-1(Kazunga and Aitken, 2000).

The change in the complex mixture of organic contaminantduring degradation process was implicated in further toxicologicalstudies done on the treated and untreated samples. Several in vitromammalian bioassays like MTT (cytotoxicity), comet (genotoxicity)and EROD (CYP1A1 induction) assays were done to evaluate thedetoxification potential of Bacillus sp. ISTPY1. EROD assay is a highlysensitive indicator of environmental alterations and allows quan-tifiable response to exposure to xenobiotics (Stegeman et al., 1992;Tabrez and Ahmad, 2010). Previous study proposed EROD as abiomarker of persistent organic pollutants in Allium cepa system(Fatima and Ahmad, 2006). Induction of EROD activity shows thepresence of dioxin or dioxin like compounds in the environment. Inthe present study, high CYP1A1 activity was found in the untreatedsample due to the presence of polycyclic aromatic hydrocarbonsand other EROD inducing chemicals. Increase in the EROD activityin 120 h treated sample observed was due to the formation ofhighly toxic quinone intermediates during the degradation processas postulated by Burczynski and Penning (2000). However, increasein EROD EC50 value after 360 h treatment clearly indicated steadymineralisation of EROD-inducing chemicals by the bacterium.

MTT assay in conjunction with EROD assay is widely used forstudying the toxicological endpoint of the CYP1A1 inducingchemical mixture (Das et al., 2012). Cytotoxicity of landfill soil wasindicated by MTT assay results. Disturbance in cell proliferation

could be explained due the presence of PAHs (Kang et al., 2010) andseveral benzamide derivatives (Jayaram et al., 1992; Braga et al.2007). The formation of toxic intermediates belonging to quinonegroup of organic compounds which are known to be intermediatemetabolites in various biodegradation pathways could be a po-tential source of cytotoxicity in the 120 h sample (O’Brien, 1991;;Bolton et al., 2000). In contrast to the above mentioned cytotoxiccompounds found in the untreated sample and 120 h sample, for-mation of aliphatic alkane and ketones in the 360 h treated sampleresulted in the increase of LC50 value.

Diverse group of chemical contaminants present in the landfillsoil like PAHs (Coughlan et al., 2002; Gamboa et al., 2008) and benzylderivatives (Demir et al., 2010) may be the reason for significantgenotoxicity observed in the untreated sample. Autooxidation orenzyme catalysed oxidation of organic compounds may result in theformation of free radicals capable of inducing genotoxicity (Li et al.,2004). Increase in the OTM value after 120 h treatment is possibledue to the formation of quinone derivatives (Gurbani et al., 2013).Removal of various contaminants after 360 h bacterial treatment canbe implicated in the reduction of OTM values showing the efficiencyof the bacterium in detoxification of landfill soil.

5. Conclusion

The study evaluated the potential of Bacillus sp. ISTPY1 forbioremediation and detoxification of organic contaminants presentin landfill soil. The bacterium is capable of degradation of a varietyof persistent organic pollutants present in landfill soil. Significantreduction in both cytotoxicity and genotoxicity in treated samplewith the bacterium for 360 h has also been shown through in vitroMTT and Comet bioassays respectively using HepG2 cell line. Itrevealed the potential of Bacillus sp. strain ISTPY1 to degrade anddetoxify organic pollutants even in the presence of range of carbonsources.

Acknowledgements

This research work was supported by fellowship grant fromCouncil of Scientific and Industrial Research (CSIR), New Delhi,India. Authors are thankful to Dr. R.K. Tyagi, S.C.M.M., JawaharlalNehru University, New Delhi, India, for HepG2 cells as generous giftand Mr. Ajai Kumar of Advanced Instrumentation Research Facility(AIRF), Jawaharlal Nehru University, New Delhi, India, for GCeMSanalysis.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ibiod.2014.03.001.

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