Bioresource Technology 110 (2012) 71–78

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Bioresource Technology

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Toxicological evaluation of paper mill sewage sediment treated by indigenousdibenzofuran-degrading Pseudomonas sp.

Mihir Tanay Das, Varaporn Budhraja, Monika Mishra, 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 16 October 2011Received in revised form 9 January 2012Accepted 16 January 2012Available online 24 January 2012

Keywords:PseudomonasBiodegradationPaper mill sewage sedimentComet assayEROD assay

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.biortech.2012.01.078

⇑ Corresponding author. Tel.: +91 11 2670 4321; faE-mail addresses: mihirdasjnu@gmail.com (M.T.

(I.S. Thakur).

a b s t r a c t

Organic pollutants present in the pulp and paper mill sewage were extracted with dichloromethane/ace-tone (1:1 v/v), and the extract was added to minimal salt medium to study the degrading and detoxifyingpotential of Pseudomonas sp. strain ISTDF1. GC–MS analysis performed after biodegradation showedremoval of chlorinated compounds like dichloroacetic acid, heptadecyl ester (RT = 28.99) and formationof simpler compounds like 4-isopropylbenzaldehyde (RT = 7.52). Methyltetrazolium (MTT) assay forcytotoxicity, 7-ethoxyresorufin-O-deethylase (EROD) assay for dioxin-like behaviour and alkaline cometassay for genotoxicity were carried out in human hepato-carcinoma cell line HuH7 before and after bac-terial treatment. The bacterium reduced toxicity as shown by a 5-fold increase in LC50 value, 2110-foldreduction in benzo (a) pyrene equivalent value and an 8-fold reduction in Olive Tail Moment after240 h treatment. The EROD assay was found to be a useful method which indicated positive correlationwith MTT and comet assay in post-biodegradation toxicity evaluation.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Pulp and paper mill effluent not only contains naturally occur-ring polymers (tannins, resin acids, stillbenes, lignin etc.), but alsoxenobiotic compounds that are formed or used during the processof pulping and paper making (chlorinated lignins, resin acids andphenols, dioxins, furans, poly aromatic compounds, plasticizersetc.) (Peck and Daley, 1994). Thacker et al. (2007) have reportedthat effluents of paper mills using chlorine for bleaching processin India were contaminated with 2,3,7,8-TCDD up to 0.12 ngL�1.1,3,6,8-TCDD, 2,5-diphenyl furan, 2,2,3,4,40,5-hexa-PCB, 2,4,4-tri-chlorobiphenyl, endosulfan, dichlorobenzene were also detectedin their study. Many of the chemicals found in paper mill effluentare toxic to aquatic life and ultimately to human health throughbioaccumulation and biomagnification in the food chain. Studieshave shown evidences of impaired liver and reproductive dysfunc-tion in fish exposed to these effluents (Orrego et al., 2010). Thereare reports of adverse health and environmental effects linked toorganochlorinated dioxin-like compounds, often related to endo-crine disruption, such as, growth retardation, thyroid dysfunction,decreased fertility, feminization or masculinisation of biota andeven tumour promotion (Mandal, 2005). The genotoxic potentialof pulp and paper mill effluent has also been studied (Mishra andThakur, 2010).

ll rights reserved.

x: +91 11 2671 7586.Das), isthakur@hotmail.com

Microbial biodegradation of persistent organic pollutant is auseful tool for removing pollutants in a cost-effective manner. Sev-eral 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 decolourise and detoxifypulp and paper mill effluent. Some of the studies have reportedmicrobial degradation of persistent toxic compounds such asdibenzofuran (Jaiswal et al., 2011) and pentachlorophenol (Chan-dra et al., 2009); however, to be practically useful, the degradationand detoxification potential of microbes for these toxic organiccompounds should be studied in a complex carbon mixture andat concentrations that are present in pulp and paper mill effluent.

Many reports have shown detoxification efficiency byperforming phytotoxicity bioassays like seed germination testsand root elongation tests (Jadhav et al., 2010). These bioassaysare time-consuming, non-specific and are more qualitative thanquantitative. Alkaline single cell gel electrophoresis (comet assay)with yeast cells has been used in some studies to demonstrate bio-remediation and detoxification efficiency (Mishra and Thakur,2010; Singhal and Thakur, 2009), but comet assays alone cannotfind out much about the different aspects of toxicity such as endo-crine disruption potential, overall cytotoxicity, and moreover, itdoes not give an idea about the chemical class of the contaminants.

In vitro models using human cancer cell lines have becomewell-established tools for rapid and accurate evaluation of toxicityat acute, chronic and sub chronic levels with fair reproducibility(Chang et al., 2007; Tai et al., 1994). Hepatocytes express many nu-clear receptor proteins that regulate the expression of xenobiotic

72 M.T. Das et al. / Bioresource Technology 110 (2012) 71–78

metabolizing enzymes, including cytochromeP450 1A1 (CYP1A1)responsible for the metabolism of multiple endogenous andexogenous chemicals making these cells ideal in vitro models fortoxicological studies (Tai et al., 1994). In this regard, the humanhepato-carcinoma HuH7 cell line has been shown to be promisingfor toxicity evaluation under in vitro condition (Chang et al., 2007).Therefore, in the present investigation, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 7-ethoxyresorufin-O-deethylase (EROD) and alkaline comet assays were carried outwith the HuH7 cell line for comparative toxicity evaluation of pulpand paper mill sewage sediment extract before and after bacterialtreatment. The MTT assay is an overall indicator of cytotoxicityand is based on the ability of living cells to reduce dissolvedMTT (yellow) into insoluble formazan (blue) in the presence ofmitochondrial succinate dehydrogenase (Mosmann, 1983). TheEROD assay has long been used for testing dioxin-like behavioursof environmental contaminants or new pharmaceuticals (Kinaniet al., 2010; Laville et al., 2004; Louiz et al., 2008). The assay isbased on the ability of certain classes of organic compounds to in-duce CYP1A1-dependent catalytic activities such as ethoxyresoru-fin-O-deethylase (EROD) and is sensitive to the presence of adiverse group of chemicals like dioxins, furans, poly chlorinatedbiphenyls (PCBs) and poly aromatic hydrocarbons (PAHs) (Tillittet al., 1991). As the pulp and paper mill sludge sediments are heav-ily contaminated with these persistent organic pollutants, theEROD assay can be an effective tool to monitor their contaminationlevel.

The present investigation was designed to study the efficiencyan indigenous Pseudomonas sp., strain ISTDF1, isolated earlier(Jaiswal et al., 2011) to degrade and reduce the toxicity of a complexorganic mixture extracted from pulp and paper mill sewage sedi-ment. The study simultaneously aims to reveal how EROD assayalong with MTT and alkaline comet assay in HuH7 cell lines canbe used to evaluate biodegradation and detoxification efficiency.

2. Methods

All chemicals were purchased from Sigma–Aldrich (St. Louis,MO, USA) or Merck (Darmstadt, Germany) unless stated otherwise,and were used without further purification.

2.1. Sediment sample collection

Paper mill sewage sediment samples were collected from M/sCentury Pulp and Paper Mill, Lalkuan (79�280E longitude and29�240N latitude), Nainital, Uttarakhand, India. The samples (tenreplicates from the site with a distance of 10 m from sample tosample) were collected in sterilized plastic containers from thebottom sewage sediment near the waste water discharge drain siteof the mill. Sediments were freeze-dried, ground and homogenizedby sieving through a stainless steel 0.2-mm sieve, and stored insealed containers at �4 �C.

2.2. Extraction of compounds from sediment

Samples of 200 g dry weight were Soxhlet extracted (US EPAmethod 3500C) for 18 h with 300 mL of dichloromethane (DCM)/Acetone (1:1 v/v), pesticide grade. The crude extract was evapo-rated to dryness at room temperature using a vacuum rotatorevaporator and dissolved in 10 mL of dimethyl sulphoxide (DMSO)for bacterial biodegradation and detoxcification experiments. Theresulting concentration of the final extract was 20 g dry sedimentequivalent mL�1 of solvent (gSedEqmL�1).

2.3. Microorganism and culture condition

One indigenous Pseudomonas sp. strain ISTDF1 (EU834943.1)isolated from the same site was shown earlier to degrade dibenzo-furan in minimal salt medium (MSM) through continuouschemostat culture (Jaiswal et al., 2011). The bacterial culture main-tained in the chemostat at 30 �C was used to inoculate MSM agarplates containing (gL�1), Na2HPO4�2H2O, 7.8; KH2PO4, 6.8; MgSO4,0.2; NaNO3, 0.085; NH4(CH3COO)3Fe, 0.01; Ca(NO3)2�4H2O, 0.05;trace element solution, 1 mLL�1 with dibenzofuran (2 mM) as car-bon source. After 4 days of growth on the selective solid medium,the bacterial colonies were inoculated into 100 mL MSM broth(pH = 10) containing 2% v/v crude sediment extract (final concen-tration: 0.4 gSedEqmL�1) and incubated up to 240 h in differentshaking flasks (125 rpm) under aerobic conditions at 30 �C. Theinoculums size at 0 h was 5 � 104 CFU mL�1 and growth wasmonitored every 24 h up to 240 h by measuring turbidity (A600).Free chloride ion release was also measured every 24 h using theargentometric method (APHA, 2005). The 0-h sample was takenas control.

2.4. Sample extraction and GC–MS analysis

Samples were prepared for GC–MS analysis according to US EPAmethod 3500C. Briefly, both control and treated samples (50 mL)were centrifuged at 2500�g for 10 min to remove bacterial bio-mass. The supernatant of each sample was extracted with threevolumes of DCM of pH = 2 and 12. The acid and base extracts werecombined, dewatered over anhydrous Na2SO4 and filtered throughWhatman No. 54 filter paper (pore size 20–25 lm). The filteredextracts were divided into two parts and both parts were evapo-rated to dryness at room temperature using a vacuum rotatorevaporator. One part was reconstituted with 1 mL of DMSO for tox-icological analysis while the other was dissolved in 1 mL DCM forGC–MS analysis. The concentration of the final extract was10 gSedEqmL�1 of solvent.

The analysis was done using a GC–MS instrument (ShimadzuQP2010 Plus) equipped with a capillary column (DB-5 MS;0.25 lm film thickness, 0.25 mm i.d., 30 m in length). One microli-ter of each sample was analyzed by GC–MS at conditions: split lessmode; initial temperature 80 �C held for 2 min; temperatureincreased from 80 to 200 �C at a rate of 10 �C min�1 held for6 min on reaching 200 �C; temperature further increased from200 to 300 �C at a rate of 5 �C min�1 held for 15 min on reaching300 �C. The head pressure of the helium carrier gas was 81.7 kPa;helium flow rate 1.21 mL min�1. Data were compared with the in-built standard mass spectra library system (NIST-05 and Wiley-8)of GC–MS.

2.5. Toxicological analysis

2.5.1. Cell cultureHuH7 cells were maintained in Dulbecco’s Modified Eagle’s

Medium (DMEM) supplemented with 10% foetal bovine serum,1% antibiotic antimycotic solution (final concentrations: penicillin,100 UmL�1; streptomycin, 0.1 lgmL�1; amphotericin B, 2.5 ngmL�1) in a humidified atmosphere of 5% CO2 at 37 �C.

For MTT and EROD assays, cells were seeded in 96-well surfacetreated polystyrene tissue culture plates (Corning 3596) whereasfor alkaline comet assay cells were seeded in 12-well plates (Cost-ar, Corning 3513) both at a density of approximately 2.5 � 105

cellsmL�1. After 24 h, at 90% confluence, cells were treated withDMSO, with positive control chemical Benzo (a) pyrene (BaP),1 mM or with test samples for 6–24 h. All positive control chemi-cals and test samples were dissolved in DMSO and were added tothe cell cultures in different dilutions to work out the dose

Time (h)0 24 48 72 96 120 144 168 192 216 240

Abso

rban

ce a

t (60

0nm

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0C

hlor

ide

ion

(mM

)

2000

4000

6000

8000

10000

Growth curveChloride ion release

Fig. 1. Growth of Pseudomonas sp. strain ISTDF1 and chloride ion release in MSMmedium containing 2% v/v crude sediment extract. Values represent the mean ± SD,n = 3.

M.T. Das et al. / Bioresource Technology 110 (2012) 71–78 73

response relationships. The final concentration of DMSO in themedium was 0.5% and all experiments were carried out in threereplicates.

2.5.2. EROD assayThe EROD assay was carried out as previously described with

some modification (Laville et al., 2004). Medium removed fromthe HuH7 cell line culture plates after 6 h was replaced by 100 lLof fresh culture medium containing 5 lM 7-ethoxyresorufin and10 lM dicumarol. Culture plates were incubated for 30 min at37 �C and then methanol (130 lL) was added to stop the reaction.Resorufin-associated fluorescence was measured at 530 nm excita-tion and 590 nm emission by using a multi-well fluorescence platereader (SpectraMax M2, Molecular Devices), and the data were fit-ted to a resorufin standard curve. Total cellular proteins were deter-mined with the bicinchoninic acid protein assay using bovineserum albumin as a standard (Smith et al. 1985). The EROD activitywas derived as pmol of resorufin produced min�1 and mg�1 of pro-teins and the results were expressed as percentage of EROD activityinduced by the positive control (BaP, 1 mM). BaP-equivalents (BaP-Eq) for each sample were determined as the ratio of the EC50 of BaPexpressed as gL�1 to that of the sample expressed as gSedEqL�1

(Kinani et al., 2010; Louiz et al., 2008). Sigmoid dose–responsecurves for BaP and different samples along with their EC50 valueswere derived from the global curve fitting analysis with fourparameter logistic curve equation. F test for dose response parallel-ism was carried out considering both shared and un-shared param-eters for four parameter logistic curve equation.

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

the cell lines was evaluated using the MTT assay (Laville et al.,2004; Mosmann, 1983). Cell viability was expressed as percentageof the corresponding control (DMSO 0.5%). The LC50 values andsigmoid dose–response curves were plotted as described in Section2.5.2.

2.5.4. Alkaline single-cell gel electrophoresis – Comet assayAlkaline comet assay was performed as described by Tice et al.

(2000). HuH7 cells treated with either test samples, positivecontrol (BaP, 1 mM) or negative control (DMSO 0.5%) for 24 h.The slides were stained with ethidium bromide (2 lgmL�1,100 lL per slide) and visualised with a 40� lens fitted to a fluores-cence microscope with an excitation and emission setting of 518/605 nm. The percentage of DNA in tail, tail moment and olive tailmoment (OTM) of 50 randomly selected cells were analyzed fromeach slide by using CometScore Freeware software (www.tritek-corp.com). The comets were divided into five classes on the basisof amount of DNA in the tail; Class I, less than 1% DNA in tail (intactnucleus); Class II, 1–20% DNA in tail; Class III, 20–50% DNA in tail;Class IV, 50–75% DNA in tail and Class V, more than 75% DNA in tail(Miyamae et al., 1998).

2.6. Statistical analysis

All experimental data were expressed as means ± standarddeviation of three replicates. All statistical analyses including glo-bal curve fitting and F test for dose response parallelism were per-formed with sigma plot 11 statistical package (Systat Software, SanJose, CA). Statistical differences between the control and treatedcells were examined with the aid of ANOVA followed by multiplecomparisons (Dunnett’s Method). Pearson correlation coefficient(r) was used to evaluate correlations among various endpoints. Avalue of P < 0.05 was used to determine significance in statisticalanalyses.

3. Results and discussion

3.1. Microbial degradation of contaminants of pulp and paper millsewage sediment

Pseudomonas sp. strain ISTDF1 is an indigenous bacterium of thesampling site that degrades dibenzofuran in minimal salt medium(MSM) (Jaiswal et al., 2011). When this strain was inoculated intoMSM containing 2% v/v crude sediment extract it exhibited a shortlag phase of 48 h and a log phase up to 120 h and thereafter a sta-tionary phase (Fig. 1). The chloride ion concentration in the med-ium increased continuously up to 120 h (7549 mM) after which itshowed no significant increase and remained almost constant tillend of the experiment (240 h). The release of chloride ions sug-gested dechlorination of chlorinated compounds present in thesediment extract. Chandra et al. (2009) reported the liberation ofsignificant amounts of chloride as the result of bacterial biodegra-dation (dechlorination) of pentachlorophenol and other chlori-nated phenolic compounds present in pulp and paper milleffluent. Diverse oxidation enzymes including dioxygenase andmonooxygenase are involved in the process of aerobic dechlorina-tion of organochlorine compounds (Janssen et al., 1994). An earlierstudy on Pseudomonas sp., strain ISTDF1; showed the involvementof angular as well as lateral pathways of dibenzofuran biodegrada-tion. Based on this result Jaiswal et al. (2011) postulated the pres-ence of diverse dioxygenation and aromatic ring oxidationenzymes in the strain. In the present study, the bacterium was ableto carry out dechlorination even in the presence of mixed carbonsource, which may be due to the activity of its diverse oxidising en-zymes. Organochlorine contaminations at high level leads to thenatural selection of bacterial strains specialized to utilize them.Dehalococcoides species are reported to reduce toxic chlorinated or-ganic compounds in the environment (Seshadri and Heidelberg,2005). Other bacterial strains can dechlorinate these substratesby developing adaptive mechanism by horizontal gene transferfrom resistant strains or by gene induction thereby generating adiversity of dechlorination processes (Seshadri and Heidelberg,2005). Apparently, dechlorination may be a necessary mechanismof the indigenous bacteria to survive in the organochlorine con-taminated environment.

3.2. EROD assay

The dose–response curves for EROD induction by different testand control samples are shown in Fig. 2a. Global curve fitting anal-

Concentration (g Sed Eq L-1)

1e-1

2

1e-1

1

1e-1

0

1e-9

1e-8

1e-7

1e-6

1e-5

1e-4

1e-3

1e-2

1e-1

1e+0

1e+1

1e+2

1e+3

1e+4

% C

ell V

iabi

lity

(100

% =

DM

SO 0

.5%

)

0

20

40

60

80

100

120

BaP (g L-1)

UT (g Sed Eq L-1)

T24 (g Sed Eq L-1)

T48 (g Sed Eq L-1)

T120 (g Sed Eq L-1)

T240 (g Sed Eq L-1)

(b)Concentration (g Sed Eq L-1)

1e-1

2

1e-1

1

1e-1

0

1e-9

1e-8

1e-7

1e-6

1e-5

1e-4

1e-3

1e-2

1e-1

1e+0

1e+1

1e+2

1e+3

1e+4

ERO

D a

ctiv

ity (1

00%

= 1

mM

BaP

)

0

20

40

60

80

100

120

BaP (g L-1)

UT (g Sed Eq L-1)

T24 (g Sed Eq L-1)

T48 (g Sed Eq L-1)

T120 (g Sed Eq L-1)

T240 (g Sed Eq L-1)

(a)

Fig. 2. Evaluation of toxicity of contaminants degraded by Pseudomonas sp. strainISTDF1. 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, T240: 240 htreated extract. Values represent the mean ± SD, n = 3. (a) EROD inductionmeasured after 6 h exposure period; 100% EROD induction was considered for1 mM BaP treatment. Global goodness of fit R2 = 0.9983. (b) Cell viability measuredafter 24 h exposure period; 100% cell viability was considered for 0.5% DMSOtreatment. Global goodness of fit R2 = 0.9926.

74 M.T. Das et al. / Bioresource Technology 110 (2012) 71–78

ysis was carried out with four parameter logistic equation to plotthe curves. The data failed the F-test for dose response parallelism(P > 0.05), indicating that the dose response curves are not parallel.Therefore, statistical results obtained without sharing any of theparameters of four parameter logistic equation were consideredfor analysis of EC50 values and further calculation of biologicalBaP-Eqs (Table 1). The un-treated (UT) sample elicited significantEROD activity shown in terms of EC50 and BaP-Eq values. Sampleresponses varied greatly depending on the duration of the bacterialtreatment. The highest BaP-Eqs values were found for the UT sam-ple (4.3 lg gSedEq�1) and the lowest values for the 240 h treated(T240) sample (0.002 lggSedEq�1). The EROD EC50 valueincreased about 1810 times and the BaP-Eq value showed a2110-fold decrease after 240 h bacterial treatment.

EROD induction by dioxin-like compounds in liver cell lines hasbeen effectively utilized to monitor such compounds in environ-mental samples. Results of this bioassay have been routinelycompared with chemical analysis methods. EROD assay derived

BaP-Eqs were positively correlated with toxic equivalents derivedfrom chemical PAH analysis (Kinani et al., 2010; Louiz et al.,2008), but Brack et al. (2000) and Kaisarevic et al. (2009) foundthat biologically derived toxic equivalents mostly exceed thechemically derived toxic equivalents. To explain this deviation,Kaisarevic et al. (2009) have suggested the presence of non-ana-lysed compounds with EROD inducing potency in the sedimentextract. Similarly, Brack et al. (2000) have postulated the presenceof heterocyclic aromatic hydrocarbons and amines, aminotoluenes,imidazoles, and other pesticides, a number of natural compounds,such as wood components, retene, and juvabione in their sample toexplain the exceeding biological equivalent value. Even though theEROD assay-derived toxic equivalent lacks general agreement withchemically derived toxic equivalents, it can be utilized to monitortoxicity of pre- and post-bioremediated samples qualitatively andquantitatively. In present study the un-treated (UT) sample elicitedsignificant EROD activity, indicating a general contamination bydioxin-like and other EROD-inducing compounds at the studiedsite. Further post biodegradation increase in EROD EC50 valueand decrease in BaP-Eq value clearly indicated steady mineralisa-tion of EROD-inducing chemicals.

3.3. Cell viability

The cell viability in terms of MTT assay derived LC50 values(Table 1) and the dose response curves (Fig. 2b) showed that thelevel of toxicity decreased with increasing duration of bacterialtreatment. The lowest value of LC50 was found for the UT sample(10.15 gSedEqL�1) and the highest for T240 (52.17 gSedEqL�1).LC50 values increased about five times after 240 h bacterial treat-ment. EROD EC50 values and MTT assay LC50 values showed posi-tive correlation with a Pearson Product Moment Correlationcoefficient r = 0.864 (p < 0.05).

A significant observation with the present study was that theEROD EC50 value increased nearly 1816-fold increase after 240 hbacterial treatment whereas the MTT assay-derived LC50 valuesshowed only about a 5-fold increase under the same conditions.Apparently, the EROD-inducing compounds were successfully min-eralised by the bacteria leading to the significant increase of theEROD EC50 value but at the same time, some other factors wereresponsible for overall cell cytotoxicity. Still, the decrease in cellcytotoxicity was quite significant after 240 h of microbial biodeg-radation. Some compounds found in paper mill sewage sedimentlike phenanthrene, anthracene, pyrene have cellular proliferativeeffect and simultaneously they demonstrate high EROD-inducingpotency (Kaisarevic et al., 2009). Aryl hydrocarbon receptor (AhR)dependent disruption of cell cycle control, followed by enhancedcell proliferation, induced by certain PAHs and their methylatedderivatives have been evidenced in rat liver epithelial WB-F344cells (Machala et al., 2008). Overall results of the MTT assay sug-gested that the pulp and paper mill sewage sediment contained asignificant load of toxicants that disturbed cell growth, in termsof cell proliferation or induction of cytotoxicity.

3.4. Comet assay

The outcome of the single cell gel electrophoresis (Comet assay)with treated and un-treated sample is shown in Fig. 3a and b. Thecomets were divided into five classes on the basis of amount ofDNA in tail (Fig. 3b). HuH7 cells treated with the UT sampleresulted in 64% comets that fell under either of the classes III, IVor V; whereas only 24% comets fell under the same classes withT240 sample. Tail moments of 50 randomly selected comets arepresented as quantile box plots. The plot shows that distributionof comets became more homogenous with very low tail moment(1.0119 ± 1.6070) with the T240 sample in comparison to UT

Table 1EROD EC50 and MTT LC50 values of un-treated and treated samples along withcorresponding BaP-Eq values.

Treatmentsa EROD EC50b R2 (EROD EC50) BaP-Eqc MTT LC50

b R2(LC50)

BaP 1.68e�6 0.9972 – 1.64e�6 0.9889UT 0.4 0.9986 4.227e�6 10.15 0.9908T24 1.27 0.9977 1.320e�6 11.24 0.9905T48 4.17 0.9991 4.021e�7 19.51 0.9981T120 164.82 0.9983 1.017e�8 38.9 0.9960T240 719.92 0.9970 2.328e�9 52.17 0.9955

a HuH7 cell lines were treated with different test samples (dilutions, 10�3 to103 gSedEqL�1) or positive control BaP (dilutions, 10�11 to 10�3 g L�1) either for 6 h(EROD assay) or for 24 h (MTT assay).

b Derived using global curve fitting model with four parameters logistic non-linear regression equation, expressed in terms of gSedEqL�1 (test samples) or gL�1

(BaP).c BaP-Eq = EC50 of BaP (g L�1) / EC50 of Sample (gSedEqL�1).

M.T. Das et al. / Bioresource Technology 110 (2012) 71–78 75

sample (tail moment = 18.8316 ± 10.5536). This indicated sifting ofthe comet class towards a lower value of tail moment after the bac-terial treatment. The olive tail moment data showed a decreasingtrend with increasing duration of bacterial treatment. T240 sampleresulted in an 8-fold decrease in DNA migration (OTM = 1.14 ± 1.4)in comparison to that of the UT sample (OTM = 9.14 ± 4.15). Statis-tically significant DNA damage (Dunnett’s Method p < 0.05)noticed in UT, T24 and T48 samples with respect to negative con-trol group (DMSO 0.05%) confirmed the genotoxic nature of thecontaminants. The OTM data showed negative correlation withLC50 data (Pearson Product Moment Correlation coefficientr = �0.875, p < 0.05) whereas it showed positive correlation withBaP-Eq data (r = 0.835, p < 0.05).

Genotoxicity can be induced by a diverse group of chemicalcontaminants present in the pulp and paper mill effluent including

Treatments

BaP 1mM UT R24 R48 R120 R240 DM

Tail

Mom

ent

0

10

20

30

40

50

Tail MomentOlive Tail Mo

*

*

*

*

(a)

(b)

Fig. 3. Genotoxicity of the contaminants before and after treatment with Pseudomonasdifferent samples. Tail moments of 50 randomly selected comets are presented as quantilline in the box presents the median value while dotted line represents mean value. Errorbeyond 5th and 95th percentiles. Olive tail moments of same 50 comets are shown as theDMSO control group (Dunnett’s Method p < 0.05) for both data sets. (b) Images of differebromide. Roman numerical indicates class of comet.

dioxins, furans, PCB and PAH. From the results of the comet assay,it is clear that bacterial treatment for 240 h was efficient in reduc-ing the genotoxic nature of pulp and paper mill sewage sediment.The comet assay can be performed with different eukaryotic cellsincluding mammalian cell lines. The assay has been carried outin yeast cells earlier to demonstrate post biodegradation toxicityreduction of pulp and paper mill effluent (Mishra and Thakur,2010; Singhal and Thakur, 2009), but mammalian hepato-carci-noma cell line HuH7 was a better option due to its sensitivity inthe EROD assay.

3.5. GC–MS analysis

The treated and un-treated (UT) samples were analyzed by GC–MS to find degradation patterns of compounds present in the papermill sewage sediment extract. The chromatograph correspondingto compounds extracted from UT and T240 samples are shown inFig. 4a and b respectively and the identified compounds are sum-marised in Table 2. The degradation products mentioned in thetable were identified from the available standards of the authenticcompounds documented in NIST-05 and Wiley-8 libraries. Com-parative analysis of total number of peaks in the GC–MS chromato-gram before and after microbial treatment qualitatively infers theextent of biodegradation. An increase in the total number of peaksfrom 48 for the UT sample to 93 for the T48 sample and a decreaseto 54 for the T240 sample was observed. It can be inferred fromthis data that initial complex compounds were broken down intosimpler compounds which were later on gradually mineralised.Earlier studies on biodegradation of complex molecules havereported an increase in the number of peaks during the course ofbiodegradation (Chandra et al., 2009; Kaushik et al., 2010; Rajet al., 2007).

SO 0.5%

Oliv

e Ta

il M

omen

t

0

5

10

15

20

25

30

ment

sp. strain ISTDF1. (a) The tail moment and the olive tail moment plotted againste box plots. The edges of the box represent the 25th and the 75th percentiles; a solidbars indicate 90th and 10th percentiles and the black circles indicate outlying points

mean ± standard deviation. The asterisk denotes the significant difference from thent classes of comets seen under fluorescent microscope after stained with ethidium

Fig. 4. GC Chromatogram before and after bacterial treatment. X-axis represents retention time in minutes and Y-axis represents relative abundance. (a) Un- treated sample,UT (b) 240 h treated, T240.

76 M.T. Das et al. / Bioresource Technology 110 (2012) 71–78

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

Peak retention time (min) Present in sample Name of identified compound

UT T24 T48 T120 T240

5.26 � � + � � Benzeneacetic acid, heptyl ester5.40 � � + � � Benzaldehyde5.59 � � � � + Decane6.82 � � � � + 3-Methyldecane7.25 + + + � � 4-tert-Butyltoluene7.41 � � � � + Dodecane7.51 � � � � + 4-Isopropylbenzaldehyde

16.7 + + + + + Naphthalene 1,2,3,4-tetrahydro-1-methyl-8-(1-methylethyl)19.49 + + + � � Octadecane24.02 + + + � � Trichloroacetic acid, tetradecyl ester25.74 � + + + � Phthalic acid, diisobutyl ester28.07 + + + � � Dibutyl phthalate28.30 � + + � � Oxalic acid, 2-ethylhexyl isohexyl ester28.99 + + + � � Dichloroacetic acid, heptadecyl ester

M.T. Das et al. / Bioresource Technology 110 (2012) 71–78 77

Result of the study indicated emergence of varied and increasednumber of peaks at early retention times in the treated samplesthat were mostly identified as aliphatic open chain compounds likedecane, dodecane and methyl decane. Most of the major peakspresent in the UT sample beyond RT at 40.05 min disappeared inthe T240 sample. Naphthalene, 1,2,3,4-tetrahydro-1-methyl-8-(1-methylethyl) (RT = 16.70) was present in all the samples, but itspeak area was reduced from 3.23 e5 in UT sample to 1.46 e5 inT240 sample which indicated a decrease in its concentration. Thisresult showed correlation with the EROD assay results. PAHs likenapthalene are EROD-inducing chemicals. A decrease in the con-centration of such compounds will lower BaP-Eq values andincrease EROD EC50 values. Chlorinated compound like dichloro-acetic acid, heptadecyl ester (RT = 28.99) and trichloroacetic acid,tetradecyl ester (RT = 24.02) detected in the UT sample were notfound after 120 h bacterial treatment which indicated dechlorina-tion and/or mineralisation of such compounds. This result was cor-related with the results of the argentometric detection of chloriderelease. Reduction of chlorinated and poly aromatic compoundsmay have attributed to the reduction of toxicity.

Some low molecular weight aromatic compounds were de-tected in T240 sample e.g. 4-Isopropylbenzaldehyde (RT = 7.52).The presence of this compound in the treated sample was due todegradation of lignin present in pulp and paper mill effluent (Rajet al., 2007). A few members of the phthalate family were also de-tected at different stages of biodegradation. Dibutylphthalate(RT = 28.08) was present in the UT sample but was not found after48 h. However, diisobutylphthalate (RT = 25.75) was formed after24 h treatment and was present till 120 h. Phthalate derivativessuch as butylphthalate and bis (2-ethylhexyl) phthalate have beenreported in pulp and paper mill effluents (Ksibi et al., 2003; Rajet al., 2007). Phthalates leach from PVC goods and are thus a majorclass of industrial contaminant (Blount et al., 2000), but phthalatesmay also be formed during microbial degradation of poly aromaticcompounds (Seo et al., 2009). Finally it was also noted that with in-crease in the duration of bacterial treatment the total peak area inthe GC–MS chromatogram gradually declined from 3.48 e7 in theUT sample to 4.85 e6 in the T240 sample which indicated steadymineralisation of organic compounds.

4. Conclusion

This report illustrates the use of different bioassay techniques toevaluate detoxification efficiency of a bioremediation process. Thethree bioassays quantitatively and qualitatively describe differentaspects of toxicity including overall cytotoxicity (MTT assay), diox-in-like behaviour (EROD assay) and genotoxicity (comet assay) in a

cost- and time-effective manner. The study also reveals the poten-tial of an earlier reported Pseudomonas sp. strain ISTDF1 to degradeand detoxify persistent organic pollutants even in the presence of arange of carbon sources. This in vitro study can further be extendedto microcosm analysis and in situ remediation process.

Acknowledgements

This paper was supported by the research grants of Departmentof Biotechnology, Government of India, and New Delhi, India. Wethank Century pulp and paper mill, Uttarakhand, India for provid-ing effluent and sewage sediment during the course of investiga-tion and Mr. Ajai Kumar of Advanced Instrumentation ResearchFacility (AIRF) Jawaharlal Nehru University, New Delhi for GC-MSanalysis. We also thank Dr. Vijay Kumar of International Centrefor Genetic Engineering and Biotechnology, New Delhi for kinddonation of HuH7 cell line. The author (Das MT) also thanks Uni-versity Grants Commission (UGC), New Delhi, Government of India,for providing Research Fellowship.

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