Effect of bacterial inoculum ratio in mixed culture for decolourization and detoxification of pulp paper mill effluent

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Research ArticleReceived: 29 June 2011 Revised: 16 September 2011 Accepted: 19 September 2011 Published online in Wiley Online Library: 9 January 2012

(wileyonlinelibrary.com) DOI 10.1002/jctb.2758

Effect of bacterial inoculum ratio in mixedculture for decolourization and detoxificationof pulp paper mill effluentRam Chandra,a∗ Rachna Singhb and Sangeeta Yadava

Abstract

BACKGROUND: Effluent released from industry is a mixture of various pollutants. For the degradation of complex pollutants,mixed bacterial cultures can be more effective than a single culture. This study investigated the balance of bacterial populationsin a mixed culture for maximum reduction of pollutants.

RESULTS: This study deals with the degradation and detoxification of pulp paper mill effluent (PPME) by three bacterial strains,i.e. Serratia marcescens, Serratia liquefaciens and Bacillus cereus in different ratios, and found that two ratios, 4 : 1 : 1 and 1 : 4 : 1,were effective for the degradation of PPME. These ratios reduced the various pollution parameters. Enzyme bioassay revealedthat more enzyme was produced during degradation for the ratio 4 : 1 : 1. High performance liquid chromatography (HPLC)analysis showed that the ratio 4 : 1 : 1 degraded 95% of lignin and related compounds, and chlorophenols up to 98%, whereasratio 1 : 4 : 1 reduced lignin by 84% and chlorophenols by 58% after 7 days incubation. Degradation products were confirmedby gas chromatography–mass spectrometry (GC-MS) analysis. A seed germination bioassay on Phaseolous mungo L. revealedthat toxicity was reduced by the ratio 4 : 1 : 1.

CONCLUSION: Due to variable potential of different bacteria show variation in their growth pattern at any contaminated site.This study shows that an appropriate ratio of mixed cultures is required for maximum degradation and detoxification of PPME.c© 2012 Society of Chemical Industry

Keywords: chlorophenols; GC-MS; inoculum ratio; lignin; peroxidases; rayon grade effluent

INTRODUCTIONIn India presently 625 paper mills are in operation, of which 25are large and the others are small. Papers are manufactured fromwood containing cellulose fiber, from recycled paper and fromagricultural residues. The pulp and paper industry breaks downthe wood to separate cellulose from non-cellulosic substances,and the fibrous mass is known as pulp. Rayon grade pulp (RGP) isproduced from long fibers of the highest quality.1 Manufacturingof RGP requires only high quality fiber containing wood chipswith an extra chemical process that involves pre-hydrolysis ofwood chips at elevated temperature and pressure followed byalkaline digestion and multistage bleaching. This process requireslarge amounts of water and energy and releases approximately47 000–80 000 gallons of toxic effluent into aquatic water bodies.The effluent contains a mixture of substances stemming fromwood and the chemicals used (particularly Cl2, ClO2) and otherchlorinated compounds arising from pulping and bleaching,i.e. lignosulphonic acids, chlorolignins, chlorinated resin acids,chlorinated phenols, chlorinated hydrocarbons, waxes, varioussurfactants, plasticizers and biocides.2,3 Thus, effluents releasedfrom these industries are heavily loaded with organic mattercontaining 200 organic and 700 kinds of inorganic compounds.4

These pollutants increase toxic substances as well as chemicaloxygen demand (COD), biological oxygen demand (BOD) andtotal dissolved solids (TDS) of receiving water bodies, which causesimbalances in the aquatic ecosystem and profoundly affects the

terrestrial ecosystem. The high chemical diversity of the organicpollutants in pulp and paper mill waste-water causes a varietyof toxic effects on aquatic communities and in recipient waterbodies.5 Discharge of untreated effluent from the pulp and paperindustry causes slime growth, thermal impact, scum formation,colour problems and loss of aesthetic beauty of the environment.The brown colour imparted to water due to addition of effluentsis detectable over long distances. It makes downstream waterunfit for domestic and irrigation purposes, thus, the adequatetreatment of effluents is necessary prior to its discharge.

The physico-chemical treatment processes used by largeindustries are expensive and not affordable by small industries.However, several reports are available on the degradation ofpulp paper mill effluent (PPME) by white rot fungus.6,7 Themain constraint on using a fungal system is the requirement

∗ Correspondenceto: Ram Chandra,DepartmentofEnvironmentalMicrobiology,Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar,Raebareli Road, Lucknow-226025, India. E-mail: rc [email protected];ramchandra [email protected]

a Department of Environmental Microbiology, Babasaheb Bhimrao AmbedkarUniversity (A Central University), Vidya Vihar, Raebareli Road, Lucknow-226025,India

b Environmental Microbiology Section, CSIR-Indian Institute of ToxicologyResearch, Post Office Box No. 80, M. G. Marg, Lucknow 226 001 (U.P.), India

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Mixed culture decolourization and detoxification of PPME www.soci.org

for low pH (4–5), difficult growth in submerged conditions, andO2 limitation. Recently, bacteria Halomonas sp. and Bacillus sp.have been used for black liquor degradation and decolourizationat high pollution load.8,9 Bacillus species,10 Serratia marcescens,11

Pseudomonas, Ancylobacter and Methylobacterium12 are also usedfor pulp paper effluent degradation. In the present study, bacteriaSerratia marcescens, Serratia liquifacines and Bacillus cereus wereused for pulp paper mill effluent degradation. Although there areseveral reports on paper mill effluent degradation, the effect ofbacterial ratio on degradation and detoxification of PPME is notreported. This present study was focused on the effect of bacterialratio on the degradation and detoxification of PPME. However,the compounds generated after degradation and their effect ontoxicity was also investigated.

MATERIAL AND METHODSSample collection and its physico-chemical analysisThe raw effluent prior to any treatment discharged from the washmachine and bleaching section was collected from the RGP unitof M/s Century Pulp Paper Mill Ltd, Lalkuan, Uttarakhand, India,located at the foothills of the Himalayas. Eucalyptus and bamboowoods are the main as raw materials and a kraft process is used forpulping, followed by multistage chlorine bleaching to make whitepaper. This process discharges wash machine effluent releasedafter pulp washing and bleached effluent separately. The millproduces 524 tons of fine quality pulp per day and discharges48 426 m3 effluent in total. The effluents were collected separatelyin pre-sterilized plastic jerry-cans (20 L capacity), brought to thelaboratory, and stored at 4 ◦C. All the physico-chemical analysiswas done within 48 h.

The physico-chemical parameters were analyzed before andafter bacterial degradation in accordance with standard methodsfor waste-water analysis.13 Physico-chemical measurements weremade as follows: total dissolved solids (TDS), BOD5, COD openreflux method, total nitrogen by the Macro Kjeldal method,total phenol by a chloroform extraction method, and sulphateby the BaCl2 precipitation method. Phosphate was measuredby a vanadomolybdo-phosphoric acid colourimetric methodperformed as the standardized method described in APHA (2005);colour was measured according to the CPPA standard methodat 465 nm by UV visible spectrophotometer (Techcomp 2300).14

pH was estimated using a pH meter, water and soil analysis kit(Model 1160), metals were detected after acid digestion by atomicabsorption spectrophotometry, and different ions (i.e. sodium,chloride, nitrate and potassium) were analyzed by Orion ion meter(Model 960, USA) using a selective ion electrode.

Bacterial cultures and culture conditionThree lyophilized bacterial cultures were obtained from ATCC*(American type culture collection) Serratia marcescens (ATCC-14 756) and Serratia liquifaciens (ATCC-27 592) known for degra-dation of high concentration chlorophenols15 and Bacillus cereus(ATCC-10 876) for lignin degradation.16 Cultures were revived onkraft lignin amended nutrient agar media and maintained onL-MSM16 agar plates.

Screening for ligninolytic activity of bacterial strainsThe purified bacterial strains were screened for detection of theirdifferent ligninolytic enzyme activity by plate assay method. Ligninperoxidase (LiP, EC 1.11.10.14) and manganese peroxidase (MnP,

EC1.11.1.13) were detected by the method described by Pangalloet al.17 while, laccase (1.10.3.2) was analyzed by the methoddescribed by D’Souza et al.18 The presence of LiP activity wasdetected by disappearance of blue colour in the media and MnPby conversion of dark pink colour to yellow; a brown colour zonesurrounding the colony indicated positive laccase activity.

Degradation of pulp paper mill effluentOptimization of carbon and nitrogen sourceThe concentration of peptone and dextrose was optimized as suit-able nitrogen and carbon sources for growth and decolourizationof pulp paper mill effluent. Carbon concentration was varied from0.5 to 1.5% w/v and peptone from 0.1% to 1% to optimize the min-imum requirement of nutrient for bacterial growth and to increaseBOD/COD ratio. Minimum concentration was optimized becauseglucose and peptone also contribute to BOD, COD increase.

Degradation of pulp paper mill effluentIn total, seven combinations of mixed culture were prepared, withS. marcescens : S. Liquifaciens : B. cereus in the ratio 1 : 1 : 1, 1 : 1 : 4,1 : 4 : 1, 4 : 1 : 1, 1 : 1 : 2, 2 : 1 : 1 and 1 : 2 : 1. These mixed cultureswere inoculated into 100 ml of autoclaved PPME containingpeptone (0.5%) and dextrose (1%), followed by incubation at35 ± 2 ◦C in a temperature controlled shaker (New Brunswick,Innova 4230, USA) at 140 rpm. The growth was monitored at620 nm, with decolourization and reduction in BOD and CODfor 7 days at 24 h intervals. Pulp paper mill effluent withoutinoculum was used as a control. Pentachlorophenol was measuredafter periodic extraction with dicloromethane by UV visiblespectrophotometry (Techcomp 2300) at 320 nm,19 while ligninwas measured according to the method described by Pearl andBenson.20

Bioassays of ligninolytic enzymesEnzyme bioassay was performed during the bacterial treatmentof PPME. LiP, MnP and laccase were assayed as described byArora et al.21 The LiP assay was done by monitoring the oxidationof dye Azure B in the presence of H2O2. The reaction mixturecontained sodium tartrate buffer (50 mmol L−1, pH 3.0), Azure B(32 µmol L−1), 500 µL of culture filterate, 500 µL of H2O2 (2 µm).After 10 min OD was taken at 651 nm. One unit of enzyme activityis equivalent to an absorbance decrease of 0.1 units min−1 mL−1.MnP assay was performed by the modified method of Arora et al.21

based on the oxidation of phenol red. Reaction mixture (4 mL)contained 1 mL of potassium phosphate buffer (pH 7.0), 1 mL ofenzyme extract, 500 µL of MnSO4 (1 mmol L−1),1 mL of phenolred (1 mmol L−1) and 500 µL H2O2 (50 µmol L−1). A 1 mL samplewas removed from the reaction mixture and 40 µL of 5 mol L−1

NaOH was added to stop the reaction. Optical density (OD) wastaken at 610 nm at 1 min intervals. One unit of enzyme activityis defined as an absorbance increase of 0.1 unit min−1 mL−1 andfor Laccase the absorbance was read at 450 nm. The completereaction mixture contained 3.8 mL acetate buffer (10 mmol L−1,pH 5.0), 1 mL guaiacol (2 mmol L−1) and 0.2 mL of enzyme extract.One unit of enzyme activity was defined as a 0.001 increase in OD.For the control, reaction mixture was prepared instead of enzymeextract, buffer was added.

Metabolites characterization by HPLC and GC-MS analysisTo assess degradability, the bacterial degraded and undegraded(control) samples of PPME were centrifuged at 5000 rpm for

J Chem Technol Biotechnol 2012; 87: 436–444 c© 2012 Society of Chemical Industry wileyonlinelibrary.com/jctb

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www.soci.org R Chandra, R Singh, S Yadav

10 min to separate bacterial biomass and particulate matter.The supernatant obtained was acidified to pH 2 using 1N HCland extracted three times with equal volumes of ethyl acetateand dichloromethane for maximum extraction of lignin andchlorophenols, respectively. The collected organic layer wasdewatered over anhydrous Na2SO4 and filtered through Whatmanno. 54 filter paper (Whatman, UK). The dried residues weredissolved in acetonitrile and samples were analysed by HPLC(Hitachi Model - Organizer, Japan) equipped with reverse phasecolumn C-18 at 27 ◦C. 20 µL of sample was injected, followed bymobile phase containing acetonitrile and water in 70 : 30 ratio at aflow rate of 1 mL min−1. For lignin the detection wavelength wasset at 280 nm according to Chandra et al.15 and chlorophenolswere detected at 254 nm.22

The analysis of pollutants and characterization of metabolitesformed after bacterial treatment was performed through GC-MS

analysis (Perkin Elmer, UK). The dried residues of ethyl acetateand dichloromethane were derivatizated by Tri methylsilyl (TMS).An aliquot of 1 µL of silylated compounds was injected into theGC-MS equipped with PE auto system XL gas chromatographinterfaced with Turbomass spectrometric mass selective detector,and equipment parameters were set as detailed by Raj et al.23

The pollutants present in the control sample and metabolicproducts were identified by comparing the mass spectra with thatof National Institute of Standard and Technology (NIST) libraryavailable with instrument by comparing retention time with thoseof available authentic compounds.

Toxicity assessment by seed germination testFor toxicity assessment, different dilutions of degraded andnondegraded PPME samples 10, 25, 50, 75 and 100% v/v were takenand tap water was kept as control (C). Seeds of Phaseolous mungo L.

Table 1. Screening of bacterial strains for different ligninolytic enzymes laccase, lignin and manganese peroxide with their respective substrates atdifferent pH

pH 6 pH 7 pH 8 pH 9 pH 10 pH 11

Enzyme I II III I II III I II III I II III I II III I II III

Manganese Peroxidase − − − + + +++ ++ + ++ +++ ++ + + +++ ++ − − −Lignin Peroxidase + +++ ++ − − +++ − − +++ − − − − − − − − −Laccase + − − ++ − − ++ − − ++ − − ++ − − − − −I- Serratia marcescens; II- Serratia liquifaciens; III- Bacillus cereus; (−) no enzyme activity; (+) slow enzyme activity; (++) moderate enzyme activity;(+++) good enzyme activity.

Table 2. Physico-chemical characteristics of pulp paper mill effluent before and after bacterial treatment

Percentage reduction by 4 : 1 : 1 Percentage reduction by 1 : 4 : 1

Parameters Control (mg L−1 except pH and colour) 1 d 3 d 7d 1 d 3 d 7d Permissible limit (EPA35)

Colour 8117.50 ± 185 35.60 59.12 65.06 30 45 55 –

COD 16400 ± 120 12.50 32.50 63.37 8 15 35.22 120.00

BOD 5850 ± 50.12 14.52 38.85 64.00 10 20 56.60 40.00

TDS 840 ± 32.45 16.90 57.62 63.69 12 45 50 –

TSS 100 ± 4.00 8.00 46.00 85.00 6 45 55 35.00

TS 940 ± 2.71 15.95 54.25 71.70 5 15 52 –

Phenol 1272 ± 30.45 10.24 47.64 63.34 12 24 30 0.0050

T. Nitrogen 571 ± 25.12 07.00 17.68 74.64 2 10 35 25.00

Lignin 614 ± 8.13 34.39 45.24 95.00 10 24 84 –

PCP 145.11 ± 4.56 10.60 39.24 98.00 7 24 58 10.00

Nitrate 41.52 ± 3.56 48.36 51.34 52.04 30 45 48 10.00

Ammonium 23.54 ± 0.89 7.45 32.35 46.81 21 26 40 –

Chloride 31.42 ± 0.86 11.43 18.67 28.16 3 12 15 1500.00

Sodium 136.56 ± 4.56 36.30 68.98 84.43 40 50 60 200.00

Pottasium 86.52 ± 2.58 57.72 66.88 86.65 15 30 40 –

Cr 0.2020 ± 0.01 21.26 50.00 99.99 10 40 70 0.01

Cu 0.5110 ± 0.10 19.60 50.00 100 15 48 80 0.50

Mn 0.8750 ± 0.03 13.62 49.92 99.79 20 50 85 0.20

Ni 0.1500 ± 0.02 18.73 49.73 99.46 24 46 78 0.10

Cd 0.2078 ± 0.09 16.007 50.00 99.89 26 48 79 0.01

Fe 1.203 ± 0.04 21.78 49.95 99.98 28 30 82 2.00

Pb 0.0148 ± 0.00 20.45 50.00 99.89 30 49 85 0.05

Zn 0.3330 ± 0.01 21.54 50.50 99.94 40 42 84 2.00

∗ All the values are mean of triplicate (n=3±SD) measurements.

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were washed in running tap water followed by surface sterilizationwith 3% (v/v) solution of H2O2 according to Liu et al.24 and thenrinsed with distilled water several times. Ten seeds of P. mungoL. were placed in different petri-dishes lined with two layer ofWhatman No-1 filter paper soaked in 5 mL of different sampleconcentrations. Seeds were incubated at 20 ◦C in a BOD incubatorfor 192 h. The toxicity of PPME was detected in terms of percentagegermination and speed of germination index according to Anjumand Bajwa,25 and the α-amylase activity of seeds. 10 seeds fromeach petri-dish were homogenized in 0.1 mol L−1 sodium acetatebuffer (pH 4.8) and crude enzyme extracts were used for theassay of α-amylase activity.26 All the experiments were performedin triplicate. The isolation, purification and characterization ofα-amylase were done according to the method described byBharagava et al.27

RESULTS AND DISCUSSIONScreening of ligninolytic enzymesThe bacterial strains S. marcescens, S. liquifaciens and B. cereus werescreened for the presence of ligninolytic enzyme, i.e. LiP, MnPand laccase. All these strains showed the presence of LiP and MnPwhereas laccase activity was shown only by S. marcescens (Table 1).These strains were further used for the degradation of PPME inaxenic (single) and a mixture of all three cultures in different ratios.

Physico-chemical characterization and degradation study ofPPMEThe physico-chemical analysis of PPME before bacterial treatmentshowed high BOD, COD, TDS, lignin, chlorophenol, total phenol,chloride, sodium, nitrate, potassium and colour, as shown inTable 2.

Optimization of carbon and nitrogen source and degradation ofPPME1% dextrose and 0.5% peptone concentration was found to bethe best combination of carbon and nitrogen source. It showedbest degradability of PPME (Fig. 1(A)). Reduction in colour andphysico-chemical property was recorded after bacterial treatmentwith axenic (data not shown) as well as the consortium after7 days of incubation. The consortium showed better degradationand decolourization of PPME. Bacterial cell numbers increaseand decrease in nature as well as in the medium accordingto the degradation capacity and compatibility of strains witheach other. In order to determine the effect of inoculums ratioon the degradation of PPME, S. marcescens, S. liquifaciens andB. cereus inoculated initially at 1 : 1 : 1 ratio showed the dominanceof S. marcescens and did not show better degradation capability,whereas two other selected ratios, i.e. 1 : 1 : 4 and 1 : 1 : 2, alsoresulted in an increase in colour compare with the control. Similarresults were also observed by Perestelo et al.28 during treatmentof kraft pine lignin by Bacillus megaterium isolated from compost,where it was explained that oxidation of α-carbon of the sidechain caused by bacterial action resulted in high molecular weightcompounds.28 However, another two ratios, 4 : 1 : 1 and 1 : 4 : 1,suggest that greater degradation, decolourization, and reductionin BOD and COD (Table 3) might be due to the chlorophenolsdegradation capability of S. marcescens and S. liquifaciens. Further,these two inoculum ratios were periodically monitored for growth,degradation parameters, enzyme activity as well as metabolitecharacterization. Bacterial ratio 4 : 1 : 1 was found to be more

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Figure 1. (A) Optimization of nitrogen and carbon source for pulp papermill effluent degradation and (B) growth and decolourization pattern ofpulp paper mill effluent by two inocula, ratio 4 : 1 : 1 and 1 : 4 : 1.

Table 3. Screening of consortia for the degradation and decolouriza-tion of pulp paper mill effluent

Consortium

Percentagecolour

reductionPercentage

BOD reduction

PercentageCOD

Reduction Growth

1 : 1 : 1 26.60 35.00 28.02 +1 : 1 : 4 (−)21.00 28.00 22.00 ++1 : 4 : 1 55.00 56.60 35.22 +++4 : 1 : 1 65.06 64.00 63.42 +++1 : 1 : 2 (−)18.40 25.00 20.44 ++2 : 1 : 1 22.90 24.80 18.26 ++1 : 2 : 1 20.80 26.00 24.42 ++(−) increase colour; (+) slow growth; (++) moderate growth; (+++)good growth.

effective in reducing BOD, COD, colour, TDS and total phenols byup to 64.0, 63.37, 65.06, 63.69 and 63%, respectively (Table 1 andFig 1(B)).

Bioassay of ligninolytic enzymesTo determine the role of extracellular enzymes inr the degradationof PPME, enzymes were measured and LiP and MnP werefound to be the dominating enzymes at the initial stage ofdegradation. Enzyme induction was greater in the ratio 4 : 1 : 1than at 1 : 4 : 1. Maximum LiP activity was recorded at day 4, and


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Figure 2. Enzyme assay of lignin peroxidase, manganese peroxidase andlaccase during the degradation of pulp paper mill effluent by two inocula,ratio 1 : 4 : 1 and 4 : 1 : 1.

was 0.178 U mL−1 min−1, MnP activity was maximum at day 3(0.247 U mL−1 min−1), and laccase activity was maximum (0.241 UmL−1 min−1) at day 4 of incubation (Fig. 2). No enzyme activity wasdetected in the control. LiP and MnP have a broad substrate rangereported for polymer degradation. These enzymes were reportedto degrade phenolic and nonphenolic polymeric compounds.However, the presence of enzymes in medium depends on theconstituents of the culture medium, availability of substrate andpotentiality of organisms to secrete a particular enzyme. Ligninis a polymeric compound which is abundantly present in PPME.Degradation of the compound and induction of the enzyme is asimultaneous process which supports each other. Measuring theenzymes shows that the loss of polymeric lignin can be attributedto the action of LiP and MnP. The production of extracellularenzyme requires H2O2, which is produced by glucose oxidation,present abundantly at the initial phase of bacterial growth29,30

and laccase was reported in later stages of degradation. Similarobservations were made by Arora et al.21 in the fungal system

(A)

(B)

Figure 3. HPLC analysis of (A) lignin and (B) chlorophenol degraded by mixed culture inoculum ratio 1 : 4 : 1 and 4 : 1 : 1, with respect to control.


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(A)

(B)

(C)

Figure 4. GC-MS analysis of bacterial degraded and undegraded pulp paper mill effluent samples extracted in ethyl acetate (lignin and relatedcompounds) from (A) control, (B) sample degraded by ratio 1 : 4 : 1 culture, (C) sample degraded by 4 : 1 : 1 culture.

Table 4. GC-MS analyzed compounds extracted by ethyl acetate from control and bacterial degraded PPME

Serial No. Compound RT C 1 : 4 : 1 4 : 1 : 1

1 2-Ethoxyethoxy-Trimethylsilane 10.439 − +2 Propylene carbonate 10.449 − + −3 Butanoic acid,2-oxo (acid) 11.190 − + −4 Methanediamine,N,N,N,N-tetramethyl 11.280 − − +5 2-Ethoxyethoxy-trimethylsilane 12.460 − + −6 Butane,2Ethoxy- 16.057 − − +7 Diphenylthiocarbazide 16.617 − − +8 1-(2,4-Diethoxy-Phenyl)Ethanone 17.112 − − +9 1,4-Dimethoxy-2-Phenylbutane(phenol) 19.013 − − +10 Oxalic acid,Cyclobutyl heptadecylester (cyclo) 19.283 − +11 8-Pentadecanone(ketone) 20.953 + −12 1,2Benzenedicarboxylic acid,Bis(2-Methylpropyl) Ester 21.824 + − −13 1-Phenyl-1-nonyne(surfactant) 21.999 − + −14 Sulphurousacid,Octadecyl 2-Propylester 22.044 + −15 Benzene,1,3-Bis(1-methylethenyl) 22.489 − +16 3-Ethenyl-6-Dimethylaminomethyleneaminobenzonitrile 22.509 − − +17 N-(3-Bromo-1-Methyloxycarbonyl-1H-Indol-2-YLmethyl)-N-(1-Methoxycarbonyl-2-methybutyl 23.249 − − +18 Proponoic acid,2-(Benzoylamino)-333 Trifluro-2-[(Trifluromethyl)Phenyl]Amino-Ethyl 23.364 − − +19 Butane,2-phenyl-3-(trimethylsilyloxy) 23.925 − +20 2-Propanoic acid,3(4-Methylphenyl)-, ethylester 24.140 − + −21 2-Propanoic acid,3- (MethylPhenyl), Ethylester 24.245 − − +22 Phthalicacid,Dodecyl 2-Ethylhexylester 26.010 − − +(+) present; (−) absent; (C) control; (D) degraded; (RT) retention time in minutes.


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Table 5. GC-MS analyzed compounds extracted by dichloromethane (DCM) from degraded and control mixed pulp paper mill effluent

Serial No. Compound RT C 1 : 4 : 1 4 : 1 : 1

1. 3- Trifluroacetoxydodecane 14.40 + − −2. 3,5-Bis (1,1-dimethylethyl)phenol 15.87 + − −3. 4-trifluroacetoxypentadecane 16.65 + − −4. N-Capric acid isopropyl ester 17.00 − + −5. 2-Methyl-dodecane 17.40 − − +6. N-Tridecan-1-ol (alkanols) 17.78 − + −7. 4,6,8-Trimethyl-1-azulenecarbaldehyde 18.10 + − −8. 5-amino2,2,4-trimethyl-cyclopentanemethanamine 18.96 − − +9. 17-pentatriacontene (alkene) 18.97 + + −10. Z-10 pentadecen-1-ol (alkenols) 19.34 + − −11. Octadecanal (aldehyde) 20.69 + − −12. N-Hexadecanoic acid(fatty acid) 21.32 + −13. Cis-1-chloro-9-octadecene (alkene) 22.79 + −14. 11-octadecanoicacid,methylester(fatty acid) 23.22 − + +15. E-11-hexadecenal (alkenals) 23.88 + − −16. Oleic acid (fatty acid) 24.23 + − −17. P,P′-DDE 24.93 + − −18. 2,4-DI-tert-butyl-6-(tert-butylamino)phenol 25.31 + − −19. 3,5-Bis(1,1-dimethyl)-phenol 28.12 + − −20. 1-Eicosanol(alkane) 28.62 + − −21. 1,2-benzenedicarboxylic acid,ditridecylester 32.65 + − −(+) present; (−) absent; (C) control; (D) degraded; (RT) retention time in minutes.

(A)

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Figure 5. GC-MS analysis of bacterial degraded and undegraded pulp paper mill effluent samples (chlorophenols and other pollutants) from (A) control,(B) sample degraded by ratio 1 : 4 : 1 culture, (C) sample degraded by ratio 4 : 1 : 1 culture.


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Table 6. Effect of bacterial treated and untreated PPME on seed germination of P. mungo L

Germination (%) Speed of germination index

Serial No. Conc. (%) C 4 : 1 : 1 1 : 4 : 1 C 4 : 1 : 1 1 : 4 : 1

1. 10 100.60 100.00 80.00 234.00 ± 7.12 236.00 ± 5.32 205.00 ± 3.33

2. 25 80.00 100.00 60.00 201.00 ± 3.65 212.00 ± 3.23 154.00 ± 2.34

3. 50 53.33 100.00 33.30 187.00 ± 2.45 214.00 ± 3.12 120.00 ± 4.23

4. 75 33.33 80.00 20.00 56.00 ± 1.20 180.00 ± 5.55 90.00 ± 1.00

5. 100 13.00 66.60 16.00 3.21 ± 0.00 175.00 ± 4.25 40.00 ± 0.80

(A)

(B)

Figure 6. (A) Effect of different concentration of untreated and bacterialtreated effluent on α-amylase enzyme. (B) SDS PAGE of purified α-amylaseenzyme.

Phlebia spp.21 Generally LiP and MnP are secreted at acidic pH, butisolated bacterial stains have the capability to tolerate a broad pHrange and continue to release enzyme.31

Metabolites characterizationUndegraded and bacterial degraded samples were extractedwith ethyl acetate for acid soluble lignin fragments and otherrelated phenolic compounds, and with dichloromethane forchlorophenols. The HPLC analysis of both types of samplesshowed marked reduction in the peak area. The comparativereduction in peak area revealed biotransformation as well asbiodegradation of lignin, chlorophenols and related compounds(Fig. 3(A) and (B)). The effluent degraded by mixed culturesshowed 94.69% reduction of lignin contents using 4 : 1 : 1 ratioand 84% using 1 : 4 : 1 ratio, and chlorophenols reduced upto 97.31% and 58%, respectively at day 7. This study clearlyindicates the effect of ratio on the degradation of pulp paper millpollutants due to enzyme activity, and the metabolites formedafter degradation by each strain. The metabolites generatedafter degradation can enhance or suppress enzyme activityas well as degradation activity as already mentioned. A fewratios increase the colour of effluent whereas in ratios 4 : 1 : 1and 1 : 4 : 1 colour is decreased and different metabolites weregenerated.

GC-MS analysis of the ethyl acetate extracted sample showedthat pollutants present in untreated samples were plant products,which might be transformed during the pulp processing toform more toxic substances, as shown in Fig. 4(A) and Table 4.The major compound detected in the control sample was 1, 2-benzenecarboxalic acid (RT 21.82); other smaller peaks detected

were 8-pentadecanone (RT 20.95) and sulphurousacid, octadecyl-2-propylester (RT 22.044). PPME degraded by inoculum ratio 1 : 4 : 1showed the formation of butanoic acid (RT 11.19) and 1-phenylnonyne (RT 22.00). The 1-phenyl nonyne was recorded as degradedproduct of surfactant (Fig. 4(B)). PPME degraded by the 4 : 1 : 1inoculum sample showed the presence of butane (RT 16.05),1,4-dimethoxy-2-phenylbutane (RT 19.03), and propionic acid (RT23.364) as major compounds (Fig. 4(C)).

Compounds extracted in dichloromethane from controlsamples belonged to different groups. The detected com-pounds were long chain alkanes, alkenes, alkanols, alkenalsand their chlorinated derivatives, detailed in Table 5 as SerialNos.(1,3,6,9,10,11,13,15,20), fatty acids (12,14,16) and phenolicgroups (2,18,19) (Fig. 5(A) and Table 5). After degradation byculture ratio 1 : 4 : 1, compounds generated were N-capric acidisopropyl ester (RT 17.00), N-tridecan-1-ol (RT 17.78) and pen-tatriacontene (RT 18.97) as metabolic products (Fig. 5(B)). Ratio4 : 1 : 1 cultures resulted in the generation of major metabolites 11-octadecanoicacid (RT 23.82) (Fig. 5(C)). 11-octadecanoicacid (RT23.82) was detected in both samples.

The possible number of compounds present in rayon gradePPME, high molecular weight compounds, di, tri terpenoids,surfactants, coagulants, resin acids, aliphatic acids which areknown toxicants.32,33 The GC-MS data showed degradation ofcomplex toxic compounds and generation of low molecular weightsimpler compounds. Larger phenolic polymers cleaved to formphenyl units, which further reduced to form aliphatic compoundsby ring cleavage, which resulted in the decolourization of PPME.

Toxicity assessment by seed germination testThe toxicity assessment was performed on P. mungo L. Toxicityassay of bacterial treated and untreated PPME showed an effecton the germination of P. mungo L. and on α-amylase activity.In the seed germination test α-amylase is an important enzymeand generally initializes the germination process by mobilizationof stored food material.34 The effect of degraded and non-degraded PPME on seed germination is shown in Table 6. Thetable clearly indicates that 100% effluent is not suitable for seedgermination as it showed only 13% seed germination and 3.21speed of germination index. After treatment with ratio 4 : 1 : 1 and1 : 4 : 1 cultures, toxicity decreased by up to 80.40 and 18.75%,respectively. In 10% untreated and treated effluent with ratio4 : 1 : 1 culture, 100% germination was observed, and ratio1 : 4 : 1showed 80% germination.

The toxicity assessment on α-amylase enzyme revealed thatseeds treated with 10% (v/v) untreated effluent showed 1.04 Ug−1 enzyme activity, which indicated that 10% concentration wasnon-toxic, showing better activity than the control, (tapwater)which was 1.0 U g−1. However, both degraded samples showed


44

4


enhanced amylase activity at 10% concentration, 1.22 and1.16 U g−1, respectively, at ratio 4 : 1 : 1 and 1 : 4 : 1. 100% (v/v)concentration showed reduction in toxicity and enhancementin α-amylase (Fig. 6(A)). The purification and characterization ofα-amylase enzyme confirmed the molecular weight, i.e. 46 KDaand the band intensity pattern also indicated the induction andinhibition of seed germination at different concentrations. 100%untreated effluent does not show any induction of enzyme andtherefore no bands were detected, whereas in degraded samplesbands appear at low intensity (Fig. 6(B)), revealing that effluentwas detoxified after bacterial treatment.

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Effect of bacterial inoculum ratio in mixed culture for decolourization and detoxification of pulp paper mill effluent