Bioresource Technology 102 (2011) 6429–6436

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Bioresource Technology

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Bacterial decolorization and detoxification of black liquor from rayon gradepulp manufacturing paper industry and detection of their metabolic products

Ram Chandra ⇑, Amar Abhishek, Monica SankhwarEnvironmental Microbiology Section, Indian Institute of Toxicology Research (CSIR), Post Box 80, M.G. Marg, Lucknow 226001, Uttar Pradesh, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 January 2011Received in revised form 16 March 2011Accepted 16 March 2011Available online 22 March 2011

Keywords:Black liquorDecolorizationLigninolytic enzymeBacteriaGC–MS

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.03.048

⇑ Corresponding author. Tel.: +91 522 2968915/2472228227/2228471.

E-mail addresses: ramchandra_env@indiatimes.co(R. Chandra).

This study deals with the decolorization of black liquor (BL) by isolated potential bacterial consortiumcomprising Serratia marcescens (GU193982), Citrobacter sp. (HQ873619) and Klebsiella pneumoniae(GU193983). The decolorization of BL was studied by using the different nutritional as well as environ-mental parameters. In this study, result revealed that the ligninolytic activities were found to be growthassociated and the developed bacterial consortium was efficient for the reduction of COD, BOD and colorup to 83%, 74% and 85%, respectively. The HPLC analysis of degraded samples of BL has shown the reduc-tion in peak area compared to control. Further, the GC–MS analysis showed that, most of the compoundsdetected in control were diminished after bacterial treatment while, formic acid hydrazide, 4-cyclohex-ane-1,2-dicarboxylic acid, carbamic acid, 1,2-benzenedicarboxylic acid and erythropentanoic acid werefound as new metabolites. Further, the seed germination test using Phaseolus aureus has supported thedetoxification of bacterial decolorized BL.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

In pulp paper industry, manufacturing of pulp from wood chipscalled pulping process where, wood chips are cooked in the solu-tion of sodium hydroxide and sodium sulfite at elevated tempera-ture and pressure to break into fiber mass. In order to manufacturerayon grade pulp (RGP), only high quality fiber containing woodchips are used with an extra chemical process that involves prehy-drolysis of wood chips at elevated temperature and pressure fol-lowed by alkaline digestion. This process ensures the removal ofhemicellulose and remaining fibers with high cellulose contentaround 92%. Thus, the effluent generated from pulping stagemainly contains lignins, hemi-cellulose, phenolics, resins, fattyacids and tannins which mixed together and make dark black alka-line wastewater known as black liquor (Zaied and Bellakhal, 2009).Black liquor (BL) contains about 40–48% lignin, which is a mixtureof polypenolic compounds having a complex chemical structureconsisting only 10–15% of total wastewater, but contributes almostabove 90–95% of the total pollution load of pulp paper millwastewater, which make it significantly toxic to the environment(Pokhrel and Viraraghavan, 2004).

In many developing countries including India, farmers areirrigating their crop plants with industrial effluents having high

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m, rc_microitrc@yahoo.co.in

level of several toxic compounds due to the non-availability ofalternative sources of irrigation. Hence, the adequate treatmentof BL prior to its final discharge into the environment is necessary.Though, recent developments in physical and chemical methods(i.e. electrocoagulation, ozonation, ultrafiltration) or combinationof different methods in series for the treatment of lignin containingwastewater has shown some encouraging results (Pokhrel andViraraghavan, 2004). To date the most effective method for themanagement of BL is the alkali recovery process in which BL is firstconcentrated and then incinerated. In this way organic substancesin wastewater is burn and alkali can be recovered (Yang et al.,2008). However, medium and small scale paper mills are graduallyclosing due to their incapability of alkali and energy recovery fromblack liquor. Acid precipitation is another alternative for ligninremoval, which facilitates the secondary treatment greatly. Largeconsumption of mineral acids and secondary pollution of chlorineand sulfur make the technology unsatisfactory. Therefore, morepractical method is needed for the treatment process for smalland medium scale industries.

Biological treatment is another practical choice in BL treatment.Among biological methods tried so far, most of the literatureconfined to a few genera of white rot fungi because of their nonspecific extracellular enzymatic system (LiP, MnP and Laccase).But, bacteria seem to be more effective than fungi for the bioreme-diation of environmental pollutants due to their immense environ-mental adaptability and biochemical versatility. Bacteria isolatedfrom compost soil, viz. Azotobacter and Serratia marcescens, werecapable of degradation and decolorization of lignin (Morii et al.,

6430 R. Chandra et al. / Bioresource Technology 102 (2011) 6429–6436

1995). Bacteria such as Bacillus subtilis and Bacillus sp. have alsobeen tested for kraft-lignin degradation (El-Hanafy et al., 2008;Abd-Elsalam and El-Hanafy, 2009). Previously, three potential bac-terial strains of Panibacillus sp. Aneurinibacillus aneurinilyticus andBacillus sp. were isolated from pulp paper sludge for degradationand decolorization of synthetic lignin at 500 mg l�1 and character-ized their metabolic products by GC–MS analysis (Chandra et al.,2007; Raj et al., 2007). However, all the above studies have beencarried out on synthetic/model compounds with very low pollutionload (COD less than 8000 mg l�1) which do not directly explain thedegradation of lignin present in pulp wastewater. However, the lit-erature on bacterial degradation of BL discharged from RGP manu-facturing industry is lacking at high pollution load. Hence, thepresent investigation has been focused on the decolorization ofBL at high pollution load discharged from RGP manufacturingindustry which will be useful for the management of lignin con-taining pulp paper mill wastewater.

2. Methods

2.1. Sample collection and isolation of bacteria

The RGP black liquor used in biodegradation study was col-lected from wash machine section of M/s. Century Pulp Paper Mill,Lalkuan, Nainital, Uttrakhand, India located (79�100E longitude and29�30N latitude) at foot hills of Himalayas. The industry produces90–100 ton per day of rayon grade pulp and discharges approxi-mately 100 m3 per day of wastewater. For the isolation of potentiallignin degrading bacterial strains, sludge samples were collectedfrom the disposal site of M/s Century Pulp Paper Mill containingdecomposed wood. The potential bacteria were isolated fromsludge sample by serial dilution method. Further, the morphologi-cally distinct bacteria were purified by plate streak method on lig-nin amended MSM (Mineral salt media) agar plates containing(g l�l) lignin, 0.2; D-glucose, 10; peptone, 5; Na2HPO4, 2.4;K2HPO4, 2.0; NH4NO3, 0.1; MgSO4, 0.01; CaCl2, 0.01 as describedpreviously (Chandra et al., 2007).

2.2. Screening, construction of consortium and identification ofpotential bacterial strains

Ten purified isolated bacterial strains designated as IITRBL1,IITRBL2, IITRBL3, IITRBL4, IITRBL5, IITRBL6, IITRBL7, IITRBL8,IITRBL9 and IITRBL10 were screened on the basis of their COD, col-or and lignin reduction potential. MSM broth having black liquorlignin (1000 mg l�1) was inoculated with individual isolates andincubated at 30 ± 1 �C, in shaking condition (120 rpm) for 192 h.Three potential bacterial strains (IITRBL1, IITRBL3 and IITRBL4)showing rapid growth and reduction of pollution parameters(COD, color and lignin) were selected for further study. For the con-struction of effective bacterial consortium, the screened potentialstrains were inoculated and tested in different combinations i.e.IITRBL1 + IITRBL4; IITRBL1 + IITRBL3; IITRBL3 + IITRBL4 and IITR-BL1 + IITRBL4 + IITRBL3. Further, the identification of strains wasestablished by 16S rRNA gene sequence analysis.

For 16S rRNA gene sequence analysis, alkaline lysis method wasused for the total DNA preparation from overnight grown pure cul-tures. The 16S rRNA gene was amplified using universal eubacterialprimers (Narde et al., 2004). The PCR reactions were performed un-der the following conditions: 30 cycles of denaturation at 94 �C(1 min), annealing at 55 �C (1.5 min), and a final extension at72 �C (1 min). A 1466 bp product was amplified using forward pri-mer 5-AGAGTTTGATCCTGGCTCAG-30 and reverse primer 50-TAC-GGTTACCTTGTTACGACT T-30. The nucleotide sequences obtainedwere subjected to BLAST search using the online option available

at www.ncbi.nlm.nih.gov/BLAST suggesting the identity of isolatedbacterial strains (Chandra et al., 2007).

2.3. Optimization of nutritional and environmental parameters

The influence of co-substrates on decolorization and degrada-tion of BL was optimized by supplementing the MSM broth withdifferent carbon (glucose, galactose, sucrose and fructose) andnitrogen (beef extract, peptone, sodium nitrate, and ammonium ni-trate) sources at 1.0% and 0.5% (w/v), respectively. In addition, theeffect of environmental parameters including inoculum ratio, inoc-ulum size (0.5–8%), shaking speed (100–200 rpm), temperature(30–35 �C) and initial pH (5–10) were examined to achieve theoptimum decolorization of black liquor.

2.4. Bacterial decolorization of BL

2.4.1. Bacterial growth pattern, pH change and enzyme activityFor degradation studies, 10% (v/v) black liquor having COD:

18,700 mg l�1, initial color: 6100 Co.pt color unit and lignin1000 mg l�1 supplemented with 1% carbon and 0.5% nitrogensource was used throughout the experiment. The batch experi-ments were performed at optimized conditions in 250 ml flaskscontaining 100 ml BL. During the decolorization period, sampleswere taken at every 24 h interval and analyzed for bacterialgrowth, pH and enzyme activity. Growth in terms of colony form-ing unit (cfu ml�1) was performed by spreading sample dilutionson the surface of nutrient agar plates (APHA, 2005). Change inpH of the medium was measured with the pH selective electrodeof Thermo Orion pH meter (Model 960, USA). Enzyme activities(Mn-peroxidase and laccase) were also determined as describedby Miyata et al. (2000).

2.4.2. Reduction in pollution parametersReduction in color (Cobalt–platinate method), COD (open reflux

method), BOD (5 day method), and other physico-chemical param-eters in control and degraded samples were accomplished as de-scribed in standard methods for the examination of water andwastewaters (APHA, 2005). For the measurement of residual lignin,samples were centrifuged at 8000g for 30 min. Supernatant (1 ml)was diluted by adding 3 ml of phosphate buffer (pH 7.6) and absor-bance was measured at 280 nm with UV–visible spectrophotome-ter (GBC Cintra-40, Australia).

2.4.3. Adsorption evaluation during BL decolorization processTo confirm whether the decolorization process was biological or

adsorption phenomenon, NaOH extraction method was adopted(Jiranuntipon et al., 2008). The living and heat killed bacterial con-sortium pellets were resuspended in equal volume of NaOH (0.1 M)to extract color substances adsorbed to the cell surface. The ex-tracts were centrifuged and absorbance was measured at 465 nm.

2.5. Metabolite characterization

2.5.1. TLC and HPLCTo assess the degradation and decolorization of complex and

colored BL, thin layer chromatography (TLC) and HPLC analysiswere done. For the extraction of lignin, 30 ml of bacterial treatedand untreated BL were centrifuged at 5000 rpm for 20 min (SigmaModel-3k30, Germany). Supernatant was acidified (pH 1–2) byusing 0.1 N HCl and extracted thrice with equal volume of ethylacetate. The organic layer was collected, pooled and dewateredover anhydrous Na2SO4. Further, the residues were dried under astream of nitrogen gas and dissolved in acetone for TLC andacetonitrile for HPLC analysis. For TLC analysis, the acetone solublesamples were spotted on pre-coated silica gel TLC plates (Merck,

Table 1Physico-chemical characteristics of RGP black liquor.

Parameters RGP black liquor

pH 9.0 ± 0.2Color intensity (Co.pt) 6100 ± 3.5COD 18,700 ± 440BOD 7360 ± 153TDS 1402 ± 1.5Total phenol 38.5 ± 2.8Lignin 1000 ± 1.1Sulfate 1800 ± 14Phosphate BDLK+ 12.2 ± 1.33Na+ 102 ± 11

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Germany). The solvent system containing toluene:methanol:aceticacid (90:16:24, v/v) were used for the separation of metabolicproduct along with control (Godden et al., 1992). The TLC plateswere observed under ultra-violet (UV) light of gel documentationsystem (Syngene, UK). Further, to confirm the decolorization ofBL the samples were analyzed through HPLC system (Waters,515 USA), equipped with 2487 UV/VIS detector, via millenniumsoftware. Samples (20 ll) were injected followed by implementa-tion of HPLC grade acetonitrile/water (70:30 v/v) at flow rate of1 ml min�1. Reverse phase column C-18 (250 mm � 4.6, particlesize 5 lm) at 27 �C was used to analyze lignin at 280 nm (Chandraet al., 2007).

Nitrate 3 ± 4.5

Heavy metalsCd BDLCr BDLCu 0.105 ± 0.013Fe 3.990 ± 0.47Ni 2.840 ± 0.38Zn 1.500 ± 0.17

All the values are in mg l�1 except color and pH; BDL:below detection limit.

Table 2Screening of isolated bacteria for the decolorization of black liquor.

S. No. Strain name % Reduction

COD Color Lignin

1 IITRBL1 51 ± 2.16 44 ± 3.40 36 ± 3.142 IITRBL2 12 ± 1.38 16 ± 2.12 7 ± 2.473 IITRBL3 32 ± 1.86 43 ± 2.37 42 ± 2.644 IITRBL4 36 ± 2.48 41 ± 3.41 39 ± 2.105 IITRBL5 17 ± 1.72 22 ± 3.47 8 ± 2.336 IITRBL6 7 ± 2.34 11 ± 2.30 3 ± 3.167 IITRBL7 28 ± 2.47 29 ± 2.03 13 ± 3.128 IITRBL8 22 ± 3.47 26 ± 2.72 11 ± 3.119 IITRBL 9 6 ± 2.47 12 ± 1.07 3 ± 2.1710 IITRBL10 27 ± 2.13 32 ± 1.99 14 ± 1.83

2.5.2. GC–MS analysisFor the characterization of metabolic products of BL, alkaline

hydrolysis method was used with slight modification in previouslydescribed method by Scalbert et al. (1985). In brief, 100 ml of bac-terial treated and untreated BL were centrifuged at 5000 rpm for20 min followed by addition of 10 ml NaOH (2 M) and left for48 h at 25 �C. Samples were acidified and extracted as mentionedin previous section. The extracted organic layer was concentratedwith liquid nitrogen before derivatization with trimethyl silyl[BSTFA (N,O-bis (trimethylsilyl) trifluoroacetamide) TMCS]. An ali-quot of 1 ll of silylated compounds was injected into the GC–MSequipped with a PE Auto system XL gas chromatograph interfacedwith a Turbomass Mass spectrometric mass selective detector. Theanalytical column connected to the system was a PE-5MS capillarycolumn (20 m � 0.18 mm i.d., 0.18 lm film thickness). Helium gaswith flow rate of 1 ml min�1 was used as carrier gas. The columntemperature was programmed as 50 �C (5 min); 50–300 �C(10 �C min�1, hold time: 5 min) as described earlier (Raj et al.,2007). The metabolic products were identified by comparing theirmass spectra with that of National Institute of Standards and Tech-nology (NIST) library available with instrument and by comparingthe retention time with those of available authentic organiccompounds.

2.6. Environmental toxicity evaluation of BL by seed germination test

Bioassay of acute toxicity test was performed to evaluate thedetoxification of BL using seed germination method of Phaseolusaureus (Wang, 2003). Toxicity was expressed in terms of inhibitionof their amylolytic activity. For evaluation of toxicity samples weretaken at the beginning and end of decolorization process of con-centrations i.e. 10, 20, 40, 60, 80 and 100% (v/v) with tap water(Bharagava and Chandra, 2010).

3. Result and discussion

3.1. Characteristics of RGP-BL and screening of potential bacterialstrains

Black liquor sample (10%) used for the bacterial decolorizationstudy showed following characteristics i.e. pH 9.0, color 6100 Co.pt,lignin 1000 mg l�1, BOD 7360 mg l�1, COD 18,700 mg l�1. Phos-phate, Cr, and Cd were noted below the detection limit (Table 1).The high COD/BOD ratio (approximately 2.8) in BL is due to pres-ence of high molecular weight compounds, i.e. lignin and its deriv-atives which contribute high COD and color instead of BOD(Esposito et al., 1991). The source of sulfate ions in effluent mightbe sodium sulfite, which is used in pulping process and the nitratesdetected in BL indicated the presence of nitrogen in lignin (Singhaland Thakur, 2009). The metals tested in this study were below thedetection limit except Fe, Ni and Zn which might be added in BL

from corrosion of digestion vessels and possibly due to bioaccumu-lation of these metals by plants which are used as raw material.

Out of ten bacterial isolates, the most efficient bacterial strainswere IITRBL1, IITRBL3 and IITRBL4 which could reduce 36%, 42%,39% lignin and 44%, 43%, 41% color and 51%, 32%, 36% COD, respec-tively. The screening results revealed different ability of individualisolates for black liquor treatment, where IITRBL3 and IITRBL4showed higher abilities for lignin and color removal, while a redpigment producing gram negative bacterium designated as IITRBL1had showed higher abilities for COD removal (Table 2). Therefore,these three isolates were selected for construction of consortium.The consortium (1%) comprising IITRBL1 (31 � 104 cfu ml�1),IITRBL3 (30 � 104 cfu ml�1) and IITRBL4 (29 � 104 cfu ml�1) werefound most efficient over the tested combination (data not shown).The data obtained from consortium showed the presence of eachbacterial strain in culture medium has cumulative enhancing effectfor growth and lignin degradation rather than inhibition. There-fore, this consortium was used in optimization and degradationexperiment.

Further, the screened bacterial strains IITRBL1, IITRBL3 andIITRBL4 were identified as Serratia marcescens (GU193982), Klebsi-ella pneumoniae (GU193983) and Citrobacter sp. (HQ873619),respectively on the basis of 16S rRNA gene sequence analysis.

3.2. Optimization of nutritional and environmental parameters

Out of various carbon and nitrogen sources tested, glucose(1.0%) and peptone (0.5%) was found the most adequate carbon

6432 R. Chandra et al. / Bioresource Technology 102 (2011) 6429–6436

and nitrogen source as it showed highest reduction of color (67%)and lignin (53%) within 192 h incubation period. However, sucroseand galactose were also found to be fairly good carbon sourceallowing up to 52–54% color and 45–46% lignin reduction.Whereas, fructose was relatively poorer carbon source allowing51% color and 42% lignin degradation only. Hence, the order ofeffective carbon sources were as glucose > galactose > sucrose >fructose. Similarly, the order of effective nitrogen sources amongtested were as peptone > beef extract > sodium nitrate. While, neg-ative contribution for color reduction was showed by ammoniumnitrate (Fig. 1A). Thus, our results revealed that organic nitrogensources favor the decolorization of BL and bacterial growth com-pared to inorganic nitrogen sources. Similar to this observation,negative effect of inorganic nitrogen source on effluent treatmenthave been also previously reported by Zhang et al. (1999). The bac-terial decolorization pulp paper effluent has been also reported byvarious authors (Morii et al., 1995; Jain et al., 1997; Gupta et al.,2001; Yang et al., 2008) but only few studies have optimized theenvironmental and nutritional parameters.

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Fig. 1. Effect of different nutritional and environmental factors during optimization ofinoculum ratio of IITRBL1, IITEBL3 and IITRBL4 (% v/v); (C) inoculum size (%); (D) shakin

Besides nutritional factors, the ratio of individual strains in con-sortium also influenced the degradation and decolorization of BL.The ratio of IITRBL1:IITRBL3:IITRBL4 of 1:1:2 in consortiumshowed maximum color and lignin reduction in comparison toother combination tested as shown in Fig. 1B. The bacterial strainIITRBL4 has showed slow growth potential compared to IITRBL1and IITRBL3 hence, to obtain an effective balance composition ofstrains for optimum decolorization of BL; inoculum size of IITRBL4was doubled in consortium. This observation was noted as newinformation regarding the effect of inoculum ratio for BL decolor-ization study which indicated the competitive behavior of differentbacterial strains in environment. Similarly, the inoculum size ofconsortium also influenced the BL decolorization. The optimumreduction of color and lignin was noted at 4% inoculum size ofconsortium and further increase in inoculum size up to 6% do notaffect the decolorization process. However, further increase ininoculum size negatively affect the decolorization process possiblydue to the depletion of oxygen and nutrient in culture media(Rahman et al., 2005) (Fig. 1C). In addition, pH of medium and

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agitation rate also influenced the bacterial growth and decoloriza-tion process. Maximum reduction in color and lignin of BL wasnoted at 180 rpm and pH 8 (Fig. 1D and E). This indicated thatthe bacteria growing in BL required high dissolve oxygen in alka-line condition because high pH favors the solubility of lignin andits derivatives. The ambient temperature (35 ± 1 �C) was also notedfor optimum decolorization of BL (data not shown).

3.3. BL decolorization studies at optimized conditions

The developed bacterial consortium consisting S. marcescens(IITRBL1), Klebsiella pneumoniae (IITRBL3) and Citrobacter sp.(IITRBL4) was capable for 74% reduction of color of BL at optimizedconditions (i.e. glucose: 1%; peptone: 0.5%; inoculum size: 4%; agi-tation rate 180 rpm and pH 8). The consistent growth was ob-served among all the three bacterial strains in constructedconsortium up to 72 h. However, the cfu count of IITRBL3 wasdominated over IITRBL4 followed by IITRBL1. Further, results re-vealed a direct co-relation of enzyme activities with bacterialgrowth, where the MnP activity was noted maximum at 72 h whenIITRBL3 and IITRBL4 were growing dominantly and highest laccase

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activity were found between 122 and 144 h of incubation periodfor strain IITRBL1 (Fig. 2A). The production of laccase and peroxi-dase activity showed direct correlation with lignin degradation.Similar observation for S. marcescens with laccase also has beenpreviously reported by Perestelo et al., 1994). Lignin induced lac-case (EC 1.10.3.2) and peroxidase (MnP, EC 1.11.1.13), were identi-fied and characterized in unicellular bacteria (Perestelo et al.,1994; Singh et al., 2007; Dick et al., 2008; Oliveira et al., 2009).Studies were reported for Klebsiella and Citrobacter sp. for the deg-radation of color containing compounds and toxic pollutants(Jiranuntipon et al., 2008; Wang et al., 2009) whereas, by the timeno reports are available regarding the ligninolytic enzyme by thesebacteria and their role in BL detoxification. During degradation theshift in pH towards acidic condition was noted within initial 48 hof bacterial growth which indicated the formation of acidic com-pounds through tricarboxylic acid (TCA) cycle, utilizing the simplerform of carbon sources present in medium by consortium (Yanget al., 2008). As the supplementary nutritional source depleted,the pH of medium shifted towards higher facilitating the lignindegradation, as lignins are uniformly soluble at high pH that iswhy the more reduction in COD, BOD and color were noted after72 h (Fig. 2B).

Low decolorization at initial phase in spite of fast bacterialgrowth might be due to the utilization of simpler form of carbonand nitrogen source available in growth media. But, subsequentstarvation of easily available nutritional source in media insistedthe bacterial consortium for utilization of BL lignin as a co-sub-strate. Similar to this study, co-metabolism mechanism in fungifor lignin containing effluent degradation has also been reportedby various authors (Zhang et al., 1999; Singhal and Thakur,2009). Additionally, to confirm that lignin degradation is a biolog-ical phenomenon not adsorption, NaOH extraction method wasadopted. It showed that the fraction of NaOH extractable color sub-stances were negligible. Hence, the results clearly indicated thatthe degradation process is biological phenomenon (Fig. 2C).

3.4. Metabolite characterization

A comparative fragmentation pattern of extracted BL is ob-served in control and bacterial degraded samples on silica coatedTLC plates. TLC analysis showed that the polymer compounds(i.e. lignin) present in BL were broken into smaller fractions duringbacterial degradation process (Fig. 3A). The degradation was fur-ther conformed by HPLC analysis which showed the reduction aswell as shifting of peaks compared to controls. This indicated the

Fig. 3. Comparative TLC (A) and HPLC (B) chromatograph of control (c) anddegraded (d) samples of black liquor.

Fig. 4. Total ion chromatogram (TIC) of compounds from black liquor control (A) and degraded sample (B) after alkaline hydrolysis.

6434 R. Chandra et al. / Bioresource Technology 102 (2011) 6429–6436

degradation with miner biotransformation (Fig. 3B). Decrease incolor intensity by bacterial culture clearly revealed the depolymer-ization of lignin by bacterial ligninolytic action (Lara et al., 2003;Chandra et al., 2007).

To confirm the degradation and identification of metabolicproducts, control and degraded samples of BL were analyzed byGC–MS. The GC–MS analysis of control sample has shown majorpeaks between 10 and 28 retention time (RT-min) (Fig. 4), detailof compounds generated from lignin and other phenolic com-pounds due to the degradation process is listed in Table 3. The re-sults showed a considerable qualitative as well as quantitativedifference in the pattern of compounds obtained after lignin degra-dation by bacterial consortium compared to control sample. Thetotal ion chromatogram (TIC) of degraded sample showed the mas-sive consumption of compounds as compared with its control indi-cated that the consortium has strong ability to utilize itsconstituents as sole source of carbon, nitrogen and energy. Manyaromatic compounds were detected in control sample of BL, suchas 2-methoxy phenol (Guaiacol) (RT 12.99 min), benzoic acid (RT13.8 min), benzene acetic acid (RT 13.9 min), (bis (2-ethylhexyl)phthalate) (RT 28.1 min) as low-molecular-weight derivatives ofphenolic units of lignin (Raj et al., 2007; Yang et al., 2008; Koet al., 2009). The phthalate derivative (bis (2-ethylhexyl) phthal-ate) had been also detected from photodegradation of black liquorlignin (Ksibi et al., 2003) as well as fungal peroxidase degradationof lignosulfonate (Shin and Lee, 1999). Apart from this finding,

other workers have also reported more acid-type compounds thanaldehyde and ketone-type due to degradation of lignin (Hernandezet al., 2001; Gupta et al., 2001). Moreover, formic acid hydrazide, 4-cyclohexane-1,2-dicarboxylic acid, carbamic acid, 1,2-benzenedi-carboxylic acid and erythropentanoic acid were produced as newmetabolites. While on the other hand, compounds such as choles-terol trimethylsilyl ether (RT 26.1 min) remain unchanged. Choles-terol trimethylsilyl ether is the derivatizing agent which is usedduring the derivatization process. Thus, this consortium showedpotential application for the biotreatment of BL with high pollutionload.

3.5. Toxicity assessment for environmental safety

Results of seed germination test revealed that the untreated BLabove 10% (v/v) inhibited sprouting of seeds as well as amylaseactivity (Fig. 5A and B). However, the seeds treated with tap waterhave shown lower amylase activity (0.3 U) than the seeds treatedwith 20% and 80% (v/v) concentration of untreated and treatedBL, respectively. This revealed that untreated BL act as growth pro-moter at lower concentration (10%) whereas treated BL was foundas growth supporter up to 80%. This indicated that toxicity hasbeen reduced significantly after bacterial treatment. The growthpromoting effects of untreated BL on amylase at lower concentra-tion might be due to the presence of optimum level of organicnutrients essential for plant growth (Kannan and Oblisami,

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control BL-UT BL-TR

B

Fig. 5. Toxicity evaluation of untreated (UT) and treated (TR) black liquor by seedgermination test (A) and amylase activity (B) on Phaseolus aurous L. at differentconcentration. TW: tap water.

Table 3Compounds identified by GC–MS analysis after alkaline hydrolysis from RGP blackliquor pre (control) and post bacterial treatment.

S. No. Compound name RT C D

1 Propanoic acid 10.08 + �2 Formic acid hydrazide 10.40 � +3 4-Cyclohexane-1,2-dicarboxylic acid 10.90 � +4 1,2 Butanediol 11.20 + �5 Carbamic acid 11.60 � +6 3-Cyclohexane 1-methanol 12.50 + �7 2-Methoxy phenol (Guaiacol) 12.99 + �8 4-Methyl benzaldehyde 13.10 + �9 Benzoic acid 13.80 + �10 Benzene acetic acid 13.90 + �11 Benzylemalonic acid 15.50 + �12 3-Hydroxy-4-methoxymandilic acid 15.70 + �13 Butylated hydroxytoluene 17.10 + �14 2,4-Bis (1,1-dietyl)-phenol 17.22 + �15 Heptadecanoic acid 18.03 + �16 2-Methoxy propanoyl chloride 18.90 + �17 4-Hydroxy-3,5-dimethoxy benzaldehyde 19.20 + �18 Tetradecanoic acid 20.50 + �19 1,2-benzenedicarboxylic acid 20.90 � +20 Dibutyle phthalate 21.57 + �21 Erythropentanoic acid 21.70 � +22 Ricinoleic acid 22.50 + �23 Phthalate 26.05 + +24 Cholesterol trimethylsilyl ether 26.11 + +25 1,1-(1,2-ethanediyl) bis[4-methoxy] benzene 26.40 + �26 2,4-Bis (1-phenylethyl)-phenol 27.77 + �27 Bis (2-ethylhexyl) phthalate 28.19 + �

RT: retention time (in min); C: control; D: bacterial treated; +: present; �: absent.

R. Chandra et al. / Bioresource Technology 102 (2011) 6429–6436 6435

1990). The reduction in amylase activity at higher concentration ofundegraded BL might be due to the high pollution content affectingvarious physiological and biochemical process during seedgermination.

4. Conclusion

This study revealed that the developed bacterial consortiumwas capable of 85% decolorization of BL from RGP plant having18,700 mg l�1 COD at optimized conditions. Further, the HPLCand GC–MS analysis of control and bacterial degraded sampleshowed that the consortium utilized the BL rather than biotrans-formation. Hence, the developed bacterial consortium was capablefor the effective decolorization and detoxification of pulp papereffluent for environmental safety.

Acknowledgement

The financial assistance from Department of Biotechnology un-der GAP-192 project and CSIR under SIP-08 is highlyacknowledged.

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