Bb

Aa

b

a

ARRA

KPPBGT

1

lfracrit(

trtwmff

f

h0

Ecological Engineering 71 (2014) 355–362

Contents lists available at ScienceDirect

Ecological Engineering

jou rn al hom ep age: www.elsev ier .com/ locate /eco leng

ioremediation and toxicity reduction in pulp and paper mill effluenty newly isolated ligninolytic Paenibacillus sp.

bhay Raja,∗, Sharad Kumara, Izharul Haqa, Sudheer Kumar Singhb

Environmental Microbiology Section, CSIR-Indian Institute of Toxicology Research (IITR), M.G. Marg, Post Box No. 80, Lucknow 226 001, UP, IndiaMicrobiology Division, CSIR—Central Drug Research Institute (CDRI), Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226 031, UP, India

r t i c l e i n f o

rticle history:eceived 5 October 2013eceived in revised form 25 May 2014ccepted 12 July 2014

eywords:aenibacillus sp. strain LD-1 (JX499920)

a b s t r a c t

The present study deals with the bioremediation of potentially hazardous pulp and paper mill efflu-ent by a laccase producing Paenibacillus sp. strain LD-1 (JX499920) isolated from contaminated soilsample by lignin enrichment method. The bacterium effectively reduced pollution parameters (colour68%, lignin 54%, phenol 86%, BOD 83% and COD 78%) after 144 h of treatment at 34 ± 1 ◦C and 120 rpm.GC–MS analysis of control and treated samples showed that concentration of most of the low molecularweight phenolic compounds like 2-methoxyphenol, 2,6-dimethoxy phenol, 2-methoxy-4-ethyl-phenol,

ulp and paper mill effluentioremediationC–MSoxicity test

3-allyl-6-methoxyphenol, ethanone 1-(-4-hydroxy-3,5-dimethoxyphenyl), benzoic acid, 2-methoxy-4-(1-propenyl) phenol and 4-methoxycinnamic acid present in control untreated effluent were reducedafter bacterial treatment. The toxicity assessments were carried out with treated and untreated effluentby studying the growth and germination of seeds of mung bean (Vigna radiata L.). The mung bean bioassayconfirmed the detoxification of effluent after bacterial treatment.

© 2014 Elsevier B.V. All rights reserved.

oeasmani

apiCmwem

. Introduction

There are about 515 paper mills in India, producing 6 mil-ion tons of paper using a variety of raw materials ranging fromorest-based wood to agricultural residues such as wheat straw,ice straw, and bagasse. Pulp and paper industry utilises largemount of chemicals especially, sodium hydroxide, solvents andhlorine compounds during paper manufacturing processes andeleases coloured effluent with high BOD, COD and also consist-ng of potentially toxic chlorinated compounds, suspended solids,annins, resin acids and sulphur compounds along with ligninsPokhrel and Viraraghavan, 2004).

Lignin and chlorinated phenols are the major environmen-al pollutants discharged from pulp and paper industry. Lignin isesponsible for offensive colour and also inhibits the growth of pho-otrophic organisms by decreasing the transmission of sunlight inater (Karrasch et al., 2006). Chlorinated phenolics are among the

ain chemical species responsible for the toxicity to both flora and

auna. Chlorinated organic compounds, which include dioxins andurans, are also suspected to cause genetic mutations in exposed

∗ Corresponding author. Tel.: +91 0522 2476051/91 0522 2476057;ax: +91 0522 2228227/+91 05222228471.

E-mail addresses: araj@iitr.res.in, abhayraj 1@rediffmail.com (A. Raj).

epuaia2e

ttp://dx.doi.org/10.1016/j.ecoleng.2014.07.002925-8574/© 2014 Elsevier B.V. All rights reserved.

rganisms (Malik et al., 2009; Theodorakis et al., 2006; Eastont al., 1997; Nestmann, 1985). Chlorinated compounds measureds adsorbable organic halides (AOXs) may bioaccumulate in fish tis-ue causing a variety of clastogenic, carcinogenic, endocrinic andutagenic effects (Savant et al., 2006), which may subsequently

lso pose problems to humans after consumption of the contami-ated fish. Exposure to dioxin and furan can cause skin disorders

ncluding skin cancer and also reproductive effects.The conventional treatment methods, such as aerated lagoons

nd activated sludge plants are ineffective in removing colour andhenolics. In most cases, this effluent (raw or treated) is discharged

nto the rivers, stream or other water bodies; resulting in high BOD,OD and also causing problems to community and environment. Inany developing countries farmers are irrigating their crop plantsith water bodies which might be severely exposed to industrial

ffluents. This leads to risks of bioaccumulation of toxicants as weove-up the food chain. Thus, it is important to treat the industrial

ffluents before their final discharge. Despite the fact that, severalhysical and chemical methods (electrocoagulation, ozonation, andltrafiltration) or combination of different methods in series arevailable for the treatment of effluent, but they are more energy

ntensive and suffer from residual effect and hence are less desir-ble than biological process which is cost-effective (Yang et al.,008). Thus, there is still a need for energy efficient, affordable andnvironment friendly technologies.

3 nginee

cAbollaKtoersiraa(a2peeefee

ldTsbg

2

2

lfl3pcH

2

aistlws

2

7gp

tCBats(

2

twpw0fspaTiTrw

2

ag1wl3tp

2

sAoom31Tiaaa1l

2

B

56 A. Raj et al. / Ecological E

Microorganisms (fungi and bacteria) are nature’s original recy-lers, converting toxic organic compounds to harmless products.lso, intensive studies have been carried out to explore the micro-ial diversity, particularly of contaminated areas in search ofrganisms that can degrade a wide range of pollutants at high pol-ution load. The white rot fungi had been found to possess goodigninolytic activity because of production of enzymes like LiP, MnPnd laccase (Minussi et al., 2007; D’Souza et al., 2006; Unal andolankaya, 2001). However, the use of fungal systems for effluent

reatment purpose has been constrained due to their requirementf narrow pH 4–5 for their growth and enzyme production. Gen-rally, pH values of pulp and paper mill are high (pH 7–9) andequirement to reduce the pH prior to the application of fungalystem adds additional cost. In contrast to fungi, bacteria grow-ng at neutral to alkaline pH (pH = 7–pH = 9) may play an importantole in decolourisation of pulp and paper mill effluents withoutny need for pH adjustment. Bacterial laccases are highly activend much more stable at high temperatures and high-pH valuesSharma et al., 2007). Several species of bacteria have been evalu-ted for lignin degradation (Chandra et al., 2007; EL-Hanafy et al.,007, 2008; EL-Hanafy and Abd-Elsalam, 2009) and for pulp andaper mill effluent treatment (Thakur, 2004; Raj et al., 2007; Singht al., 2011). However, there was a gap in knowledge regardingnzymes used by these systems for lignin degradation or in efflu-nt treatment. Similarly, the laccases purified and characterisedrom bacteria were not evaluated for their paper and pulp efflu-nt treatment abilities (Wang et al., 2011; Singh et al., 2007; Hullot al., 2001).

Hence, in the present study we tried to address this gap by iso-ating ligninolytic laccase producers and studied their suitability forecolourisation and detoxification of pulp and paper mill effluent.he laccase producing bacteria were isolated from contaminatedite. The phenolics removal from treated effluent was analyzedy GC–MS while toxicity assessment was performed using seedermination bioassay.

. Materials and methods

.1. Chemicals

The reagents used in this study were of analytical grade. Kraftignin, guaiacol, trimethylsilyl (BSTFA (N,O-bis (trimethylsilyl) tri-uoroacetamide) TMCS), phenylmethylsulfonyl fluoride (PMSF),,5-dinitrosalicylic acid, 2-methoxyphenol and benzoic acid wererocured from Sigma-Aldrich (USA). All other reagents for bacterialulture media such as glucose, peptone, agar and salts were fromi-Media (Mumbai, India).

.2. Collection of soil and effluent samples

The soil and effluent samples were collected from M/s. Star Pulpnd Paper mill located at Saharanpur (Uttar Pradesh, India) forsolation of bacteria and effluent decolourisation studies. The soilamples were collected from effluent channels in presterilised testubes using a sterile spatula. The final effluent samples were col-ected at the main outlet in presterilised plastic bottles. The samples

ere transported to the laboratory in iceboxes and immediatelytored at 4 ◦C after their arrival.

.3. Characterisation of wastewater samples

The pH of the effluent was measured using pH meter (Model44, Metrohm). Chemical oxygen demand (COD), biological oxy-en demand (BOD), total dissolved solids, total nitrogen, sulphate,hosphate, nitrate and total phenol were estimated according

owiu

ring 71 (2014) 355–362

o American Public Health Association guidelines (APHA, 2005).olour and lignin were measured using CPPA (1974) and Pearl andenson (1940) methods. Heavy metals like copper, iron, nickel, zincnd manganese were analysed with atomic absorption spectropho-ometer (Model A. analyst-300, Perkin Elmer) after digestion ofamples (100 ml) in a digestion mix (5:1) of nitric–perchloric acidAPHA, 2005).

.4. Isolation of lignin-degrading bacteria

Selective enrichment culture technique was used for the isola-ion of lignin degrading (LD) bacteria. Briefly, 1.0 gm soil sampleas added to 99 ml of sterile mineral salt medium (MSM) sup-lemented with 500 mg/l lignin. The composition of MSM (g/l)as: Na2HPO4, 2.4; K2HPO4, 2.0; NH4NO3, 0.1; MgSO4, 0.01; CaCl2,

.01; d-glucose, 5.0 and peptone, 5.0. The flasks were incubatedor 7 days in a rotary shaker (Innova 4230 Refrigerated Incubatorhaker, New Brunswick, USA) at 120 rpm and 34 ± 1 ◦C. The sam-les from flasks exhibiting decolourisation were serially dilutednd spread on MSM agar plate supplemented with 500 mg/l lignin.he colonies grown were sub-cultured on MSM-agar plate contain-ng increasing concentration of lignin i.e. 1000, 1500 and 2000 mg/l.he isolates showing growth at 2000 mg/l lignin were purified byepeated streaking on nutrient agar plates. The isolated coloniesere also assessed microscopically after Gram staining.

.5. Screening of ligninolytic activity on agar plate-test

Selection of laccase producing bacteria was done on guaiacol-gar plates. The guaiacol-agar plate medium consisting of 1.0%lucose, 0.2% peptone, 0.1% yeast extract, 0.5% NaCl, 0.0025% Cu2+,.6% agar (pH = 7.6) was autoclaved and afterwards supplementedith filter sterilised 0.02% guaiacol (Sigma). Each LD bacterial iso-

ate was spot-inoculated on plates and plates were incubated at4 ± 1 ◦C for 7 days. Laccase activity was visually scored for forma-ion of brown coloured zone around the colonies on guaiacol-agarlate (Coll et al., 1993).

.6. Quantitative assay

Bacterial isolates showing positive reaction in the platecreening were grown in liquid guaiacol medium (devoid of agar).t first, the positive bacteria were grown in nutrient broth forvernight (18 h) and from this 1.0 ml inoculum with an absorbancef 0.5 OD (A600; 1 cm cuvette) was used to inoculate 99 ml ofedium (pH = 7.6) in 250 ml Erlenmeyer flasks and incubated at

4 ± 1 ◦C, 120 rpm for 120 h. The culture broth was centrifuged at0,000 × g for 15 min and supernatant was taken for laccase assay.he assay was performed by adding 1.0 ml of culture supernatantn 10 mM guaiacol (prepared in 100 mM phosphate buffer, pH 7.0)nd incubating for 30 min at 30 ◦C. The absorbance was measuredt 470 nm (Arora and Sandhu, 1985). One unit (U/ml) of enzymectivity was defined as the amount of enzyme required to oxidise

�mol substrate per min under assay conditions. The promisingaccase producing isolate LD1 was used for further studies.

.7. Bacterial identification

The selected laccase producing isolate was inoculated in Luriaertani broth (Hi-Media, Mumbai, India) at 37 ◦C and 180 rpm for

vernight (18 h) growth. The overnight grown bacterial cultureas used for total DNA isolation (UltraClean Microbial DNA

solation Kit, MO BIO, USA). The 16S rRNA gene was PCR amplifiedsing universal primers: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′)

ngineering 71 (2014) 355–362 357

ape3sacTBtsa

2

pNws13adrgnw

2

cSwwaS(oGitcuHc(p3meiaw

2

gLsfew

Table 1Physicochemical characteristics of pulp and paper mill effluent. Values aremean ± SD of triplicate samples.

Parameters Values (mg/l, except colour and pH)

pH 8.2 ± 1.0TDS 850 ± 30COD 792 ± 70BOD 385 ± 12Colour (CU) 2242 ± 56Lignin 436 ± 18Total nitrogen 116 ± 32Sulphate 993 ± 6Phosphate 8.3 ± 0.3Nitrate 73.3 ± 6Total phenol 42 ± 2.5Heavy metals

Cu 0.09 ± 0.1Fe 10.22 ± 9

ioPii�1irupt

3

3

ece(t(a(pCfl1do(snabmp

3

A. Raj et al. / Ecological E

nd 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) at annealing tem-erature of 56 ◦C (35 cycles). The PCR product was purified by gelxtraction (Gel extraction Kit, Qiagen) and was sequenced in an ABI130 genetic analyzer using Big Dye Terminator version 3.1 cycleequencing kit. The 16S rRNA gene sequences were compared withvailable sequences using NCBI-BLAST. A phylogenetic tree wasonstructed using Bioinformatics Bacterial Identification (BIBI)ool (http://umr5558-sud-str1.univ-lyon1.fr/lebibi/lebibi.cgi).IBI relies on the use of BLAST and CLUSTAL W programs appliedo different subsets of sequences extracted from GenBank. Theseequences are filtered and stored in a new database, which isdapted to bacterial identification (Devulder et al., 2003).

.8. Effluent decolourisation study

Erlenmeyer flasks (250 ml) containing 99 ml of effluent sup-lemented with mineral salts (Na2HPO4, 2.4 g/l; K2HPO4, 2.0 g/l;H4NO3, 0.1 g/l; MgSO4, 0.01 g/l; CaCl2, 0.01 g/l), glucose (0.5%,/v) and peptone (0.25%, w/v) were pH adjusted (pH = 7.2) and

terilised for 20 min, at 121 ◦C. The flasks were inoculated with ml of overnight (18 h) grown culture having an inoculums size of0.4 × 105 cfu/ml. The control and inoculated flasks were incubatedt 34 ± 1 ◦C and 120 rpm for 144 h. The samples (5 ml) were with-rawn at every 24 h interval for the analysis of bacterial growth,eduction in colour, lignin, total phenol, COD and BOD. Bacterialrowth was monitored by colony forming unit (cfu/ml) counts onutrient agar plate by serial dilution technique. The experimentsere conducted in triplicate and data presented are mean ± SD.

.9. GC–MS analysis

Control and bacterial treated effluent samples (50 ml) wereentrifuged (8000 × g for 15 min) to remove suspended solids.upernatants were acidified to pH 1–2 and then extracted thriceith equal volume of ethyl acetate. The ethyl acetate extractas vacuum dried. The ethyl acetate extracts were analyzed

s trimethylsilyl (TMS) derivatives (Lundquist and Kirk, 1971).amples were derivatised using trimethylsilyl (BSTFA (N,O-bistrimethylsilyl) trifluoroacetamide) TMCS). An aliquot of 1 �lf silylated compounds was injected in the injector port ofC–MS equipped with a PE Auto system XL gas chromatograph

nterfaced with a Turbomass Mass spectrometric mass selec-ive detector (Perkin Elmer, Waltham, MA, USA). The analyticalolumn connected to the system was a PE-5MS capillary col-mn (20 m × 0.18 mm internal diameter, 0.18 �m film thickness).elium gas with flow rate of 1 ml/min was used as carrier gas. Theolumn temperature was programmed as 50 ◦C (5 min); 50–300 ◦C10 ◦C min, hold time: 5 min). The transfer line and ion source tem-eratures were maintained at 200 and 250 ◦C. A solvent delay of.0 min was selected. In the full-scan mode, electron ionisation (EI)ass spectra in the range of 30–550 (m/z) were recorded at electron

nergy of 70 eV. The metabolic products were identified by compar-ng their mass spectra with that of National Institute of Standardsnd Technology (NIST) library and by comparing the retention timeith those of available authentic organic compounds.

.10. Toxicity evaluation by seed germination tests

Acute toxicity of treated samples was assessed using seedermination bioassay (Wang, 2003). Mung bean (Vigna radiata. var. K-851) was purchased from local shop and beans were

urface-sterilised with 0.1% HgCl2. The seeds were soaked withour different dilutions of (0, 25, 50 and 100% v/v) of the efflu-nt. Ten seeds were kept on filter paper in each petridish soakedith respective test solution. The plates were incubated at 28 ± 1 ◦C

lc

Ni 5.03 ± 1Zn 9.83 ± 1Mn 0.04 ± 0

n dark condition for 48 h. Toxicity was expressed in termsf inhibition of their sprouting length and �-amylase activity.roteins were extracted from seeds in extraction buffer contain-ng 1 mM of phenylmethylsulfonyl fluoride (PMSF) as proteasenhibitors. The crude �-amylase was prepared after inactivating-amylase, debranching enzyme, and �-glucosidase at 70 ◦C for5 min. The �-amylase was assayed by quantifying the reduc-

ng sugars (maltose equivalent) liberated from starch using DNSAeagent (3,5-dinitrosalicylic acid) at 540 nm (Bernfeld, 1955). Onenit of enzyme activity was defined as the amount of enzyme thatroduced 1 �mol of reducing sugar per min under the test condi-ions.

. Results and discussion

.1. Wastewater characterisation

The physico-chemical characteristics of pulp and paper millffluent are detailed in Table 1. The effluent had a dark brownolour and was slightly alkaline in nature (pH = 8.2). The analysis offfluent showed presence of high colour (2242 ± 56 CU) and lignin436 ± 18 mg/l) in effluent. Apart from this the various parame-ers were: COD (792 ± 70 mg/l), BOD (385 ± 12 mg/l), phosphate8.3 ± 0.3 mg/l), nitrate (73.3 ± 6 mg/l), total phenol (42 ± 2.5 mg/l)nd heavy metals like Fe (10.22 mg/l), Ni (5.03 mg/l) and Zn9.83 mg/l). Lignin and phenolics are plant constituents and majorollutants in pulp and paper industry effluent. Lignin causes highOD and colour in pulping effluent. Phenols are harmful to bothora and fauna, even at relatively low concentration. Phenol at.0 �g/ml or higher concentration inhibited the photosynthesis ofiatoms and blue algae. Also, phenol concentration in the rangef 100–400 �g/ml caused complete inhibition of photosynthesisKostyeav, 1973). The source of sulphate ions in effluent might beodium sulphite, which is used during pulping process and theitrates detected in effluent possibly come from lignin (Singhalnd Thakur, 2009). Metal content of the effluent might be due toioaccumulation of these metals by plants which are used as rawaterial as well as from various chemicals used during the pulping

rocess.

.2. Isolation and characterisation and ligninolytic bacteria

Five different types of lignin degrading (LD) bacterial iso-ates growing on MSM agar plate were isolated from effluentontaminated soil by kraft lignin enrichment. All five bacterial

358 A. Raj et al. / Ecological Engineering 71 (2014) 355–362

Fg7

i(optdpe

mtfioLoabBda

3

aoatawoHsawtsSIdls2

Fig. 2. Phylogenetic tree of the strain LD1 and their related genera has been linkedbs

otTwlprtsocatatt

bfiipa2tltoa

be

ig. 1. Screening test for selecting laccase producing bacteria. Laccase activity onuaiacol-agar plate (a) and maximum laccase activity in guaiacol medium (b) after2 h.

solates formed brown coloured zones on guaiacol-agar plateFig. 1a) indicating laccase production. Guaiacol oxidation is onef the most convenient qualitative assays for ligninolytic enzymeroduction by microorganisms. Brown colour zone under or aroundhe colonies on guaiacol-agar plate medium suggests laccase pro-uction. The formation of reddish brown zone is due to oxidativeolymerisation of guaiacol by laccase (D’Souza et al., 2006; Collt al., 1993).

The enzyme production studies were conducted in liquidedium and highest enzyme producer was selected. Fig. 1b shows

he production of maximum laccase by isolates for 72 h. Among theve isolates, the isolate LD 1 was found to give highest productionf laccase (6.5 IU/ml) and was selected for further study. IsolateD1 was identified using 16S rRNA gene analysis. BLAST analysisf isolate LD1 partial 16S rRNA gene sequence (1407 bp, GenBankccession no. JX499920) showed 99% identity with several Paeni-acillus species. The phylogenetic tree (Fig. 2) constructed usingIBI software showed clustering of isolate LD1 with Paenibacillusendritiformis (accession no. AY359885). Hence, present strain waslso inferred to be belonging to P. dendritiformis.

.3. Decolourisation of pulp and paper mill effluent

The decolourisation study was conducted for 144 h on pulpnd paper mill effluent containing lignin 436 mg/l with a CODf 792 mg/l at pH = 7.2. The cfu profile along with changes inbsorbance due to colour at 465 nm during course of decolourisa-ion is depicted in Fig. 3a. The initial increase in cfu count plateauedt 120 h of incubation followed by a slight decline thereafter,hereas significant reduction in absorbance due to colour started

nly after 48 of incubation and reached a maximum (68%) on 144 h.owever, not much decolorisation was observed up to 48 h. This

uggests that during initial phase of growth bacteria utilised readilyvailable carbon (glucose) and nitrogen (peptone) sources whichere already supplemented in the growth medium. During the ini-

ial growth bacterial also adapted to lignin and lignin degradationtarted afterwards with consequent increase in colour reduction.imilar findings were also reported by Singhal and Thakur (2009).n a similar study by Chandra et al. (2007) rapid depletion of glucose

uring initial growth was observed with subsequent utilisation of

ignin. Also, EL-Hanafy et al. (2008), while using lignin as a soleource of carbon observed slow lignin degradation during initial4 h. This might be due to lack of adaptation of strain as well as lack

4anp

ased on partial 16S rDNA sequence comparisons. Their names and respective acces-ion numbers are given in the tree.

f alternative carbon sources in the medium. Once, the cells startedo use lignin as a carbon source the degradation started after 48 h.he removal of colour by biological treatment could be correlatedith degradation of lignin and its related phenolic compounds. The

ignin and related compounds are major components of pulp andaper mill effluent and are cause of higher COD. The lignin and CODemoval were found to be 54% and 78% respectively after 144 h ofreatment (Fig. 3b). This showed that the bacterium was capable ofignificant reduction in pollutants load of the effluent. The BOD loadf the effluent was reduced by 83% after treatment. Phenols andhlorinated phenols in effluent cause harmful effect on both florand fauna even at relatively low concentration of 10–12 mg/l. Theotal phenol content of untreated and treated effluent was 42 mg/lnd 5.9 mg/l. This accounted for around 86% reduction after 144 hreatment (Fig. 3b). The reduction in phenol content may be due tohe ability of bacterium to use it as a carbon source.

During decolourisation, the pH of the effluent changed due toacterial metabolic activity. Initially, there was a decrease in pHrom pH = 7.2 to pH = 4.6 after 48 h, and thereafter gradually itncreased up to pH = 6.3 at the end of the experiment (Fig. 3c). Whilen the control flasks the medium pH remained constant. The shift inH towards acidic condition during decolourisation might be due tocetate efflux along with other TCA cycle intermediates (Yang et al.,008). As the simple carbon sources deplete, the bacteria shifts backo utilise the excreted metabolic intermediates including acetate,eading to a gradual increase in pH. This facilitates lignin degrada-ion as lignins are uniformly soluble at high pH as well as the needf using lignin as a carbon source. This explains reduction in colour,nd COD after 48 h of incubation period (Fig. 3a and b).

The Fig. 3d shows level of laccase enzyme induced duringacterial decolourisation of pulp and paper mill effluent. Thenzyme activity was increased up to 96 h with laccase activity of.4 IU/ml and thereafter gradually declined. Laccases (EC 1.10.3.2),

re copper-containing oxidase enzymes that oxidise both phe-olic and nonphenolic lignin-related compounds. The pulp andaper mill effluents contain phenolics and lignin which can induce

A. Raj et al. / Ecological Engineering 71 (2014) 355–362 359

F (c) anT at 12

llm1id(lam

eeo8(6cetBpa

tos(ppr

3

paeimpm(

ig. 3. Growth and colour (a), reduction in pollutant parameters (b), pH of mediumhe effluent was inoculated with isolate and incubated at 34 ± 1 ◦C in orbital shaker

igninolytic enzymes in microorganisms. Induction of LiP, MnP andaccase in white-rot fungi during decolourisation of pulp and paper

ill wastewaters has been well documented (Alessandro et al.,999; Wu et al., 2005). Although laccase activity from bacteria

ncluding Bacillus licheniformis (Koschorreck et al., 2008), Pseu-omonas putida F6 (MacMahon et al., 2007), �-proteobacterium JBSingh et al., 2007), Bacillus subtilis (Hullo et al., 2001) and Azospiril-um lipoferum (Diamantidis et al., 2000) has been reported but theirpplication in decolorisation and detoxification of pulp and paperill effluent has not been studied.Lignin and phenolics are major pollutants in pulp and paper mill

ffluents and their removal from effluents is highly significant fromnvironmental safety perspective. The earlier study on treatmentf pulp and paper mill effluent using Aeromonas formicans reported5%, 80% and 70% reduction of colour, lignin and COD after 8 daysGupta et al., 2001). Similarly, Raj et al. (2007) reported removal of1%, 53%, 82%, 78% and 77% of colour, lignin, BOD, COD and phenolontents by Bacillus sp. within 6 days of incubation. Also, Chandrat al. (2009) reported Bacillus cereus and Serratia marcescens for

reatment and found reduction in colour (45–52%), lignin (30–42%),OD (40–70%), COD (50–60%) and phenol (32–40%), in a 7-dayeriod. By using sulphate reducing bacteria, Hao and Man (2006)ble to remove COD up to 70–75% after 3 weeks and increase

ceRp

d laccase activity (d) during course of effluent decolourisation by Paenibacillus sp.0 rpm. Values are mean ± SD of three replicates.

o 82–88% by subsequent aerobic treatment for 48 h. The levelf colour, lignin, BOD, COD and phenol reduction in the presenttudy within 144 h is higher than that of B. cereus and S. marcescensChandra et al., 2009) and Bacillus sp. (Raj et al., 2007). The com-arison of present study with earlier reports suggests that in theresent study higher reduction of phenolic content along witheduction of colour, lignin, BOD and COD was observed.

.4. GC–MS analysis of effluent

The GC–MS analysis of ethyl acetate extractable com-ounds from the acidified supernatant obtained from controlnd treated sample showed, that in control sample, sev-ral low molecular weight phenolics were identified, whereas,n treated sample they were not identified using the NIST

ass spectral database (Fig. 4a and b and Table 2). Thehenolic compounds identified in control sample were 2-ethoxyphenol (guaiacol) RT = 14.68, 2,6-dimethoxy phenol

syringol) RT = 15.96, 2-methoxy-4-ethyl-phenol (4-ethyl guaia-

ol) RT = 16.38, 3-allyl-6-methoxyphenol (m-eugenol) RT = 19.04,thanone 1-(-4-hydroxy-3,5-dimethoxyphenyl) (acetosyringone)T = 19.97, benzoic acid RT = 20.17, 2-methoxy-4-(1-propenyl)henol (isoeugenol) RT = 22.26 and 4-methoxycinnamic acid

360 A. Raj et al. / Ecological Engineering 71 (2014) 355–362

Fig. 4. GC–MS chromatograms of compounds extracted with ethyl acetate from control (a) and Paenibacillus sp. treated effluent (b). The MS-identified compounds withrespect to their retention times are listed in Table 2.

Table 2Compound identified as trimethylsilyl (TMS) derivatives in ethyl extract from con-trol (a) and Paenibacillus sp. treated (b) effluent samples as given in Fig. 3.

Retention time(in min)

Present/absent in Compounds

a b

11.4 + + Acetic acid12.97 + − 2-Hydroxymethyl cyclopropane

carboxylic acid14.68 + − 2-Methoxyphenola

15.7 + − Phthalic anhydride15.96 + − 2,6-Dimethoxy phenol16.38 + − 2-Methoxy-4-ethyl-phenol16.6 − + Unidentified17.58 + − Benzene acetic acid19.04 + − 3-Allyl-6-methoxyphenol19.97 + − Ethanone, 1-(-4-hydroxy-3,5-

dimethoxyphenyl)20.17 + − Benzoic acida

20.95 + − 1,2-Benzenedicarboxylic acid21.68 + + Hexadecanoic acid22.26 + − 2-Methoxy-4-(1-propenyl) phenol22.29 + − Clionasterol acetate23.07 − + Octadecane23.29 + − Octadecanoic acid24.50 + − 4-methoxycinnamic acid26.1 + + Trimethyl-salyl

a Confirmed by match of retention time (RT) with known standards.

Rl(tGaRodRigaetetebstmawac

T = 24.50. These compounds are degradation products of threeignin units (p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S))Gutierrez et al., 2006) which may possibly be coming fromhe industrial pulping process (Hernandez-Coronado et al., 1998;upta et al., 2001). Apart from these phenolic compounds,cidic compounds (acetic acid RT = 11.4, benzene acetic acidT = 17.58, hexadecanoic acid RT = 21.68, octadecane RT = 23.07 andctadecanoic acid RT 23.29), phthalate derivatives (phthalic anhy-ride RT = 15.70 and 1,2-benzenedicarboxylic acid (phthalic acid)T = 20.95) and extractive (clionasterol acetate RT = 22.29) were

dentified. These compounds had been reported earlier during fun-al peroxidase degradation of lignosulfonate (Shin and Lee, 1999)nd also from bacterial degradation of black liquor lignin (Chandrat al., 2011). The GC–MS analysis result reveals marked reduc-ion in the intensity of peaks of compounds identified in untreatedffluent after treatment. This strongly suggests the ability of bac-erium to utilise effluent constituents as carbon, nitrogen andnergy source. Trimethylsilyl (RT 26.1, min) remain unchangedecause it is a derivatising agent which is used during the derivati-ation process. A new compound appeared in extract of bacterialreated effluent at RT-16.6 min that could not be identified by

ass spectrometry. The GC–MS analysis in the present study was

ble to conclusively demonstrate the metabolism of low moleculareight compounds released from lignin degradation and has been

method of choice for analysis of volatile low molecular weightompounds (Hernandez-Coronado et al., 1998).

A. Raj et al. / Ecological Engineering 71 (2014) 355–362 361

Fig. 5. Effect of the untreated (UT) and bacterial treated (BT) effluent at the concentration of 0, 25, 50 and 100% on seed germination (a), �-amylase activity (b) and sproutinglength (c).

3

t�wcseieTemereesrtdcp2mti

4

itcaseigp

A

cs(

R

A

.5. Toxicity assessment for environmental safety

The effects of untreated and treated effluent at the concentra-ion of 0, 25, 50 and 100% on seed germination, sprouting length and-amylase activity are presented in Fig. 5. The seeds initially treatedith undiluted and untreated effluent (100% v/v, without biologi-

al treatment) failed to germinate, while, after bacterial treatmenteeds were able to germinate when treated with undiluted efflu-nt (100% v/v) (Fig. 5a). The �-amylase is key enzyme involvedn seed germination and its titres during seed germination afterxposing them to different levels of effluent are detailed in Fig. 5b.he �-amylase titres were decreased when exposed to increasingffluent concentrations as compared to control. Also, effects wereore pronounced when seeds were exposed to untreated efflu-

nt as compared with biologically treated effluent (Fig. 5b). Theelative sprout length of 48 h seeds with biologically treated efflu-nt was more than the sprout length of seed exposed to untreatedffluent (Fig. 5c). At 25% treated effluent the sprouting length waslightly larger than the control seeds. This suggests that toxicityeduction took place after treatment of effluent with bacterial cul-ure, which was possibly due to degradation of possible toxicantsuring biological treatment. The reduction in toxicity by biologi-al treatment had earlier been reported for olive mill effluent andulp paper effluent (Singhal and Thakur, 2009; Quaratino et al.,

007) and it further confirms our assumption that biological treat-ent with the present isolate can lead to significant reduction in

oxicity associated with effluent along with overall improvementn effluent characteristics.

A

A

B

. Conclusions

Paenibacillus sp. strain LD1, a laccase producing bacterium wassolated from effluent contaminated site. The bacterium was usedo treat pulp and paper mill effluent. The overall removal efficien-ies of 68%, 54%, 86%, 83 and 78% for colour, lignin, total phenol, BODnd COD were observed after 144 h of incubation. GC–MS of treatedample showed utilisation of phenolic compounds as carbon andnergy source. Further, the toxicity of treated effluent was signif-cantly reduced and the treated effluent was able to support therowth of mung bean even in undiluted form suggesting improvedroperties of biologically treated effluent.

cknowledgements

Author AR is thankful to Director, CSIR-Indian Institute of Toxi-ology Research (IITR), Lucknow (India) for his encouragement andupport. The financial support of CSIR TASK FORCE project INDEPTHBSC0111) is acknowledged.

eferences

lessandro, D.A., Silvia, R.S., Vittorio, V., Elena, D.M., Giovanni, G.S., 1999. Charac-terization of immobilized laccase from Lentinula edodes and its use in olive millwastewater treatment. Process Biochem. 34, 697–706.

PHA, 2005. Standard Methods for the Examination of Water and Wastewater, 21thed. American Public Health Association, Washington, DC.

rora, D.S., Sandhu, D.K., 1985. Laccase production and wood degradation by white-rot fungus Daedoalea flavida. Enzyme Microb. Technol. 7, 405–408.

ernfeld, P., 1955. Amylase alpha and beta. Methods Enzymol. 1, 149–158.

3 nginee

C

C

C

C

C

D

D

D

E

E

E

E

G

G

H

H

H

K

K

K

L

M

M

M

N

P

P

Q

R

S

S

S

S

S

S

T

T

U

W

W

62 A. Raj et al. / Ecological E

handra, R., Abhishek, A., Sankhwar, M., 2011. Bacterial decolorization and detoxifi-cation of black liquor from rayon grade pulp manufacturing paper industry anddetection of their metabolic products. Bioresour. Technol. 102, 6429–6436.

handra, R., Raj, A., Purohit, H.J., Kapley, A., 2007. Characterisation and optimizationof three potential aerobic bacterial strains for Kraft lignin degradation from pulppaper waste. Chemosphere 67, 839–846.

handra, R., Raj, A., Yadav, S., Patel, D.K., 2009. Reduction of pollutants in pulp papermill effluent treated by PCP-degrading bacterial strains. Environ. Monit. Assess.155, 1–11.

oll, P.M., Fernandez-Abalos, J.M., Villanueva, J.R., Santamaria, R., Perez, P., 1993.Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971). Appl. Environ. Microbiol. 59,2607–2613.

PPA, 1974. Technical Section Standard Method H5P. Canadian Pulp and Paper Asso-ciation, Montreal, Canada.

’Souza, D.T., Tiwari, R., Sah, A.K., Raghukumar, C., 2006. Enhanced production oflaccase by a marine fungus during treatment of colored effluents and syntheticdyes. Enzyme Microb. Technol. 38, 504–511.

evulder, G., Perrière, G., Baty, F., Flandrois, J.P., 2003. BIBI, a bioinformatics bacterialidentification tool. J. Clin. Microbiol. 41, 1785–1787.

iamantidis, G., Effosse, A., Potier, P., Bally, R., 2000. Purifcation and characterizationof the first bacterial laccase in the rhizospheric bacterium Azospirillum lipoferum.Soil Biol. Biochem. 32, 919–927.

L-Hanafy, A.A., Abd-Elsalam, H.E., 2009. Lignin biodegradation with ligninolyticbacterial strain and comparison of Bacillus subtilis and Bacillus sp. isolated fromEgyptian soil. American-Eurasian J. Agric. Environ. Sci. 5, 39–44.

L-Hanafy, A.A., Abd-Elsalam, H.E., Hafez, E.E., 2007. Fingerprinting for the lignindegrading bacteria from the soil. J. Appl. Sci. Res. 3, 470–475.

L-Hanafy, A.A., Abd-Elsalam, H.E., Hafez, E.E., 2008. Molecular characterization oftwo native Egyptian ligninolytic bacterial strains. J. Appl. Sci. Res. 4, 1291–1296.

aston, M.D.L., Kruzynski, G.M., Solar, I.I., Dye, H.M., 1997. Genetic toxicity of pulpmill effluent on juvenile Chinook salmon (Onchorhynchus tshawytscha) usingflow cytometry. Water Sci. Technol. 35, 347–355.

upta, V.K., Minocha, A.K., Jain, N., 2001. Batch and continuous studies on treatmentof pulp mill wastewater by Aeromonas formicans. J. Chem. Technol. Biotechnol.76, 547–552.

utierrez, A., Rodriguez, I.M., del-Rio, J.C., 2006. Chemical characterization of ligninand lipid fractions in industrial hemp bast fibers used for manufacturing highquality paper pulps. J. Agric. Food Chem. 54, 2138–2144.

ao, D.T., Man, T.D., 2006. Study on treatment of alkaline black liquor using sulphatereducing bacteria. Adv. Nat. Sci. 7, 139–144.

ernandez-Coronado, M.L., Hernandez, M., Rodriguez, J., Arias, M.E., 1998. Gas chro-matography/mass spectrometry as a suitable alternative technique to evaluatethe ability of Streptomyces to degrade lignin from lignocellulosic residues. RapidCommun. Mass Spectrom. 12, 1744–1748.

ullo, M.F., Moszer, I., Danchin, A., Martin-Verstraete, I., 2001. CotA of Bacillus subtilisis a copper-dependent laccase. J. Bacteriol. 183, 5426–5430.

arrasch, B., Parra, O., Cid, H., Mehrens, M., Pacheco, P., Urrutia, R., Valdovinos, C.,Zaror, C., 2006. Effect of pulp and paper mill effluents on the microplanktonand microbial self-purification capabilities of the Biobio River Chile. Sci. TotalEnviron. 359, 194–208.

oschorreck, K., Richter, S.M., Ene, A.B., Roduner, E., Schmid, R.D., Urlacher, V.B.,

2008. Cloning and characterization of a new laccase from Bacillus licheni-formis catalyzing dimerization of phenolic acids. Appl. Microbiol. Biotechnol.79, 217–224.

ostyeav, V.Ya., 1973. The Effect of Phenol on Algae, in the Effect of Phenol onHydrobionts. Leningrad Nauka Publications, Moscow, pp. 98–113.

W

Y

ring 71 (2014) 355–362

undquist, K., Kirk, T.K., 1971. Acid degradation of lignin. Acta Chem. Scand. 25,889–894.

alik, M.K., Kumar, P., Seth, R., Rishi, S., 2009. Genotoxic effect of paper mill effluenton chromosomes of fish Channa punctatus. Curr. World Environ. 4, 353–357.

acMahon, A.M., Doyle, E.M., Brooks, S., O’Connor, K.E., 2007. Biochemical char-acterisation of the coexisting tyrosinase and laccase in the soil bacteriumPseudomonas putida F6. Enzyme Microb. Technol. 40, 1435–1441.

inussi, R.C., Pastore, G.M., Duran, N., 2007. Laccase induction in fungi andLaccase/N-OH mediator systems applied in paper mill effluent. Bioresour. Tech-nol. 98, 158–164.

estmann, E.R., 1985. Detection of genetic activity in effluent from pulp andpaper mills: mutagenicity in Saccharomyces cerevisiae. In: Zimmerman, F.K.,Taylor-Mayer, R.E. (Eds.), Testing in Environmental Pollution Control. Horwood,London, pp. 105–117.

earl, I.A., Benson, H.K., 1940. The determination of lignin in sulphite pulping liquor.Paper Trade J. 111, 35–36.

okhrel, D., Viraraghavan, T., 2004. Treatment of pulp and paper mill wastewater—areview. Sci. Total Environ. 333, 37–58.

uaratino, D., D’Annibale, A., Federici, F., Cereti, C.F., Rossini, F., Fenice, M., 2007.Enzyme and fungal treatments and a combination thereof reduce olive millwastewater phytotoxicity on Zea mays L. seeds. Chemosphere 66, 1627–1633.

aj, A., Reddy, M.M.K., Chandra, R., 2007. Decolourisation and treatment of pulp andpaper mill effluent by lignin-degrading Bacillus sp. J. Chem. Technol. Biotechnol.82, 399–406.

avant, D.V., Abdul-Rahman, R., Ranade, D.R., 2006. Anaerobic degradation ofadsorbable organic halides (AOX) from pulp and paper industry wastewater.Bioresour. Technol. 97, 1092–1104.

harma, P., Goel, R., Capalash, N., 2007. Bacterial laccases. World J. Microbiol.Biotechnol. 23, 823–832.

hin, K.S., Lee, Y.J., 1999. Depolymerisation of lignosulfonate by peroxidase of thewhite-rot basidiomycetes, Pleurotus ostreatus. Biotechnol. Lett. 21, 585–588.

ingh, G., Capalash, N., Goel, R., Sharma, P., 2007. A pH-stable laccase fromalkali-tolerant �-proteobacterium JB: purification, characterization and indigocarmine degradation. Enzyme Microb. Technol. 41, 794–799.

ingh, Y.P., Dhall, P., Mathur, R.M., Jain, R.K., Thakur, V.V., Kumar, V., Kumar, R.,Kumar, A., 2011. Bioremediation of pulp and paper mill effluent by tannic aciddegrading Enterobacter sp. Water Air Soil Pollut. 218, 693–701.

inghal, A., Thakur, I.S., 2009. Decolorization and detoxification of pulp paper efflu-ent by Cryptococcus sp. Biochem. Eng. J. 46, 21–27.

hakur, I.S., 2004. Screening and identification of microbial strains for removal ofcolour and adsorbable organic halogens in pulp and paper mill effluent. ProcessBiochem. 39, 1693–1699.

heodorakis, C.W., Lee, K.L., Adams, S.M., Law, C.B., 2006. Evidence of altered geneflow, mutation rate, and genetic diversity in redbreast sunfish from a pulp-mill-contaminated river. Environ. Sci. Technol. 40, 377–386.

nal, A., Kolankaya, N., 2001. Dechlorination of bleached kraft pulp by laccaseenzyme produced from some white-rot fungi. Turk. J. Biol. 25, 67–72.

ang, C., Zhao, M., Lu, L., Wei, X., Li, T., 2011. Characterization of spore laccase fromBacillus subtilis WD23 and its use in dye decolorization. Afr. J. Biotechnol. 10,2186–2192.

ang, W., 2003. The use of plant seeds in toxicity tests of phenolic compounds.Environ. Int. 11, 49–55.

u, J., Xiao, Y.Z., Yu, H.Q., 2005. Degradation of lignin in pulp mill wastewaters bywhite-rot fungi on biofilm. Bioresour. Technol. 96, 1357–1368.

ang, C., Cao, G., Li, Y., Zhang, X., Ren, H., Wang, X., Feng, J., Zhao, L., Xu1, P., 2008.A constructed alkaline consortium and its dynamics in treating alkaline blackliquor with very high pollution load. PLoS One 3, e3777.