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International Biodeterioration & Biodegradation 96 (2014) 1e8

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International Biodeterioration & Biodegradation

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

Dual application of agricultural residues for xylanase production anddye removal through solid state fermentation

Prachi Kaushik*, Abhishek Mishra, Anushree MalikApplied Microbiology Laboratory, Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India

a r t i c l e i n f o

Article history:Received 25 June 2014Received in revised form14 August 2014Accepted 15 August 2014Available online

Keywords:Agro-residuesCellulaseDye removalCharacterizationXylanase

* Corresponding author. Tel.: þ91 11 26596011; faxE-mail address: kaushik.prachi@yahoo.com (P. Kau

http://dx.doi.org/10.1016/j.ibiod.2014.08.0060964-8305/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The present study is a novel attempt to utilize agricultural residues for addressing the dual challengefaced by pulp and paper industries to control pollution caused due to pulp bleaching and release ofcolored waste water. Fungal isolate, Aspergillus lentulus, was utilized for the production of xylanasethrough solid state fermentation utilizing various low-cost agro-residues as substrate. Maximum xyla-nase production was obtained on the 4th day of incubation using wheat bran as the substrate (158.4 U/g)followed by corn cob (153.0 U/g), sugarcane bagasse (129.9 U/g) and wheat straw (49.4 U/g). Theseactivities were accompanied by very low cellulase activities. The enzyme exhibited good stability at highpH and temperature (>75% activity retained at pH 9 and 70 �C). Later, the left over spent fermented slurrywas utilized to remove anionic (>85.0% removal) and cationic (>96.0% removal) dyes. Results indicatecellulase-free; pH and thermo stable nature of the xylanase enzyme which is required during bio-bleaching process. Moreover, successful utilization of spent residues from fermentation in dye removalprocess signify that the proposed technology can be utilized to meet the requirements of pulp andbleaching industries through an effective and sustainable approach.

© 2014 Elsevier Ltd. All rights reserved.

Introduction

Pulp and paper industries have been considered as majorpolluting units across the world (Sumathi and Hung, 2006; Ratiaet al., 2012). Different processes such as the pulp bleaching,deinking, production of colored paper etc. contribute to the pollu-tion caused by these industries. Last decade has seen an increase inthe number of studies focusing on the possible pollution reduction,alternative waste treatment technologies, and waste management(Monte et al., 2009).

Recently, utilization of microbial enzyme xylanase for pulp-bleaching has been intensely researched and adopted commer-cially (Polizeli et al., 2005). This results in cheaper and cleanerproduction process by avoiding the use of chlorine and significantlyreducing the discharge of the pollutants. To make the enzymeapplication in industries more cost effective, its production usingnegative value substrates like agro-wastes has been recommendedby many workers (Dhillon et al., 2011). Utilization of these agro-residues in bioprocesses has dual advantage of providing alterna-tive substrates as well as solving their disposal problems.

: þ91 11 26591121.shik).

Researchers have been utilizing sugarcane bagasse (Song and Wei,2010), wheat bran (Garai and Kumar, 2013), coba husk (Fang et al.,2010), jatropha cake (Chaturvedi et al., 2010) etc. as the substratesfor the production of enzymes through fermentation.

Kraft pulping requires high temperatures and high pH and asmost of the available xylanases are produced from mesophilic or-ganisms, they rapidly lose activity at temperatures above 50 �C andat pH above 7. Currently efforts are being made to producecellulase-free xylanases from thermophilic/thermotolerant micro-organisms which can retain their activity at alkaline pH and hightemperatures (Collins et al., 2005; Nigam, 2013). Also, duringxylanase production through solid state fermentation, after theextraction of enzyme, a lot of slurry comprising of degraded sub-strate and fungal biomass is produced which again has to bedisposed off. If this spent slurry can be utilized for the dye removalprocess (another pollutant from paper industries) followed bycomposting of the dye laden slurry, a zero waste objective could beaccomplished. Vermicomposting of such dye laden fermentedslurry by Kaushik et al. (2013) have been successfully demonstratedwhere the end product could be used as a soil conditioner.Although economically and ecologically favourable, such in-tegrations have not been tested practically at a larger scale.

The fungal isolate A. lentulus is an alkali, thermo and halotolerant fungus (Kaushik and Malik, 2010) and has already been

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implicated for dye (Kaushik and Malik, 2013) and metal (Mishraand Malik, 2012) remediation processes. The ability of this strainfor treatment of dye bearing wastewaters from textile and pulp andpaper industries has also been established. Thus in the presentstudy, the fungal isolate was tested for the production of xylanaseenzyme utilizing various low cost substrates and nitrogen sourcethrough solid state fermentation. Further the potential for appli-cation of this enzyme in bleaching process in terms of cellulase-freenature, pH and temperature optima and pH and thermo stabilityprofiles was evaluated. Simultaneously, utilization of spent fer-mented slurry (slurry left over after the enzyme elution) for theremoval of anionic (Acid Navy Blue) and cationic (Methylene Blue)dye was also attempted.

Materials and methods

Test organism

The experiments on xylanase productionwere performedwith astrain of A. lentulus FJ172995 previously isolated (Sharma, 2009)from the textile effluent procured from Baddi (Himachal Pradesh,India). The fungal isolate was maintained on slants of PotatoDextrose Agar. Freshly revived cultures were used for all theexperiments.

Screening of A. lentulus for xylanase production

The fungal isolate A. lentulus was initially screened for xylanaseproduction on xylan-agar plates containing 1% xylan and 0.1%Congo Red (Bajaj and Singh, 2010). Plates were spot inoculated withthe fungal spores and incubated at 30 �C for 5 days. The diameter ofthe clear zone formed around the fungal colony was measured ondaily basis.

Xylanase production through solid state fermentation

Xylanase production by A. lentulus through solid state fermen-tation of wheat bran was recorded at various time intervals. Six250 mL flasks were taken and 5 g of wheat bran was added to eachof them. The substrate was moistened with 15 mL of compositemedia (CM) comprising, Yeast extract 2.5 g/L, MgSO4 0.1 g/L,K2HPO4 0.5 g/L, NH4NO3 0.5 g/L and NaCl 1 g/L. The flasks wereautoclaved at 121 �C and 15 psi for 20 min. Sterile flasks containingsubstrate were then inoculated with 5% spore suspension andincubated at 30 �C. One flask was removed after every 24 h for sixdays and 100 mL of 0.05 M citrate buffer (pH 5.3) and Tween 80(0.1%) were added to it and it was agitated at 180 rpm for 1 h in anorbital shaker. The contents of the flasks were filtered throughWhatman No. 1 filter paper and the filtrate was centrifuged at10,000 rpm and 4 �C for 10 min and supernatant was collected andtested for crude endo-b-1,4-xylanase activity. Crude endo-b-1,4-xylanase enzyme activities were determined spectrophotometri-cally at 540 nm using di-nitrosalicylic (DNS) acid method based onthe release of reducing sugars from oat spelt xylan (Bailey et al.,1992) at pH 5.3 and temperature 50 �C utilizing 1% xylan as thesubstrate. For the sake of convenience and as per the literature, thisactivity has been termed as xylanase activity throughout the text.One unit of xylanase activity is defined as the amount of enzymerequired to produce 1 mmol of xylose per minute under standardassay conditions. Crude cellulase activity was determined throughfilter paper unit assay (Ghosh, 1987). One unit of filter paper unitassay is defined as the amount of enzyme required to release1 mmol of glucose equivalents under standard assay conditions(50 �C and pH 4.8). Total protein was estimated by Lowry's methodusing Bovine Serum Albumin as standard (Lowry et al., 1951).

Zymogram analysis

The crude enzyme extract obtained on the 4th day of solid statefermentation was partially purified through ammonium sulphateprecipitation. Ammonium sulphate was added to the crude enzymeextract to 40 and 75% saturation and stirred at 4 �C overnight. Thesaturated solution was later centrifuged and dissolved in citratebuffer (0.05 M). The dialyzed sample was then analyzed by nativePAGE utilizing 10% separating gel using BIORAD electrophoresisapparatus. The native protein markers and the partially purifiedenzyme extract were loaded. Electrophoresis was performed at aconstant voltage of 100 V for 3 h. After the run was completed, thegel containing the sample band was cut and rinsed with citratebuffer twice and incubated in 1% oat spent xylan for 5 min at 60 �C.The gel was then submerged in 0.1% Congo Red solution for 10 minand later washed with 1 M NaCl till the enzyme band appearedvisually. The rest of the gel containing protein markers was visu-alized through Coomassie Brilliant Blue R-250 staining. The mo-lecular weight of the active enzymewas estimated using the plot oflog value of weight of the markers versus the Rf value of themarkers. Rf value is calculated as the ratio of distance migrated bythe marker or sample to that migrated by the marker dye-front.

Effect of different substrates and nitrogen sources

Different agro-residues like wheat bran, wheat straw, corn coband sugarcane bagasse were taken as the substrates for productionof xylanase enzyme through solid state fermentation. Wheat branwas procured from the Yusuf Sarai Market (New Delhi, India)whereas wheat straw, corncob and sugarcane bagasse were pro-cured from agricultural fields of Bulandshahar, Uttar Pradesh (In-dia). The agricultural residues were characterized for water soluble,hemicellulose, cellulose and lignin content using the sequentialacid fractionation and gravimetric method described by Datta(1981). For solid state fermentation, 5 g of the substrate wastaken in 250 mL flask and 15 mL CM was added to the flask andmixed well. The flasks were autoclaved at 121 �C and 15 psi for20 min. Sterile flasks containing 5 g substrate were then inoculatedwith 5% spore suspension and incubated at 30 �C for 4 days. After 4days, 100 ml 0.05 M citrate buffer (pH 5.3) and Tween 80 (0.1%)were added to the flasks and the flasks were agitated at 180 rpm for1 h in an orbital shaker. The contents of the flasks were filteredthroughWhatman No.1 filter paper and the filtrate was centrifugedat 10,000 rpm and 4 �C for 10 min and supernatant was collectedand tested for crude xylanase and cellulase activity. Total protein inthe supernatant was also estimated. Effect of nitrogen source onxylanase production was tested by replacing the CM by the modi-fied media (MM) containing urea and ammonium chloride (Urea0.3 g/L, NH4Cl 0.2 g/L, MgSO4 0.1 g/L and K2HPO4 0.5 g/L).

Characterization of crude xylanase

The temperature optima of the crude xylanase was determinedby carrying out the enzyme reaction with substrate (1% xylan) atdifferent temperatures ranging from 50 to 90 �C for 5 min afterwhich the reaction was stopped by adding DNS reagent. The opti-mum pH of crude xylanase was determined at 50 �C by carrying outthe enzyme reaction at different pH values using the followingbuffers: 0.05 M citrate buffer (pH 4e6); 0.05 M phosphate buffer(pH 7e10).

The thermo-stability of the xylanase was studied at pH 5.3 byexposing the crude enzyme to a given temperature (ranging from50 to 80 �C) for varying time intervals up to 3 h and assaying theresidual xylanase activity under standard reaction conditions. ThepH stability of crude enzyme was studied by diluting the crude

Fig. 1. (a) Zymogram analysis of partially purified xylanase obtained from A. lentulus.Lane AeE represents the native marker, A: catalase (240.0 KDa), B: bovine serum al-bumin (67.0 KDa), C: Ovalbumin (43.0 KDa), D: Soyabean inhibitor (20.1 KDa), E:Lactoglobulin (18.4 KDa); Lane F: Zymogram of partially purified enzyme extract ofA. lentulus (b) Plot of Rf value against molecular mass of standard protein marker andpartially purified enzyme.

Table 1Composition of various agricultural residues used as the substrates for solid statefermentation.

Substrate Hot watersolubles (%)

Hemicellulose(%)

Cellulose(%)

Lignin (%)

Corn cob 10.8A ± 1.67 36.9C ± 1.25 26.0B ± 2.41 26.3B ± 2.97Sugarcane bagasse 23.1B ± 1.81 25.2D ± 1.96 23.2A,B ± 2.09 28.5B ± 1.69Wheat bran 12.1A ± 1.50 52.7A ± 2.70 20.6A ± 1.56 14.6A ± 2.67Wheat straw 10.2A ± 1.70 20.7B ± 1.23 33.8C ± 2.22 35.3C ± 3.29

aValues are represented as mean ± SD; n ¼ 3. Values within a column with super-scripts of same alphabet are statistically insignificant at p < 0.05.

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enzyme filtrate with buffers of respective pH values (ranging frompH 4e9), incubating for 3 h and estimating the residual xylanaseactivity under standard reaction conditions.

Application of spent slurry in dye biosorption

The test dyes taken in the study, Acid Navy Blue (C.I. Acid Blue120) and Methylene Blue (C.I. Basic Blue 9), were procured fromDepartment of Textile Technology, IIT Delhi, India. The stock solu-tion of 10,000 mg/L for both the dyes was prepared in distilledwater. The respective dye solutions were scanned over the visiblerange and absorption maximum (lmax) for both the dyes was esti-mated. Biosorption studies using the spent fermented slurry (left-over slurry after enzyme elution) were conducted in 100 mlErlenmeyer flask with a working volume of 50 mL at pH 6.5 andkept in an orbital shaker at 30 �C and 150 rpm. Initial concentrationof Acid Navy Blue and Methylene Blue dye was kept at 200 mg/Land 50 mg/L, respectively, while the biosorbent loading was 10 g/Lon dry weight basis. Suitable controls with raw agricultural resi-dues as the biosorbent were also run simultaneously.

Statistical analysis

All the studies were conducted in triplicates and the results arepresented as means of the replicates along with standard error ofthe mean (represented as error bars) unless stated otherwise. Thedatawas analyzed through DMRT (Duncan'sMultiple Range Test) atp � 0.05 in one-way ANOVA (Analysis of Variance) using SPSS(version 17.0) software.

Results and discussion

Screening of A. lentulus for xylanase production

The fungal isolate A. lentulus was initially screened for theproduction of xylanase enzyme through spot inoculation on xylan-agar plates containing 0.1% Congo Red. The formation of clear haloaround the fungal colony confirmed the secretion of extracellularxylanase by the fungal isolate. Diameter of 2.5 mm was obtainedafter 48 h of incubation which increased to 3.1 mm after 96 h. Thisis the first study reporting the production of xylanase enzyme byA. lentulus. Previous literature reports the production of thisenzyme by various other species of Aspergillus genus such asA. terrestris, A. fumigatus, A. niger, A. fischeri, A. ochraceus, A. ustus, A.kawachii, A. aculeatus, A. nidulans, A. sojae, A. terreus, A. sydowii, A.versicolour etc. (Beg et al., 2001; Polizeli et al., 2005).

Xylanase production through solid state fermentation andzymogram analysis

Initially, experiments were set to determine the time whenA. lentulus showed highest xylanase production in solid statefermentation using wheat bran as the substrate and yeast extract asthe nitrogen source. The time profile showed a gradual increase inxylanase production from 24 h (2.1 U/g) to 72 h (76.3 U/g) whichreached its maximum value after 96 h (135.9 IU/g). After this theactivity started to decline (123.3 U/g after 120 h) exhibiting 90.3 U/gactivity after 144 h of incubation.

Zymogram analysis of the partially purified enzyme extract ofA. lentulus grown on wheat bran is given in Fig. 1a. Lane AeE rep-resents the protein markers (18.4, 20.1, 43.0, 67.0, 240.0 KDa in size)obtained after the Coomassie Brilliant Blue R-250 staining of thegel. Lane F represents the zymogram of the partially purifiedenzyme obtained after Congo Red staining. Themolecular weight ofthe active enzyme, as calculated using the Rf value (Fig 1b) was

estimated to be 141.2 KDa. Souza et al. (2012) obtained multiplebands of varying sizes (94.9, 73.4 and 35.3 KDa) for endo-b-1,4-xylanase produced from A. fumigatus.

Effect of different substrates and nitrogen source on xylanaseproduction

Effect of various substrates on xylanase production by A. lentuluswas tested using various agricultural residues such as wheat bran,wheat straw, corn cob and sugarcane bagasse. The composition ofthese substrates obtained through acid fractionation and gravi-metric method has been tabulated in Table 1. Highest amount ofhemicellulose was present in wheat bran (52.7%) whereas the leastamount of hemicellulose was present in wheat straw (20.7%).Fig. 2a shows the profile of crude xylanase production by A. lentuluson various agro-residues as substrates for solid state fermentation.

Fig. 2. (a) Crude xylanase and cellulase activities (b) Total protein and specific xylanase activities obtained during solid state fermentation of different agro-residues by A. lentuluswith yeast extract (CM) and urea and ammonium chloride (MM) as the nitrogen source [temperature: 30 �C, incubation: 4 days; bars followed by same alphabets (in case ofxylanase activity and total protein) and numbers (in case of cellulase activity and specific xylanase activity) are statistically insignificant at p � 0.05].

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Although fungal growth was apparently abundant on all substrates,there were large differences in the production of xylanase. Highestxylanase activity (158.4 U/g) was obtained when wheat bran wasused as the substrate. The xylanase activity obtained with corn cob(153.0 U/g) was only marginally lower than wheat bran (which isstatistically insignificant at p� 0.05) followed by sugarcane bagasse(129.9 U/g) and wheat straw (49.4 U/g). The xylanase activitiesobtained with different substrates increased with the increasinghemicellulose content of the substrate. These xylanase activitieswere accompanied by very low cellulase activities; wheat straw(2.0 fpu/g) < wheat bran (1.9 fpu/g) < corn cob (1.4 fpu/g) < sugarcane bagasse (1.3 fpu/g). Highest amount of cellulaseenzyme was produced when wheat straw (which had significantlythe highest amount of cellulose) was used as the substrate. Secondhighest amount of cellulase was produced when wheat bran wasused as the substrate in spite of having low cellulose content ascompared to corn cob and sugar cane bagasse. This can be

attributed to the fact that wheat bran had significantly loweramount of lignin as compared to corncob and sugarcane bagassewhich could have hindered the exposure of cellulose towardsdegradation (Perez et al., 2002). The obtained values of cellulaseactivities are very low in proportion to the xylanase activities andthus xylanase produced on these substrates utilizing yeast extractas the nitrogen medium could be utilized for the applicationswhere cellulase-free xylanase is required such as for bleaching ofthe pulp in paper industries. Fig. 2b shows the total protein andspecific xylanase activity obtained with each of the substrate.Highest specific xylanase activity was obtained with corn cob as thesubstrate (4.9 U/mg).

Quiroz-Casta~neda et al. (2011) reported oak sawdust as the mostsuitable substrate among various agro-residues such as the cedarsawdust, wheat straw, rice husk, jatropha seed husk and cornstubble for the production of xylanase enzyme by Bjerkanderaadusta (0.4 U/mg) and Pycnoporus sanguineus (0.3 U/mg). Antoine

Fig. 3. (a) Relative xylanase activity obtained at different pH ranges (highest activity obtained at pH 5 taken as 100%; reaction conditions: temperature: 50 �C, time 5 min); barsfollowed by same alphabets are statistically insignificant at p � 0.05 (b) pH-stability profile for xylanase obtained from A. lentulus at different time intervals (activity obtained at 0 htaken as 100%; reaction conditions: temperature: 50 �C, pH: 5.3, time 5 min); bars for respective time interval followed by same alphabets are statistically insignificant at p � 0.05.

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et al. (2010) studied the production of extracellular xylanases byPenicillium canescens over various agro-industrial substrates suchas soya oil cake, soya meal, wheat bran, whole wheat bran and pulpbeet through solid state fermentation. Soya oil cake was observedto be the best substrate for xylanase production. Various otheragro-residues such rice bran, coconut coir pith, dry banana leaves,tea exhausts (Kar et al., 2013), cantaloupe, orange peel, banana peel,watermelon rinds (Mohammed et al., 2013) and Jatropha curcasseed-cake (Ncube et al., 2012) have been investigated as substratesfor enzyme production.

Effect of nitrogen source on xylanase production was alsostudied by replacing yeast extract containing CM with the previ-ously optimized modified media (MM) containing urea andammonium chloride (Kaushik and Malik, 2011). Fig. 2a shows thecrude xylanase activity obtained with different substrates whennitrogenwas provided by MM in place of CM. Comparable xylanaseactivities were obtained with urea and ammonium chloride as ni-trogen source with maximum amount of production onwheat bran(130.3 U/g) followed by corn cob (114.0 U/g), sugarcane bagasse(102.5 U/g) and wheat straw (57.5 U/g). Lower cellulase activity was

reported with each of the substrate in presence of MM as comparedto yeast extract. Following trend was observed for cellulase activity;wheat bran (1.9 fpu/g) < wheat straw (1.5 fpu/g) < sugarcanebagasse (1.3 fpu/g) < corn cob (1.2 fpu/g). Fig. 2b shows the totalprotein and specific xylanase activity obtained using differentsubstrates with MM. As the case with yeast extract, highest specificxylanase activity was obtained with corn cob. Thus corn cob wasselected as the substrate for conducting further characterizationexperiments. Since the crude xylanase activities obtained with MMwere comparable to those obtained with yeast extract containingCM, it was concluded that this low cost media could well be usedfor xylanase production. In a similar study, while studying the ef-fect of different media on xylanase production by Thermomyceslanuginosus through solid state fermentation, Sonia et al. (2005)tested the combination of various nitrogen sources such as yeastextract, proteose peptone and corn steep liquor with ammoniumsulphate as the nitrogen source. They observed maximum xylanaseproduction when ammonium sulphate was used as the sole nitro-gen source. The production decreased when ammonium sulphatewas combined with yeast extract, proteose peptone and corn steep

Fig. 4. (a) Relative xylanase activity obtained at different temperature ranges (highest activity obtained at 60 �C taken as 100%; reaction conditions: pH: 5.3, time 5min); bars followedby same alphabets are statistically insignificant at p� 0.05 (b) Thermo-stability profile for xylanase obtained fromA. lentulus at different time intervals (activity obtained at 0 h taken as100%; reaction conditions: temperature: 50 �C, pH: 5.3, time 5 min); bars for respective time interval followed by same alphabets are statistically insignificant at p � 0.05.

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liquor. Ninawe and Kuhad (2005) reported highest xylanase pro-duction by Streptomyces cyaneus with peptone (720 U/mL) as thenitrogen source followed by yeast extract (648 U/mL) while cornsteep liquor acted as the strong inhibiter for xylanase production.Amongst the inorganic sources, sodium nitrate induced maximumxylanase production (492 U/mL) while (NH4)2H2PO4 ceased thexylanase production totally. NH4Cl performedmoderately with 344U/mL xylanase production.

Characterization of crude xylanase produced by A. lentulus

The pH and thermal stabilities of cellulase-free xylanases areimportant attributes for their potential industrial applications. Thecrude xylanase enzyme obtained from A. lentuluswas characterizedfor its optimal pH and temperature range. The relative xylanaseactivities obtained at different pH ranges (4e10) are presented inFig 3a. The pH was adjusted with 0.5 M citrate buffer (pH 4e6) or0.5 M phosphate buffer (pH 7e10). The highest xylanase activitywas reported at pH 5 which reduced to 92% at pH 4. The decline inenzyme activity was marginal till neutral pH range (91.6% residual

activity at pH 7) however; it declined sharply in alkaline pH rangeswith 38.5% activity at pH 10 where statistically significant differ-ences were observed at p � 0.05. Although a sharp decline atalkaline pH was observed in the xylanase activity, still 77.2% and59.1% activity was retained at pH 8 and pH 9 which is the prevailingpH during the bleaching process of the pulp. The pH stability profilefor crude xylanase is represented in Fig. 3b. The data shows that theenzyme was more stable at acidic pH ranges. Good stabilities wereseen in the pH range of 4e7 even after 3 h of incubation. Enzymewasmost stable at pH 5, retaining more than 98% activity even after3 h of incubation. Even at pH 8 and 9, more than 85% and 75%enzyme stability, respectively, was observed after 3 h of incubation.

Fig. 4a represents the relative xylanase activity of the xylanaseproduced by A. lentulus at different reaction temperatures rangingfrom 50 to 90 �C. The optimum temperature for xylanase activitywas found to be 60 �C, with 94.1% activity obtained at 50 �C and86.4% at 70 �C. The enzyme activity declined sharply with furtherincrease in temperature (19.4% activity at 90 �C). Fig. 4b shows thethermo-stability profile of crude xylanase from A. lentulus. Forthermo-stability studies, the diluted crude enzyme was incubated

Fig. 5. Biosorption of (a) Acid Navy Blue (b) Methylene Blue dye using spent fermented slurry of different substrates after solid state fermentation with A. lentulus (bars followed bysame alphabets (dye removal) and numbers (biosorption capacity) are statistically insignificant at p � 0.05).

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at respective temperatures (50e80 �C) for different time intervalsand their activity was checked. Results show that the enzyme wasmost stable at 50 �C retaining 85.4% activity even after 60 min ofincubation. The enzyme had good stability at higher temperaturesfor initial 0.5 h (more than 85% activity retained at 60 and 70 �C).However, beyond 0.5 h, activity declined sharply at 70 �C (70.1%activity after 0.75 h min and 49.2% activity after 1 h). The declinewas steeper when enzyme was incubated at 80 �C, retaining only35.2% activity after 1 h of incubation. At the end of 3 h, 15.4% and4.8% enzyme activity was observed at 70 and 80 �C, respectively.The thermostability profile of endoxylanases could be furtherimproved using recombinant technology. Fedrova et al. (2012) re-ported the optimum activity of mutant endoxylanase at pH 6.0 andtemperature 70 �C with half inactivation period of 7h at 60 �Cwhenthe gene encoding endo-1,4 b-xylanase, gene xylE, from Penicilliumcanescens was expressed under the control of strong promoter, bgaS (gene encoding b-galactosidase).

Application of spent slurry in dye biosorption

Gupta and Suhas (2009) has advocated the use of low cost ad-sorbents such as waste industrial and agricultural products forremoving dye fromwastewater as they are present in abundance andare not recycled or used further. In the present study, after the

enzyme elution, the left over fermented slurry was utilized for thebiosorptionofAcidNavyBlue andMethyleneBluedyes. Fig. 5a showsthe removal of Acid Navy Blue and biosorption capacity obtainedwhen spent slurry from different agricultural residues was used.Results show more than 50% removal of Acid Navy Blue dye (initialconcentration 200mg/L)within 4 h of contact time obtainedwith allthe agricultural residues. Highest removal (65.0%)was obtainedwithwheat bran (biosorption capacity 13.2 mg/g), while the least dyeremoval (53.8%) was obtained with wheat straw (biosorption ca-pacity 10.7 mg/g). Considerably higher removal efficiencies wereobtainedwhen fermented slurrywasused as thebiosorbent in all thecases (the difference is statistically insignificant at p� 0.05). Highestefficiency was recorded for corn cob (88.9% removal in 4 h withbiosorption capacity of 17.9 mg/g) and wheat bran slurry (87.9%removal in 4 h with biosorption capacity of 17.7 mg/g).

When Methylene Blue dye was utilized in biosorption studies(Fig. 5b), marginally higher removal (62.8e68.0%) was obtainedwith biosorption capacity varying between 3.8 and 4.0 mg/g whenraw agricultural residues were used. Considerably higher efficiencywas obtained when spent fermented slurry of the respective resi-dues was utilized for the biosorption of Methylene Blue(96.3e97.9% removal in 4 h). Biosorption capacity also increasedand ranged from 5.6 to 5.8 mg/g (the difference was statisticallyinsignificant at p � 0.05). This observation assumes great

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significance asMethylene Blue dye poses problems evenwhen purefungal biomass is used (Kaushik and Malik, 2013), which otherwiseis an efficient biosorbent for awide variety of dyes belonging to azo,disazo and triphenylmethane class (Kaushik and Malik, 2010).

The results suggest the superiority of spent fermented slurryover raw residues in biosorbing synthetic dyes. Fermentation ofagricultural wastes with A. lentulus degrades the residues andmakes them more receptive for the dye molecules. Also, spentfermented slurry has an inherent advantage due to presence ofboth fungus as well as the agricultural residue, which might over-come the limitation posed by the individual biosorbent. Also, beinga by-product during the xylanase production for industrial appli-cations, the spent fermented slurry can prove to be an ideal bio-sorbent for dye removal processes especially for pulp and paperindustries that has to deal with colouredwastewater as well as havethe requirement for cellulase-free xylanase for pulp bleaching. Thistechnology thus can serve to meet the dual environmental chal-lenges that are frequently faced by such industries.

Fermentation of agricultural residues leads to their degradationdue to the production of various hydrolytic enzymes and makingthem more receptive for the biosorption of dye molecules. More-over, presence of fungi in the fermented slurry also provides theadditional sites for the binding of the dye molecules.

Conclusions

A. lentulus is an effective producer of xylanase enzyme whichcould be grown on various ago-residues yielding good enzymeactivities even with low cost nitrogen sources. Xylanase producedwas cellulase-free; exhibited alkali and thermo stability which isessential for bio-bleaching process. The left over spent slurry fromdifferent residues have the potential of removing >85% Acid NavyBlue and >96% Methylene Blue dye in 4 h. Xylanase production onagro-residues enhances the suitability of the bioprocess for pulpand paper industries as it can target two major polluting steps of:bleaching and production of coloured waste water.

Acknowledgements

CSIR Research Associateship (Grant no 9/86(1127)/12-EMR-I) toone of the authors (PK) is gratefully acknowledged. Mr. VinodKumar, Mr. Sabal Singh and Mr. Vikram Singh (Project Staff, IITDelhi, India) are thankfully acknowledged for their assistance inexperimental work.

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