Bioresource Technology 101 (2010) 7563–7569

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Production and extraction optimization of xylanase from Aspergillus nigerDFR-5 through solid-state-fermentation

Ajay Pal *, Farhath KhanumBiochemistry and Nutrition Discipline, Defence Food Research Laboratory, Siddarthanagar, Mysore 570 011, India

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

Article history:Received 7 December 2009Received in revised form 9 April 2010Accepted 13 April 2010Available online 15 May 2010

Keywords:OptimizationStatistical designXylanaseSolid-state-fermentationRecovery

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.04.033

* Corresponding author. Tel.: +91 821 2474676; faxE-mail address: ajaydrdo@rediffmail.com (A. Pal).

The effects of solid substrates, initial moisture content, moistening medium, temperature and incubationtime on xylanase production by Aspergillus niger DFR-5 was studied and the highest activity (2596 IU/gdry substrate (gds)) was achieved in medium that contained wheat bran (WB) and soybean cake (SBC)at a ratio of 70:30, was moistened to 70% with MSS-2 mineral salt solution, and incubated for 6 daysat 40 �C. Water at 37 �C was suitable for efficient recovery of enzyme from moldy WB–SBC medium.The extraction parameters for xylanase were optimized with respect to minimum volume of extractantusing a central composite rotatable design (CCRD). The maximum recovery of xylanase (4465 ± 52 IU/gds) with 92.5% desirability was obtained employing water (10 ml/gds) as extractant at 200 rpm for60 min. The result shows that an overall 5.4-fold increase in xylanase production was obtained in concen-trated form by optimizing medium components and extraction conditions.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Solid-substrate-fermentation (SSF) has recently gained impor-tance for the production of microbial enzymes due to several eco-nomic advantages over conventional submerged fermentation(SmF) (Maciel et al., 2008). Among the various groups of microor-ganisms used in SSF, filamentous fungi are the most widelyexploited because of their ability to grow on complex solid sub-strates and production of a wide range of extracellular enzymes(Badhan et al., 2007). Filamentous fungi have been widely usedto produce hydrolytic enzymes of industrial importance, includingxylanases, whose levels in fungi are generally much higher thanthose in yeast and bacteria. Applications of xylanases can be foundin the food and beverage industries (bakery goods, coffee, starch,plant oil and juice manufacture), feedstock improvement (increas-ing animal feed digestibility) and the quality improvement of lig-nocellulosic residues (Bakri et al., 2008).

On an industrial scale, xylanases are produced mainly by Asper-gillus and Trichoderma spp. (Kulkarni et al., 1999). The cost of anenzyme is one of the main factors determining the economics ofa process. Reducing the costs of enzyme production by optimizingthe fermentation medium and the process is the goal of basic re-search for industrial applications (Maciel et al., 2008). Other thanmedium optimization, recovery of enzyme is also an important as-pect of solid-state-fermentation technology. A wealth of informa-

ll rights reserved.

: +91 821 2473468.

tion is available on medium optimization for xylanase production(Gawande and Kamat, 1999; Park et al., 2002; Maciel et al., 2008)but studies on xylanase recovery from optimized medium arescarce (Heck et al., 2005). Therefore, we studied production andrecovery of xylanase from Aspergillus niger DFR-5. The solid-statemedium was optimized based on the ‘change-one-factor-at-a-time’ approach and detailed extraction studies were carried outusing response surface methodology (RSM) to optimize the recov-ery of xylanase from optimized medium. The RSM is an empiricalstatistical technique used to find the optimum conditions of a pro-cess response variable when the mechanism underlying the pro-cess is either not well understood or is too complicated to allowthe exact model to be formulated from theory. It evaluates the rela-tion existing between a group of controlled experimental factorsand the observed results of one or more selected variables (Parket al., 2002; Maciel et al., 2008).

2. Methods

2.1. Microorganism

The xylanase producing microorganism was isolated from asoil sample collected near fruit and vegetable debris in Mysore,India, and identified as A. niger, based on morphology, at IndianType Culture Collection (ITCC), New Delhi, India. The isolate,named A. niger DFR-5, was grown on potato dextrose agar(PDA) slants at 30 �C for 5 days and subsequently stored at4 �C. Inoculum was prepared by suspending the spores from

7564 A. Pal, F. Khanum / Bioresource Technology 101 (2010) 7563–7569

PDA slants by adding sterile 0.1% tween-80 to give a final countof �1 � 106 spores/ml.

2.2. Optimization strategies for xylanase production in SSF

2.2.1. Solid-state-fermentation and enzyme assaySugarcane bagasse (SB), aloe-vera skin (AS), wheat bran (WB),

musambi peel (MP), pineapple peel (PP), banana peel (BP), ricebran (RB) and maize involucre (MI) were evaluated for their poten-tial as substrate in SSF for xylanase production. Static experimentswere conducted in 500-ml flasks containing 10 g (dry weight)powdered solid substrates moistened at 58.33% level with distilledwater. The flasks were sterilized at 121 �C for 30 min and, aftercooling, inoculated with 1 ml of spore suspension followed byincubation at 37 �C for the 5 days. The initial moisture content,determined gravimetrically, was adjusted to 60%. All liquid addedto the flask was taken into consideration in calculating the mois-ture content.

To extract the enzyme, fermented matter was mixed with cit-rate buffer (0.05 M, pH 5.0) at a solid/liquid ratio of 1 g initial drysubstrate/12 ml buffer. The mixture was stirred at 37 �C and150 rpm for 50 min. Subsequently, solids were separated fromthe extract by centrifugation at 13,500g for 15 min. The superna-tant was filtered through Whatman No. 1 filter paper to obtain aclear extract and assayed for xylanase activity by the method ofKhanna and Gauri (1993) with slight modifications. The solutionof xylan and the enzyme extract at appropriate dilution was incu-bated at 37 �C for 30 min and the reducing sugars were determinedby the dinitrosalicylic acid method described by Miller (1959),with xylose as the standard. The released xylose was measuredspectrophotometrically at 540 nm. One unit of xylanase is definedas the amount of enzyme required to release 1 lmol of reducingsugar as xylose equivalent/min under the assay conditions. Theunit ‘IU/gds’ denotes the xylanase activity in international unitper gram dry substrate. All the experiments were done in triplicate.

2.2.2. Selection of solid substrates and their ratio for xylanaseproduction

The effect of supplementation of four nitrogenous substrates(powder of cottonseed (CSC), rapeseed (RSC), soybean (SBC) andpeanut cake (PNC) separately mixed with wheat bran) on xylanaseproduction was studied. The proportions of the bran to the cakeswere 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6 (w/w).

2.2.3. Selection of initial moisture content (IMC) and moisteningmedium

The effect of IMC (55%, 60%, 65%, 70%, 75%, 80% w/w), adjustedby adding double distilled water (DDW), was investigated. All li-quid added to the flask was taken into consideration in calculatingthe moisture content.

The effect of mineral salt solutions MSS-1 (Badhan et al., 2007),(g/l: (NH4)2SO4 3.0, KH2PO4 3.0, CH3COONH4 6.0, pH 6.0), MSS-2(Camassola and Dillon, 2007), (g/l: KH2PO4 20.0, (NH4)2SO4 13.0,CO(NH2)2 3.0, MgSO4 3.0, CaCl2 3.0, FeSO4 0.05, MnSO4 0.015,ZnSO4 0.014, CoCl2 0.002, pH 6.0), MSS-3 (Seyis and Aksoz, 2005),(g/l: FeSO4 0.05, ZnSO4 0.014, CoCl2 0.02, pH 6.0), MSS-4 (Nairet al., 2008), (g/l: KCl 0.5, MgSO4 0.5, (NH4)2HPO4 2.5, NaH2PO4

0.5, CaCl2 0.01, FeSO4 0.01, ZnSO4 0.002, pH 6.0) and tap water(TW) as a moistening agent at 70% IMC was determined as compar-ison to DDW.

2.2.4. Optimization of incubation temperature and periodThe yields of xylanase in five batch fermentations conducted at

different temperatures (30, 33, 37, 40 and 43 �C) were measuredfrom 2 to 9 days at an interval of 24 h.

2.3. Xylanase recovery from optimized solid-state medium

2.3.1. Selection of extraction solvent and temperatureFive solvents (DDW pH 7.0, 0.05 M Tris–HCl buffer pH 8.0,

0.05 M citrate buffer pH 5.0, 0.1% tween-80 and 1% NaCl) pre-incu-bated at preset temperatures (7, 25, 37 and 45 �C) were added toErlenmeyer flasks containing the cultivated solids at solid/liquidratios of 1 g initial dry substrate/12 ml of solvent. The mixturewas stirred for 50 min (150 rpm) for enzyme extraction. Theextraction process was studied at 7, 25, 37 and 45 �C for eachsolvent.

2.3.2. Interactive effect of extraction parameters on the recovery ofxylanase

A CCRD comprising of three variables (extraction time (min),agitation (rpm) and volume of extractant (ml/g initial dry sub-strate)) at five levels (preliminary experiments were carried outto determine the parameters range) was used to study their inter-active influence on xylanase recovery from optimized solid-statemedium. The result of CCRD with six replicates at the center pointwas used to fit the second order response surface. In developingthe regression equation the test factors were coded according tothe following equation:

xi ¼ Xi � X0=DXi

where, xi is the dimensionless coded value of the ith independentvariable; Xi the natural value of the ith independent variable; X0

the natural value of the ith independent variable at the center pointand DXi the step change value. Once the experiments were per-formed, the experimental results were fitted with a 2nd order poly-nomial function:

Y ¼ b0 þ b1x1 þ b2x2 þ b3x3 þ b11x21 þ b22x2

2 þ b33x23 þ b12x1x2

þ b13x1x3 þ b23x2x3

where, Y is the predicted response; b0 the intercept; b1, b2, b3 thelinear co-efficient; b11, b22, b33 the squared co-efficient and b12,b13, b23 the interaction co-efficients.

2.3.3. Optimization of extraction parameters and data analysisThe extraction parameters were numerically optimized with

the constraint of ‘minimum’ volume of extractant while keepingthe other variables ‘in range’. All the statistical experimental de-signs and results analysis were carried out using Design expert7.1.4 (Stat-ease, Inc., Minneapolis, USA) and Minitab 15 (MinitabInc., State College, PA, USA) softwares. In experiments other thanRSM, the significance of differences among mean values was calcu-lated according to Student’s t-test (Fisher and Yates, 1963).

2.4. Laboratory level scale up of xylanase production

Xylanase production by A. niger DFR-5 under optimized solid-state fermentation conditions was attempted by growing the cul-ture in different sized flasks, i.e. 500 ml (10 g dry substrate) to5000 ml (100 g dry substrate).

3. Results and discussion

3.1. Screening of agro-industrial residues for xylanase production inSSF

Among the agro-industrial residues, wheat bran was the mosteffective substrate (Fig. 1). Enzyme activity was significantly high-er (p < 0.05) in wheat bran-based SSF medium than in the nextmost effective substrate, pineapple peel. There are several reportsdescribing wheat bran as the potent substrate for xylanase produc-

600

675

750

825

900

SB AS WB MP PP BP RB MIAgroindustrial residues

Xyla

nase

act

ivity

(IU

/gds

)

Fig. 1. Potential of agro-industrial wastes for xylanase production by A. niger DFR-5.

1350

1425

1500

1575

A

B

1650

1725

55 60 65 70 75 80

Initial moisture content (%)

Xyla

nase

act

ivity

(IU

/gds

)

1800

1875

gds)

A. Pal, F. Khanum / Bioresource Technology 101 (2010) 7563–7569 7565

tion by Arthrobacter sp. and Rhizopus stolonifer in SSF (Khandepar-kar and Bhosle, 2006; Goulart et al., 2005).

3.2. Selection of solid substrates and their ratio for xylanase production

The selection of a suitable combination of carbonaceous (C) andnitrogenous (N) substrates is a critical factor for a SSF process. Thesubstrates should not only provide nutrient for the microorgan-isms, but also allow oxygen transfer and heat dispersion. A suitableprotein rich material should be in powder form to increase sub-strate consumption rate and allow easy mixing with the C sub-strate. The ratio between C and N sources is also an importantfactor because an overabundance of the N source decreased theporosity of medium (Jian et al., 2005). Wheat bran is a suitable sup-porter and carrier because of its porosity and cheapness. Xylanaseactivities varied markedly with the substrate proportions tested(Table 1). The lowest value (820 IU/gds) was obtained when onlyWB was used for fermentation and the highest xylanase produc-tion (1545 IU/gds) was achieved when the ratio between WB andSBC powder was 7:3 (w/w). These results showed that the amountof nitrogen present in wheat bran is too low to support goodgrowth and enzyme production. A similar finding was made byPark et al. (2002), working with rice straw. Maciel et al. (2008) alsoshowed that highest xylanase activity from A. niger was achievedwhen sugarcane bagasse was mixed with soybean meal in a ratioof 1.86:1.

3.3. Selection of IMC and moistening medium

The IMC has been shown to affect the production of hydrolyticenzymes under SSF conditions by influencing the growth of the

Table 1Effect of substrates ratio on xylanase production by A. niger DFR-5.

Ratio Xylanase activity (IU/gds)*

WB:CSC WB:RSC WB:SBC WB:PNC

10:0 820 ± 10Ai 820 ± 10Ai 820 ± 10Ai 820 ± 10Ai

9:1 938 ± 12Aj 974 ± 12Bj 1008 ± 12Cj 945 ± 12Aj

8:2 1080 ± 14Ak 1120 ± 14Bk 1290 ± 13Ck 1080 ± 13Ak

7:3 1165 ± 15Al 1372 ± 16Bl 1545 ± 14Cl 1192 ± 14Al

6:4 1280 ± 16Am 1315 ± 15Bm 1415 ± 16Cm 1325 ± 16Bm

5:5 1170 ± 15Al 1205 ± 16Bn 1322 ± 17Cn 1440 ± 18Dn

4:6 1025 ± 16An 1098 ± 17Bk 1210 ± 16Co 1278 ± 16Do

Means ± SD with different small letters as superscripts in column and capital lettersas superscripts in row differ significantly (p < 0.05).* Values are Mean ± SD of three experiments.

organism (Nishio et al., 1979; Ramesh and Lonsane, 1990). In thepresent investigation, six moisture levels ranging from 55% to80% were established to study their effect on xylanase productionand the results obtained are shown in Fig. 2A. The highest produc-tion of xylanase was obtained when the IMC was 70%. Either low orhigh initial moisture significantly (p < 0.05) decreased the enzymeproduction for the reason that low moisture substrates reducemass transfer process and high moisture substrates reduce theporosity of the WB–SBC medium (Adinarayana et al., 2003). The ef-fect of moisture content on xylanases production has previouslybeen studied (Kheng and Omar, 2005; Yang et al., 2006) and arequirement of �43% and 83% initial moisture content for maxi-mum xylanases production by A. niger USM AI 1 and Paecilomycesthermophila J18, respectively, was determined.

Besides DDW, other moistening agents consisting of mineralsalt solutions and tap water were examined, and results (Fig. 2B)showed that the composition of the moistening media profoundlyaffected xylanase yield. Mineral salt solutions and tap water re-sulted in higher xylanase production as compared to DDW alone.Enzyme production using MSS-2 was significantly higher(p < 0.05) than that of the nearest most effective moistening agent,MSS-4 and therefore selected as the moistening agent in further re-search. In addition, when MSS-2 with pHs between 4.5 and 8 wasused, no significant differences were found in the enzyme yield

1575

1650

1725

DDW TW MSS-1 MSS-2 MSS-3 MSS-4

Moistening agent

Xyla

nase

act

ivity

(IU

/

Fig. 2. (A) Effect of initial moisture content on xylanase production by A. nigerDFR-5, (B) Effect of moistening agent on xylanase production by A. niger DFR-5.

7566 A. Pal, F. Khanum / Bioresource Technology 101 (2010) 7563–7569

(data not shown). This could be explained by the fact that wheatbran possesses excellent buffering capacity (Pandey et al., 1999,2001). Similar observations were made by Nampoothiri et al.(2004) and Sandhya et al. (2005) for chitinase and neutral proteaseproduction in wheat bran-based medium.

3.4. Optimization of incubation temperature and period

The highest production of xylanase (2596 IU/gds) was obtainedat 40 �C after 6 days and at higher temperatures, its production de-creased sharply (Fig. 3). Although the physiological changes in-duced by high temperatures during enzyme production are notcompletely understood, it has been suggested that at high temper-atures, microorganisms may synthesize only a reduced number ofproteins essential for growth and other physiological processes(Gawande and Kamat, 1999). With prolonged incubation, enzymeactivity decreased sharply suggesting that the end-point of fer-mentation should be carefully controlled because synthesizedxylanase could be degraded by non-specific proteases secreted bythe fungus.

3.5. Xylanase recovery from optimized solid-state medium

3.5.1. Selection of extraction solvent and temperatureRecovery of enzyme from the solid medium is another impor-

tant aspect of SSF. An ideal solvent would extract the enzyme

0

500

1000

1500

2000

2500

3000

0 2 3 4 5 6 7 8 9

Period (days)

Xyla

nase

act

ivity

(IU

/gds

)

30°C

33°C

37°C

40°C

43°C

Fig. 3. Effect of incubation temperature and period on xylanase production by A.niger DFR-5.

Table 2Effect of extraction solvent and temperature on xylanase recovery.

Xylanase activity (IU/gds)*

7 �C 25 �C 37 �C 45 �C

Citrate buffer(0.05 M, pH 5.0)

2295 ± 35Ai 2515 ± 38Bi 2596 ± 34Ci 2615 ± 40Ci

Tris buffer (0.05 M,pH 8.0)

1869 ± 25Aj 1786 ± 28Bj 1705 ± 29Cj 1324 ± 26Dj

DDW (pH 7.0) 2450 ± 34Ak 2770 ± 40Bk 2885 ± 39Ck 1995 ± 37Dk

Tween-80 (0.1%) 1685 ± 24Al 1992 ± 30Bl 2105 ± 32Cl 2015 ± 27Bk

NaCl (1%) 2185 ± 28Am 2350 ± 33Bm 2485 ± 36Cm 2212 ± 29Al

Means ± SD with different small letters as superscripts in column and capital lettersas superscripts in row differ significantly (p < 0.05).* Values are Mean ± SD of three experiments.

selectively and completely at room temperature with minimal con-tact time. In the initial solvent screening experiments, water(2885 IU/gds at 37 �C) proved suitable for efficient recovery ofthe enzyme from moldy WB–SBC medium (Table 2). Extractionswith all other solvents showed significantly lower (p < 0.05) en-zyme recovery. Since water has a higher dielectric constant thanthe other extractants used, the interactive forces between xylanaseand substratum are reduced and therefore recovery of xylanasewas increased (Heck et al., 2005). The hydrophobic/hydrophilicnature of fungal mycelia, ionic bonds, hydrogen bonds and van-der Waal forces also determine the efficacy of the extractant aspointed out by Fernendez-Lahore et al. (1998). In earlier studieson solid-state production of xylanases, water has been used as sol-vent (Liu et al., 1998; Heck et al., 2002).

3.5.2. Interactive effect of extraction parameters on the recovery ofxylanase

After selecting water as the best extractant for xylanase activityfrom optimized solid substrate medium, a full factorial CCRD wasapplied to study the interactive effect of extraction time (min), agi-tation (rpm) and volume of extractant (ml/gds) on the recovery ofxylanase and to derive a statistical model for their effects. Therange of the independent variables is presented in Table 3.

A second order polynomial model was fitted to theobtained xylanase recovery. The statistical combinations of thecritical components with the observed recovery of xylanase arelisted in Table 4. The application of the RSM yielded the followingregression equation which is an empirical relationship betweenthe xylanase recovery and the test variables in coded units:

Xylanase activity ðIU=gdsÞ¼ 2903:52þ 512:28 Timeþ 736:76 Agitation

� 490:91 Volume of extractant� 401:24 Time2

þ 149:59 Agitation2 � 361:11 Volume of extractant2

� 159:5 Time� Agitation� 46:0 Time� Volume of extractant� 595:0 Agitation� Volume of extractant

Run number 12 showed the highest level of xylanase recovery(5235 IU/gds). The xylanase recovery obtained with these condi-tions was more than double of the recovery obtained earlier(2596 IU/gds at 50 min, 150 rpm and 12 ml citrate buffer/gds) indi-cating the necessity to optimize the extraction parameters. Analy-sis of variance (ANOVA) was employed to determine thesignificance of the regression model (Table 5). The computed F-va-lue (18.41) was almost 6-fold higher than the tabulated value (F9,10

(1%) = 3.02). The Fisher F-test with a very low probability value(0.0001) demonstrates a very high significance for the regressionmodel and confirms the adequacy of the quadratic model (Franciset al., 2003). The regression equation obtained indicated the R2 va-lue of 0.9431 (a value of R2 > 0.75 indicates the aptness of the mod-el). This value ensured a satisfactory adjustment of the quadraticmodel to explain the experimental data and indicated that modelcould explain �94% of the variability in the response.

Table 3The experimental domain.

Independent variables Symbol Range and level�a* �1 0 +1 +a

Time (min) x1 16.36 30 50 70 83.64agitation (rpm) x2 65.91 100 150 200 234.09Volume of extractant

(ml/gds)x3 8.64 10 12 14 15.36

* a = 1.682.

Table 4Experimental design and results.

Runnumber

Coded level Response (IU/gds) Relative deviation(%)

x1 x2 x3 Observed* Predicted

1 �1 �1 �1 1046 ± 13 732 �42.892 +1 �1 �1 2278 ± 34 2168 �5.073 �1 +1 �1 3431 ± 40 3715 7.644 +1 +1 �1 4296 ± 47 4512 4.785 �1 �1 +1 1079 ± 11 1032 �4.556 +1 �1 +1 2398 ± 35 2284 �4.997 �1 +1 +1 1355 ± 17 1635 17.128 +1 +1 +1 1765 ± 20 2248 21.489 �a 0 0 946 ± 12 907 �4.29

10 +a 0 0 2831 ± 39 2630 �7.6411 0 �a 0 1658 ± 17 2088 20.5912 0 +a 0 5235 ± 59 4566 �14.6513 0 0 �a 2671 ± 39 2708 1.3614 0 0 +a 1333 ± 18 1056 �26.2315 0 0 0 2924 ± 41 2904 �0.6816 0 0 0 2870 ± 39 2904 1.1717 0 0 0 2880 ± 37 2904 0.8218 0 0 0 2914 ± 41 2904 �0.3419 0 0 0 2892 ± 40 2904 0.4120 0 0 0 2900 ± 41 2904 0.13

Relative deviationð%Þ ¼ Predicted response�observed responsePredicted response � 100.

* Values are Mean ± SD of three experiments.

A. Pal, F. Khanum / Bioresource Technology 101 (2010) 7563–7569 7567

The significance of each co-efficient was determined by Stu-dent’s t-test (Table 6). The smaller the p- and larger the t-value,the more significant is the corresponding co-efficient (Myers andMontogomery, 2002). Student’s t-test showed that all the linearco-efficients were highly significant (p 6 0.01). The second orderterms of time and volume of extractant as well as the interactionterm of agitation and volume of extractant were also equallysignificant while remaining terms were insignificant. The highsignificance of these terms indicates that they can act as limitingfactors and even small variations in their values will alter xylanaserecovery to a considerable extent (Adinarayana et al., 2003). Thesign and magnitude of the co-efficients indicate the effect of thevariable on the response.

The recovery of xylanase for different variables could also bepredicted by the contour plots. The interaction between agitation

Table 5Regression analysis (ANOVA) for xylanase recovery.

Source SS DF MS F-value p-Value

Model 2.186E* + 07 9 2.429E + 06 18.41 0.0001Residual 1.320E + 06 10 1.320E + 05Total 2.318E + 07 19

SS, Sum of Square; DF, Degree of Freedom; MS, Mean Square.R2 = 94.31%; Table F9,10 (1%) = 3.02.* E represents times ten raised to the power.

Table 6Co-efficients of the regression equation.

Term Co-efficient t-Value p-Value

Constant 2903.5 19.598 0.000Time 512.3 5.212 0.000Agitation 736.8 7.495 0.000Volume of extractant �490.9 �4.994 0.000Time2 �401.2 �4.193 0.002Agitation2 149.6 1.563 0.149Volume of extractant2 �361.1 �3.774 0.004Time � Agitation �159.5 �1.242 0.243Time � Volume of extractant �46.0 �0.358 0.728Agitation x Volume of extractant �595.0 �4.633 0.000

and volume of extractant is statistically significant and the contourcurve indicates that xylanase recovery is affected dominantly andpositively by agitation without being much influenced by volumeof extractant (Fig. 4). The maximum enzyme recovery was ob-tained at highest agitation rate under study with low volume ofextractant. The interaction between extraction time and volumeof extractant revealed that an extraction time lower than 60 minand a volume of extractant more than 11 ml/gds is not suitablefor total solubilization of enzymes present in the cultivated solids(data not shown). The contour plot indicated an optimum enzymerecovery in the range of time �60–70 min and a volume of extract-ant of �10–11 ml/gds. Extraction times of more than 70 min re-sulted in low enzyme recovery. According to Ghildyal et al.(1991), prolonged agitation and extraction of denaturing agentscan cause a loss of enzyme activity. The effect of variations in thelevel of all three independent variables on xylanase recovery isshown in the perturbation graph (Fig. 5). The plot revealed thatxylanase recovery was sensitive to changes in all variables. Xylan-ase recovery increased with increase in agitation and decreasedwith increased volume of extractant. The extraction time also in-creased xylanase recovery linearly up to center value and with fur-ther increase, there was no discernable effect on xylanase recovery.

3.5.3. Numerical optimization of the xylanase extraction conditionsThe extraction parameters were numerically optimized using

Design-Expert software. The criteria used for optimization alongwith predicted and actual (observed) response value are presentedin Table 7. Our aim was to ‘minimize’ the volume of extractantwhile keeping the extraction time and agitation ‘in range’.

By using the given criteria, a solution having maximum desir-ability of 92.5% was selected and experiments were conducted.The observed response (4465 ± 52 IU/gds) was 96.79% to the pre-dicted outcome (4613 IU/gds). This finding is extremely importantbecause concentrated crude extract ease downstream processes,mainly the purification steps, reduce time and cost of enzymerecovery. The use of small volumes of solvent for xylanase extrac-tion greatly reduces energy requirements, equipment size and pol-lution but, volumes that are too low could lead to unsatisfactoryrecoveries, since a significant fraction of the xylanase could be re-tained in the cultivated mass (Singh et al., 1999). In our study, themodel developed with a low volume of extractant gave satisfactoryxylanases recovery.

DESIGN-EXPERT Plot

Actual Factors:

X = Agitation

Y = Volume of extractant

Actual Constants:

Time = 50.00

Xylanase activity

Agitation

Volu

me

of e

xtra

ctan

t

100.00 125.00 150.00 175.00 200.0010.00

11.00

12.00

13.00

14.00

2768.74

3250.01

2986.89

3932.8

3567.99

2563.85

2387.71

2179.53

2179.53

4239.43

6

Fig. 4. Effect of volume of extractant and agitation on xylanase recovery from solidmedium at fixed value of extraction time.

Perturbation

Deviation from Reference Point

-1.000 -0.500 0.000 0.500 1.000

1989.995

2439.964

2889.933

3339.902

3789.871

A

A

B

B

C

C

Time

Agitation

Volume of extractant

Xyla

nase

act

ivity

Fig. 5. Perturbation graph showing the effect of time, agitation and volume ofextractant on xylanase recovery from solid medium.

Table 7Constraints, criteria for optimization, solution along with predicted and observedresponse value.

Constraints Goal Importance Solution Observedresponse*

Time (min) In range 3 60.0 –Agitation (rpm) In range 3 200 –Volume of extractant

(ml/gds)Minimize 4 10 –

Xylanase activity(IU/gds)

Maximize 4 4613 4465 ± 52

* Values are Mean ± SD of three experiments.

7568 A. Pal, F. Khanum / Bioresource Technology 101 (2010) 7563–7569

3.6. Laboratory level scale up of xylanase production

To scale up the xylanase production at laboratory level, theculture was grown in flasks ranging from 500 to 5000 ml capac-ity containing 10–100 g dry substrate. Almost the same level ofxylanase (4460 ± 60 IU/gds) was produced in all the flasks indi-cating that xylanase production can easily be scaled up usingoptimized conditions. Further work on the purification and de-tailed characterization of xylanase from A. niger DFR-5 is inprogress.

4. Conclusions

Xylanase production could be improved significantly by incor-porating SBC powder into the fermentation mixture along withWB. Under optimized conditions, the maximum recovery(4465 ± 52 IU/gds) was obtained employing water (10 ml/gds) asthe extractant at 200 rpm for 60 min, with 92.5% desirability. Otherstrategies such as strain mutation may also be applied to enhancethe xylanase activity. Further scaling-up of xylanase productionand subsequent recovery deserves more attention to reach com-mercial feasibility.

Acknowledgement

The authors are grateful to Dr. A.S. Bawa, Director, DefenceFood Research Laboratory, Mysore, for providing all the necessary

facilities, constant guidance and encouragement during thisinvestigation.

References

Adinarayana, K., Ellaiah, P., Srinivasulu, B., Devi, R.B., Adinarayana, G., 2003.Response surface methodological approach to optimize the nutritionalparameters for neomycin production by Streptomyces marinensis under solid-state fermentation. Process Biochem. 38, 1565–1572.

Badhan, A.K., Chadha, B.S., Kaur, Jatinder., Saini, H.S., Bhat, M.K., 2007. Production ofmultiple xylanolytic and cellulolytic enzymes by thermophilic fungusmyceliophthora sp. IMI 387099. Biores. Technol. 98, 504–510.

Bakri, Y., Al-Jazairi, M., Al-Kayat, G., 2008. Xylanase production by a newlyisolated Aspergillus niger SS7 in submerged culture. Pol. J. Microbiol. 57 (3),249–251.

Camassola, M., Dillon, A.J.P., 2007. Production of cellulases and hemicellulases byPenicillium echinulatum grown on pretreated sugarcane bagasse and wheat branon solid state fermentation. J. Appl. Microbiol. 103 (6), 2196–2204.

Fernendez-Lahore, H.M., Fraile, E.R., Cascone, O., 1998. Acid protease recovery froma solid-state fermentation system. J. Biotechnol. 62, 83–93.

Fisher, R.A., Yates, F., 1963. Statistical Analysis for Biological, Agricultural andMedical Research. Oliver and Boyd, Edinburgh.

Francis, F., Sabu, A., Nampoothiri, K.M., Ramachandram, S., Ghosh, S., Szakacs, G.,2003. Use of response surface methodology for optimizing process parametersfor the production of alpha-amylase by Aspergillus oryzae. Biochem. Eng. J. 15,107–115.

Gawande, P.V., Kamat, M.Y., 1999. Production of Aspergillus xylanase bylignocellulosic waste fermentation and its application. J. Appl. Micorbiol. 87,511–519.

Ghildyal, N.P., Ramakrishna, M., Lonsane, B.K., Karanth, N.G., 1991. Efficient andsimple extraction of mouldy bran in a pulsed column extractor forrecovery of amyloglucosidase in concentrated form. Process Biochem. 26,235–241.

Goulart, A.J., Carmona, E.C., Monti, R., 2005. Partial purification and properties ofcellulase free alkaline xylanase produced by Rhizopus stolonifer in solid-statefermentation. Braz. arc. biol. Technol. 48 (3), 327–333.

Heck, J.X., Hertz, P.F., Ayub, M.A.Z., 2002. Cellulase and xylanase production byisolated amazon Bacillus strains using soybean industrial residue based solid-state cultivation. Braz. J. Microbiol. 33, 215–220.

Heck, J.X., Hertz, P.F., Ayub, M.A.Z., 2005. Extraction optimization of xylanasesobtained by solid-state cultivation of Bacillus circulans BL 53. Process Biochem.40, 2891–2895.

Jian, X., Shouwen, C., Ziniu, Y., 2005. Optimization of process parameters for poly c-glutamate production under solid state fermentation from Bacillus subtilisCCTCC202048. Process Biochem. 40, 3075–3081.

Khandeparkar, R.D.S., Bhosle, N.B., 2006. Isolation, purification and characterizationof the xylanase produced by Arthrobacter sp. MTCC 5214 when grown in solid-state fermentation. Enzyme microb. technol. 39, 732–742.

Khanna, S., Gauri, 1993. Regulation, purification and properties of xylanase fromcellulomonas fimi. Enzyme Microb. Technol. 15, 990–995.

Kheng, P.P., Omar, I.C., 2005. Xylanase production by a local fungal isolate,Aspergillus niger USM AI 1 via solid state fermentation using palm kernel cake(PKC) as substrate. Songklanakarin J. Sci. Technol. 27 (2), 325–336.

Kulkarni, N., Shendye, A., Rao, M., 1999. Molecular and biotechnological aspects ofxylanases. FEMS Microbiol. Rev. 23, 411–456.

Liu, W., Zhu, W., Lu, Y., Kong, J., Ma, G., 1998. Production, partial purification andcharacterization of xylanase from Trichosporon cutaneun SL409. ProcessBiochem. 33, 331–336.

Maciel, G.M., Vandenberghe, L.P.S., Haminiuk, C.W.I., Fendrich, R.C., Bianca, B.E.D.,Brandalize, T.Q.S., Pandey, A., Soccol, C.R., 2008. Xylanase production byAspergillus niger LPB 326 in solid-state fermentation using statisticalexperimental designs. Food Technol. Biotechnol. 46 (2), 183–189.

Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducingsugar. Anal. Chem. 31, 426–428.

Myers, R.H., Montogomery, R.C., 2002. Response Surface Methodology: Process andProduct Optimization using Designed Experiments. Wiley, New York.

Nair, S.G., Sindhu, R., Shashidhar, A., 2008. Fungal xylanase production under solidstate and submerged fermentation conditions. African J. Microbiol. Res. 2, 82–86.

Nampoothiri, K.M., Baiju, T.V., Sandhya, C., Sabu, A., Szakacs, G., Pandey, A., 2004.Process optimization for antifungal chitinase production by Trichodermaharzianum. Process Biochem. 39, 1583–1590.

Nishio, N., Tai, K., Nagai, S., 1979. Hydrolase production by Aspergillus niger in solidstate cultivation. Eur. J. Appl. Microbiol. Biotechnol. 8, 263–270.

Pandey, A., Selvakumar, P., Soccol, C.R., Nigam, P., 1999. Solid-state fermentation forthe production of industrial enzymes. Curr. Sci. 77, 149–162.

Pandey, A., Soccol, C.R., Rodriguez-Leon, J.A., Nigam, N. (Eds.), 2001. Solid-stateFermentation in Biotechnology: Fundamentals and Application. AsiatechPublishers, New Delhi, p. 17.

Park, Y., Kang, S., Lee, J., Hong, S., Kim, S., 2002. Xylanase production in solid-statefermentation by Aspergillus niger mutant using statistical experimental designs.Appl. Microbiol. Biotechnol. 58, 761–766.

Ramesh, M.V., Lonsane, B.K., 1990. Critical importance of moisture content ofthe medium in alpha amylase production by Bacillus licheniformis M 27 ina solid state fermentation system. Appl. Microbiol. Biotechnol. 33, 501–505.

A. Pal, F. Khanum / Bioresource Technology 101 (2010) 7563–7569 7569

Sandhya, C., Sumantha, A., Szakacs, G., Pandey, A., 2005. Comparative evaluation ofneutral protease production by Aspergillus oryzae in submerged and solid-statefermentation. Process Biochem. 40, 2689–2694.

Seyis, I., Aksoz, N., 2005. Xylanase production from Trichoderma harzianum 1073 D3with alternative carbon and nitrogen sources. Food Technol. Biotechnol. 43 (1),37–40.

Singh, S.A., Ramakrishna, M., Rao, A.G.A., 1999. Optimisation of downstreamprocessing parameters for the recovery of pectinase from the fermented bran ofAspergillus carbonarius. Process Biochem. 35, 411–417.

Yang, S.Q., Yan, Q.J., Li, L.T., Tian, H.M., Wang, Y.Z., 2006. High level of xylanaseproduction by the thermophilic Paecilomyces thermophila J18 on wheat straw insolid-state fermentation. Biores. Technol. 97, 1794–1800.