SHORT COMMUNICATION

A novel and cost effective methodology for qualitative screeningof alkalo-thermophilic cellulase free xylano-pectinolyticmicroorganisms using agricultural wastes

Amanjot Kaur • Ritu Mahajan • Avtar Singh •

Gaurav Garg • Jitender Sharma

Received: 3 March 2010 / Accepted: 19 May 2010 / Published online: 30 May 2010

� Springer Science+Business Media B.V. 2010

Abstract Qualitative screening of alkalo-thermophilic

cellulase free xylano-pectinolytic microorganisms was

done on agricultural residues. Since xylan is an expensive

substrate for the isolation of xylanase producing microor-

ganisms, the possibility of using wheat bran for screening

of these microorganisms was investigated. Screening was

carried out on wheat bran for the selection of xylanolytic

microorganisms, on waste paper for the evaluation of cel-

lulase free xylanolytic microorganisms, and on citrus peel

for screening of pectinolytic microorganisms. Qualitative

analysis of xylanase, pectinase and cellulase activities

depicted that the zones obtained on nutrient agar medium

containing agricultural residues were apparent and com-

parable with the zones obtained on nutrient agar medium

containing commercial substrates. A strategy of using cost

effective wheat-bran, wastepaper and citrus-peel for the

isolation of cellulase free xylano-pectinolytic microorgan-

isms is a novel and promising method and will ultimately

bring down the cost of screening of these enzyme pro-

ducing microorganisms.

Keywords Alkalothermophilic � Xylano-pectinolytic �Wheat bran � Citrus peel

Introduction

Alkalothermophilic cellulase free xylanases and pectinases

have gained a unique place in the Biotechnological sector

due to their potential application in paper-pulp industry

(Kirk and Jefferies 1996). But, the technology should be

cost effective for its commercial viability. In this concern,

use of agricultural residues for the production of xylanase

and pectinase have been reported by several workers

(Ahlawat et al. 2007; Cui et al. 2008; Azeri et al. 2010).

The earlier studies concentrate on low cost enzyme pro-

duction rather than cost effective screening. Literature has

shown the use of xylan (Nair et al. 2008; Yasinok et al.

2008; Gupta et al. 2009), pectin (Boccas et al. 1994;

Mellon and Cotty 2004; Ahlawat et al. 2007), and cellulose

(Baharuddin et al. 2010) for screening purposes. The use of

these purified substrates would be too expensive for qual-

itative estimation of enzyme activity during screening.

No report has been found till date regarding the use of

agricultural residues for the qualitative screening of xylan-

opectinolytic isolates. Here, we report for the first time a

novel and cost effective protocol for the isolation of cellu-

lase free xylano-pectinolytic microorganisms. Attempts

have been made to replace the expensive commercial sub-

strates, especially xylan, pectin and cellulose, with agricul-

tural residues i.e., Wheat-bran, citrus-peel and waste-paper,

respectively, for screening of cellulase free xylano-pectin-

olytic microorganisms. This new method may be potentially

applicable in large-scale microbial screening purposes.

Materials and methods

Qualitative screening of xylano-pectinolytic

microorganisms

Soil contaminated with effluents of paper and pulp industry

was collected from Yamuna Nagar, Haryana, India. One

gram of soil was suspended in 25 ml sterile deionized

water, pH 9 containing 2% wheatbran and citrus peel and

A. Kaur � R. Mahajan (&) � A. Singh � G. Garg � J. Sharma

Department of Biotechnology, Kurukshetra University,

Kurukshetra 136 119, India

e-mail: ritupanipat@rediffmail.com

123

World J Microbiol Biotechnol (2011) 27:459–463

DOI 10.1007/s11274-010-0457-9

kept on shaker for 48 h at 50�C for the enrichment of xy-

lano-pectinolytic micro-organisms. For primary screening,

a 100 ll aliquot of clear suspension of soil sample was

plated onto nutrient-agar wheat bran medium, pH 9 con-

taining (g/l): Peptone 5.0, Beef extract 3.0, Agar 15.0 and

wheat bran 10.0. The colonies found on the plates were

spotted onto fresh nutrient agar-wheatbran plates. After

24 h incubation at 50�C, replica plates were made and

incubated under the same conditions. Xylanase producing

strains were selected by flooding replica plates with 0.5%

(w/v) Congo red for 15 min followed by repeated washing

with 1 M NaCl for zone analysis (Gessesse and Gashe

1997).

In secondary screening step, the above microbial colo-

nies were transferred to nutrient agar plates containing

waste paper, pH 9. The medium was composed of (g/l):

Peptone 5.0, Beef extract 3.0, Agar 15.0 and Waste paper

10.0. Plates were incubated at 50�C for 24 h. After pro-

viding the same incubation conditions to replica plates,

these were treated with Congo red for zone analysis.

Simultaneously, to prove the efficiency and sensitivity of

this new protocol, the isolates were also cultured individ-

ually on both 0.25% of commercial birch wood xylan

instead of 1% wheat-bran and 0.25% of cellulose in place

of 1% waste paper. The remaining protocol was exactly

same and the zone formation was observed.

In final screening step, the isolates were spotted indi-

vidually on a nutrient-agar citrus peel medium, pH 9. The

medium was composed of (g/l): Peptone 5.0, Beef extract

3.0, Agar 15.0 and Green citrus peel 10.0. Before use,

citrus-peel was washed, dried at 45�C and then grinded.

Plates were incubated at 50�C. After 24 h, replica plates

were made and incubated under same conditions. The zone

formation was observed after treating replica plate with

Congo red. To evaluate results, the pectinase positive and

negative colonies were sub-cultured on 0.25% of pectin

instead of 1% citrus peel. Same parameters were employed

for the zone analysis.

The selected cellulase free xylano-pectinolytic isolates

were transferred to the nutrient agar plates containing

wheat bran and citrus peel, pH 9. The medium was com-

posed of (g/l): Peptone 5.0, Beef extract 3.0, Agar 15.0,

Wheat-bran 10.0 and Citrus-peel 10.0. After 24 h incuba-

tion at 50�C, replica plates were made and Congo red

treatment was given. Results were evaluated by using

0.25% xylan and 0.25% pectin instead of wheat-bran and

citrus-peel under the same conditions.

Quantitative screening of xylano-pectinolytic

microorganisms

Enzymes were produced under submerged fermentation

in 250 ml erlenmeyer flasks containing 50 ml of basal

medium (g/l: peptone, 5.0; yeast extract, 5.0; KNO3, 5.0;

KH2PO4, 1.0; MgSO4.7H2O, 0.1) supplemented with 2%

wheat bran and citrus peel. The flasks were inoculated

with 2% of inoculum (24 h old) and incubated at 37�C for

48 h under shaking conditions (200 rpm). Crude enzymes

were harvested by centrifuging at 10,000 g for 20 min at

4�C and the clear supernatant was used as the source of

enzymes.

Birchwood xylan, 1%; polygalacturonic acid, 0.5%; and

carboxymethyl cellulose, 1% were used as substrates for

assaying the activity of xylanase, pectinase, and cellulase,

respectively. The reaction mixture for xylanase, pectinase

and cellulase assay contained 400 ll of respective substrate

(prepared in glycine NaOH buffer of pH 9) and 100 ll of

appropriately diluted enzyme and was incubated at 55�C

for 10 min. The reaction was terminated by adding 1.5 ml

of 3, 5- dinitrosalicylic acid reagent (Miller 1959). Control

for the enzyme assay was run simultaneously that con-

tained all the reagents but the reaction was terminated prior

to the addition of heat inactivated enzyme. The contents

were boiled for 15 min and after cooling, the colour

developed was read at 540 nm. One unit of enzyme activity

is defined as the amount of enzyme that liberates 1 lmol of

reducing sugar (equivalent to xylose, galacturonic acid and

glucose for xylanase, pectinase, and cellulase, respectively)

per minute under the assay conditions.

Results and discussion

Wheat bran consists of *58% non-starch carbohydrates,

among that arabinoxylans contributes to 70%, cellulose

24% and glucan 6% (Sun et al. 2008). It is the rich source

of xylan, hence was selected for screening of xylanolytic

micro-organisms. Citrus peel is an enriched source of

pectin. It contains 30% of pectin (Huang and Luyen 1989)

and therefore, was used for the screening of pectinase

producers. In view of the high cost of commercial sub-

strates (from Sigma): Birchwood xylan (100 g) 220$,

Pectin (100 g) 51$ and Carboxy-methyl Cellulose (100 g)

18$ approximately, the alternative cost effective agrowaste

substitutes—wheat bran (costs 0.02$ per 100 g), citrus peel

and waste paper (collected domestically) were used.

Keeping in mind, the potential application of alkalo-ther-

mophilic xylanase and pectinase in paper pulp industry

(Kirk and Jefferies 1996), isolation was carried out at 50�C

and pH 9.

In primary screening, formation of a clear zone around

the colonies, after Congo red staining of nutrient agar—

wheatbran plates, indicated the colonies to be the xylanase

producers. Efficiency of this method was proved by ana-

lyzing zones on nutrient-agar medium containing com-

mercial xylan. An isolate showing clear zone on nutrient-

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agar plate containing wheatbran (Fig. 1a) gave comparable

halo of substrate hydrolysis on birchwood xylan (Fig. 1b),

where as, an isolate giving no zone of clearance on nutri-

ent-agar plate containing wheatbran, did not show any zone

of substrate clearance on nutrient-agar plate containing

birchwood xylan (Table 1). Use of commercial xylan

containing nutrient agar plates for the screening of xylan-

ase producing strains through the formation of zone of

substrate hydrolysis have been reported by several workers

(Gessesse and Gashe 1997; Cordeiro et al. 2002; Nair et al.

2008; Yasinok et al. 2008; Gupta et al., 2009). In secondary

screening, colonies showing no zone of clearance on

wastepaper-nutrient agar medium were specified to be the

cellulase free microorganisms. Results were also evaluated

by using commercial cellulose. Baharuddin et al. (2010)

detected cellulase positive isolates qualitatively using agar-

plate containing 1% carboxymethylcellulose. In final

screening, when these isolates were transferred onto

nutrient-agar medium containing citrus-peel, colonies

forming a clear zone were indicated to be the pectinase

producers. Isolate forming a clear zone on nutrient-agar

plate containing citrus-peel (Fig. 1c) also gave comparable

zone on commercial pectin (Fig. 1d). Many workers have

assayed the pectinase activity qualitatively by spotting the

isolates on pectin containing culture plates and then ana-

lyzing the pectin digestion zone (Boccas et al. 1994;

Mellon and Cotty 2004; Ahlawat et al. 2007). Spotting of

the cellulase free xylano-pectinolytic isolate on nutrient-

agar medium containing both wheatbran and citrus peel

gave a zone (Fig. 1e) comparable to that obtained on

combination of xylan and pectin (Fig. 1f). The zone of

hydrolysis on wheat bran-citrus peel combination is rather

bigger than the zone obtained solely on wheatbran and

citrus-peel. Results have shown that the zones obtained

using agro-wastes and commercial substrates were com-

parable (Table 1). In addition to qualitative determinations,

the results were confirmed by performing quantitative

enzyme assays for all the isolates (Table 1). The best xy-

lanopectinolytic bacterial isolate AJK2, screened on agri-

cultural waste containing culture plates by analyzing the

zone of substrate hydrolysis have shown the xylanase and

pectinase activities of 395 and 45 IU/ml, respectively, and

has been identified as alkalothermophilic microorganism

from the Microbial Type Culture Collection Centre of

Institute of Microbial Technology (IMTECH) Chandigarh,

India.

In order to verify the efficiency of this agricultural

residues based method for the screening of xylano-pectin-

olytic microorganisms, a known cellulase free xylanase

producer Bacillus stearothermophilus SDX (MTCC 8508)

Fig. 1 Zone of substrate hydrolysis shown on, a wheat-bran; b birch-

wood xylan; c citrus peel; d pectin, e wheat-bran and citrus peel; fbirch-wood xylan and pectin by xylano-pectinolytic isolate AJK2.

Zone of substrate hydrolysis produced by, a known xylanase producer

Bacillus stearothermophilus on g wheat-bran; h birch-wood xylan;

and a known pectinase producer Bacillus subtilis on i citrus peel; jpectin

c

World J Microbiol Biotechnol (2011) 27:459–463 461

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deposited at Institute of Microbial Technology, India

(Dhiman et al. 2008) and a known pectinase producer

Bacillus subtilis SS (MTCC 8509) (Ahlawat et al. 2007)

were used as control microorganisms in this study. It was

noticed that Bacillus stearothermophilus produced same

size zones i.e., 37 ± 2 mm diameter on nutrient-agar

medium containing wheat-bran (Fig. 1g) and nutrient-agar

medium containing birchwood xylan (Fig. 1h). In the same

way, a pectinase producer Bacillus subtilis gave same size

zones i.e., 23 ± 1 mm diameter on nutrient agar-citrus

peel medium (Fig. 1i) and nutrient agar-pectin medium

(Fig. 1j). Results of this study indicates that the use of

agricultural residues is an attractive and promising

alternative for the cost effective qualitative screening

of alkalo-thermophilic cellulase free xylano-pectinolytic

microorganisms.

Acknowledgments Authors greatfully acknowledge the financial

assistance from University Grant Commission, New Delhi, India.

University Research Scholarship awarded to Amanjot Kaur by Ku-

rukshetra University is kindly acknowledged.

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Table 1 Qualitative and quantitative analysis of xylanase, pectinase and cellulase activities

Isolates Qualitative analysis of xylanase, pectinase and cellulase Quantitative analysis

Diameter (mm) of zone of substrate hydrolysis

on agricultural wastes

Diameter (mm) of zone of substrate

hydrolysis on commercial substrates

Enzyme activity (IU/ml)a

Wheat-bran Citrus peel Waste paper Xylan Pectin Cellulose Xylanase Pectinase Cellulase

AJK1 30 ± 1 Nil Nil 31 ± 1 Nil Nil 200 ± 5 Nil Nil

AJK2 38 ± 1 17 ± 2 Nil 41 ± 2 19 ± 2 Nil 395 ± 2 45 ± 1 Nil

AJK3 36 ± 1 13 ± 1 6 ± 2 37 ± 1 13 ± 1 5 ± 2 332 ± 1 20 ± 1 2.1 ± 0.5

AJK4 8 ± 1 16 ± 1 4 ± 1 7 ± 1 15 ± 1 4 ± 1 3.1 ± 0.5 39 ± 0.7 0.7 ± 0.02

AJK5 5 ± 2 Nil Nil 5 ± 2 Nil Nil 2.7 ± 0.06 Nil Nil

AJK6 33 ± 1 6 ± 1 4 ± 1 34 ± 1 7 ± 1 4 ± 1 257 ± 5 3.8 ± 1 0.9 ± 0.1

AJK7 30 ± 1 11 ± 1 4 ± 1 30 ± 1 10 ± 1 4 ± 1 181 ± 1.4 9 ± 0.2 1.6 ± 0.01

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AJK22 22 ± 1 11 ± 1 Nil 23 ± 1 13 ± 1 Nil 100 ± 0.2 10 ± 1.6 Nil

AJK23 19 ± 1 6 ± 1 6 ± 1 17 ± 1 6 ± 1 6 ± 1 40 ± 1 5 ± 0.9 5 ± 1

AJK24 21 ± 3 11 ± 1 12 ± 1 20 ± 2 11 ± 1 12 ± 1 72 ± 0.5 10 ± 1 15 ± 0.5

AJK25 35 ± 1 17 ± 1 Nil 36 ± 1 17 ± 1 Nil 300 ± 0.02 45 ± 0.8 Nil

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