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INTRODUCTIONCellulose is biologically renewable resource abundantly

found in agriculture waste. The cellulosic waste materialcanbe hydrolysed to glucose and other soluble sugars by usingcellulase enzymes of bacteria and fungi. The reducing sugarsobtained can be further used for the production of ethanolas biofuel (Eriksson et al., 2002). The potential celluloseproducing bacteria are Cellulomonas, Pseudomonas,Thermoactinomycetes and Bacillus spp.(Godana, 2007).

It has been estimated that 7.2 x 1011 tonnes of celluloseis reserved in plants and that the yearly production ofcellulose is 4 x 1010 tonnes (Coughlan, 1985). Cellulasesinclude three main types of enzymes, endoglucanases,cellobiohydrolases or exoglucanases and â-glucosidases.These enzymes can either be free or grouped in amulticomponent enzyme complex (cellulosome) found inanaerobic cellulolytic bacteria (Mosier et al., 1999).

Enzymatic hydrolysis is an economic process in theconversion of cellulose to easily fermentable low cost sugars(Muthuvelayudham and Viruthagiri, 2006). Thermo philicfilamentus fungi, and actinomycetes are widely used forindustrial production of specific, stable and active enzymes(Mach and Zeilinger, 2003; Haki and Rakshit, 2003).

Present study aimed to isolate and screen potentialthermophilic cellulolytic microorganisms from compost piles.

METHODOLOGYThe present study was conducted at the laboratory of

Nepal Academy of Science and Technology (NAST) fromJanuary 2009 to October 2010.

Enrichment, Isolation and Screening ofThermophilic Cellulytic Bacteria

A total of 13 different compost samples (temperature >50 °C) were collected in sterile containers. The compost pileswere air dry and heated at 550C for 1 week and air drying toreduce the mesophiles and anaerobic isolates. Two protocolswere used for isolation of thermophilic cellulase producer’sbacteria. One gm of compost piles was serially diluted inorder to reduce the initial number of microorganisms.Appropriate dilution was then both direct spread plated onCMC agar medium or enriched Cellulose broth and plating(spread) on (CMC) Agar. All incubations were done at 55°Cfor 2-4 weeks with shaking at 120 rpm in a controlledenvironment shaker (Ibrahim and El-diwany, 2007).

Screening of Potent Cellulytic ThermophilicIsolates

1% congo red indicator (El-Sersy et al., 2010) wasapplied for screening of cellulolytic bacterial isolates. Theplates were flooded with 1% congo red indicator and left for15mins followed by adding 1M NaCl solution and again leftfor another 15 min. then the development of halo zone aroundthe colony indicated the cellulose hydrolysis. If not clear,0.1N HCL was added to make further clearer zone (Wood andBhat, 1988 and El-Sersy et al., 2010).

Identification of Thermotolerent andThermophilic Cellulytic Microorganism

Representative bacterial isolates exhibiting highcellulytic activity were selected for identification accordingto method described by Bergey’s Manuals of Systematic

ISOLATION AND SCREENING OF THERMOPHILICCELLULOLYTIC BACTERIA FROM COMPOST PILES

Author for Correspondence: A. Acharya, Central Department of Microbiology, Tribhuvan university, Kirtipur, Nepal. Email: acharya_amrit@hotmail.com.

A. Acharya*, D.R. Joshi*, K. Shrestha** and D.R. Bhatta**Central Department of Microbiology, Tribhuvan university, Kirtipur, Nepal.

** Natural Product Chemistry Research Laboratory, Nepal Academy of Science and Technology (NAST) Khumaltar,Lalitpur, Nepal.

Abstract: Cellulose, a major polysaccharide found in agricultural residues and industrial and municipal wastes. In the presentstudy thermophilic cellulolytic microorganisms were isolated. The isolates were tested for their cellulytic activity. The enzymeproduction from potent isolates was optimised using cellulose basal broth medium. Activity of partially purified enzyme wasdetermined. The most potent thermophilic cellulolytic isolates were identified as Bacillus subtilis. The crude cellulaseenzyme concentrated at 80-85% ammonium sulfate produced highest zone of hydrolysis. The enzymatic degradation ofcellulose waste has been suggested as a feasible alternative for the conversion of lignocelluloses substrate into fermentablesugars and application for biofuel production.

Keywords: Thermophilic cellulolytic bacteria; Cellulase enzyme; Compost piles.

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Bacteriology and Determinative Bacteriology (Ghazifard etal., 2001; Altai et al., 1989).

Enzyme Production and activity determinationBatch fermentation was adopted for enzyme production

was done by inoculating 5 ml of each pre-fermenter inoculuminto 500 ml-Erlenmeyer flasks containing the same 100 mlcellulose basal broth medium and maintained the finalinoculums size 5%. The flasks were shaken at 150 rpm andincubated at 500C for 5- 7 days (Omojasola et al., 2008). Thesubmerged cultures were carried out in duplicate. Thepreviously studied different Basal mediums were tested formaximum production of cellulase enzyme. The fermentationwas monitored for cellulase activity, reducing sugar andprotein content. Activities of cellulase enzyme systemsecreted into the culture medium of potent isolates wereestimated in accordance with following methods listed byWood and Bhat (1988). The evaluation of crude enzymeCellulolytic activity was carried out for measuring zone ofhydrolysis on 1% CMC agar medium by Plate diffusion assaymethod (Oyekola, 2003). Fermentation was monitored day-wise by estimating reducing sugars by DNS method (Miller,1959).

Enzyme activity was determined spectrophotometricallyas adapted from the recommendations of the Commission ofBiotechnology (Wood and Bhat, 1988).

Protein estimationThe protein content of the culture supernatant was

determined by Bradford et al., (1976).

Partial Purification and Evaluation of CellulaseEnzyme

Partial Purification of the enzyme from culturesupernatant was done by Ammonium sulfate concentrationmethod (Aboul-Enein et al., 2010; Wood and Bhat (1988).

Characterization of Enzyme

Thermal Stability:To determine the thermal stability of the enzyme, the

respective isolates of enzyme was incubated in 0.05Mphosphate buffer of pH 7.0 and 0.05 M sodium citrate pH 4.8for 1 hour at temperature ranging from 30-80ºC (Oyekola,2003; Ariffin et al., 2006).

Table 1: Colony morphology and Microscopic characteristics of cellulytic Bacterial isolates.

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Table 2: Cellulytic activity of bacterial isolates

*= Thermophilic and **= Thermotolerant ?= suspected

Effect of Temperature:The temperature profile between 300C and 80°C, for

Cellulase activity, at optimum pH was determined. The solubleenzyme extract was incubated with the substrate (CMC) atvarious temperatures (30-80°C) (Oyekola, 2003 and Ray etal., 2007; Ariffin et al., 2006).

Effect of pH:In order to determine the optimum assay pH, for

cellulase activity, the assay was carried out at differentbuffers used at various pH were: 0.05 M sodium acetate (pH3-4.5), 0.05 M sodium citrate (pH 5-5.5) and 0.05 M sodiumphosphate buffer (pH 6-8) (Oyekola, 2003; Ray et al., 2007;Ariffin et al., 2006).

RESULTIn the present study 10 thermophilic and 15

thermotolerant bacteria were isolated from compost piles.They were identified as Bacillus subtilis, Bacilluslicheniformis, Bacillus spp., Cellulomonas cellulans,Geobacillus spp. and Penibacillus spp. were commonbacteria. The most potent thermophilic cellulolytic isolates

was Bacillus subtilis (Table 1) McCaig et al., (2001); Song etal., (2001).

Production Optimization of Cellulase EnzymeThe Bacillus subtilis produced maximum reducing

sugar and also showed highest zone of hydrolysis as 21mmin 8th day at optimal cellulose basal medium.

In all four different cellulose basal media used forenzyme production Bacillus subtilis, produced highestenzyme activity in the medium as described by Ray et al.,(2007). The zone of hydrolysis was 30 mm.

Optimum pH for maximum activity of PartialPurification of Enzyme

The highest cellulolytic activity of crude enzyme (1:10diluted) was given by the residue obtained by at 80%ammonium sulfate precipitation at 40C. proteinconcentration of the residue was 0.865 mg/ml (Aboul-Eneinet al., 2010)

The enzyme activity of Bacillus subtilis, was maximumat pH 7.2 at 500C. However, Bacillus subtilis, retained itsactivity from 6.6 upto 9.0 pH.

Thermostability of enzymeCellulase enzyme activity of Bacillus subtilis was

optimum at 500C. However, the result was similar as reportedpreviously (Marques et al., 2003 and Murashima et al., 2002).

DISCUSSIONBacillus subtilis, was potent thermophilic cellulytic

bacteria. Cellulase enzyme activity of B. subtilis was optimumat pH 7.2 at 500C at 1% CMC substrate concentration. Thepotential enzymatic degradation of cellulosic wastes by thethermotolent cellulase enzymes has been suggested as afeasible alternative for the conversion of lignocellulosics intofermentable sugars and fuel ethanol.

ACKNOWLEDGEMENTSWe acknowledge support of Central Department of

Microbiology, Tribhuwan University (CDM-TU) and NepalAcademy of Science and Technology (NAST), Khumaltar,Lalitpur

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