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American-Eurasian J. Agric. & Environ. Sci., 13 (4): 553-564, 2013ISSN 1818-6769© IDOSI Publications, 2013DOI: 10.5829/idosi.aejaes.2013.13.04.15513

Corresponding Author: Mervat Abo-State, National Center for Radiation Research and Technology (NCRRT),Nasr City, Cairo, Egypt.

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Optimization of Cellulase(s) and Xylanase Production byThermophilic and Alkaliphilic Bacillus Isolates

M.A.M. Abo-State, M.F. Ghaly and E.M. Abdellah1 2 2

Department of Microbiology, 1

National Center for Radiation Research and Technology, Cairo, Egypt.Faculty of Science, Zagazig University, Egypt2

Abstract: Two Bacillus isolates (MAM-29 and MAM-38) isolated from agriculture wastes had been used forcellulases (CMCase, FPase and avicelase) and xylanase production on rice straw (RS) by SSF. Different buffers(acetate, citrate, phosphate and mcllvaine), distilled and tap water were used as moisting agents. Wide rangeof pH values (3.0-8.0) were used for evaluating their influence on enzymes and extracellular protein production.The highest CMCase (460 U/ml) was found at phosphate buffer with pH (6.4) by isolate MAM-29. However,the highest CMCase (455 U/ml) was produced at pH (5.6) when mcllvaine was used as moisting agent by isolateMAM-38. The highest FPase (80 U/ml) by isolate MAM-29 with citrate buffer pH (5.0), while (65 U/ml) by isolateMAM-38 with acetate buffer pH (4.0) were produced. The highest avicelase (93 and 77U/ml) have beenproduced by MAM-29 and MAM-38, respectively when mcllvaine buffer was used as moisting agent.Meanwhile, the highest xylanase (373 U/ml) produced by MAM-29 was found in case of using citrate bufferat pH (5.0), while 353U/ml of xylanase was produced in case of mcllvaine pH (4.0). The results also revealed thatenzyme production were higher when using buffers as moisting agents than using both distilled and tap water.Also distilled water gave higher enzyme production than tap water. Optimizing the SSF conditions increasedall the enzyme production and extracellular protein.

Key words: Cellulases Xylanase Bacillus spp. Buffers pH Incubation temperature

INTRODUCTION conversion in converting cellulose to glucose because of

Lignocellulosic biomass is the Earth's most abundant non-pollution of the bio-process [4]. The bioconversionand renewable organic material with great potential for of cellulose to fermentable sugars requires the synergisticproduction of bioenergy and commodity chemicals [1]. action of complete cellulase system comprising ofLignocellulosic materials are found in abundance in nature endoglucanase (EC 3.2.1.4) which act randomly on solublein the form of agricultural and industrial residues, they and insoluble cellulose chains, exoglucanasecould be exploited as potential substrates for growing (cellobiohydrolases, EC 3.2.1.91) which liberate cellobiosethe microorganisms [2]. Rice straw (RS) is one of the from the reducing and non-reducing ends of cellulosemost abundant lignocellulosic crop residues in the world. chains and -glucosidases (EC 3.2.1.21) which liberateIts annual production is about 731 million tons which glucose from cellobiose [5, 6]. The hemicellulaseis distributed in Africa, Asia, Europe and America. system involves among other endo-1, 4- -D-xylanaseThis amount of RS can potentially produce 205 billion (EC 3.2.1.8), which cleaves internal bonds in the xylanliters of bio-ethanol per year [3]. RS is composed of chain, -xylosidases (EC 3.2.1.37), which cleavescellulose (41%) and hemicellulose (20%) which is xylooligosaccharides to produce xylose [7]. Xylanasebound to lignin (12%) by hydrogen and covalent bonds. (endo-1, 4- -D-xylan xylanohydrolase, EC 3.2.1.8)The content of lignin in RS is less than that in other catalyzes the hydrolysis of xylan to produce acommon feedstocks, such as corn stover and wheat straw. mixture of shorter xylo-oligosaccharides, xylose andBiodegradation is more attractive than chemical xylobiose [2].

its advantages such as low investment and low or

Am-Euras. J. Agric. & Environ. Sci., 13 (4): 553-564, 2013

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There is an increasing demand for cellulases in the prime importance because an improper optimization ofmarket for various applications, among which the these factors leads to a lower production of the enzymebioconversion of lignocellulosic biomass for ethanol [2].production is the major one [8]. Besides this, cellulases Recently, much attention has been paid to thehave many other potential applications as well, for thermophilic bio-processing of cellulosic biomass toexample, formulation of washing powder, animal feed biofuels which, due to the use of elevated temperatures,production [9], textile industry, pulp and paper industry, offers several potential advantages such as improvedstarch processing, grain alcohol fermentation, malting hydrolysis of cellulosic substrates, higher mass transferand brewing, extraction of fruits and vegetable juices [10]. rates leading to better substrate solubility, lowered risk ofThe thermostability characteristics of cellulases enzyme potential contamination and increased flexibility withsystem is a key to industrial interest of cellulose respect to process design thus improving the overallhydrolysis. Thermostable cellulolytic enzymes have wide economics of the process. However, the celluloseapplications in food and sugar industries where high recalcitrance to biodegradation poses several major bottletemperature process such as pasteurization is used [11]. necks in the thermophilic digestion of biomass with oneSolid state fermentation (SSF) systems have generated being the lack of robust cellulases that can functionmuch interest in recent years because they offer several efficiently at high temperatures and at a broad range ofeconomical and practical advantages over submerged pH. Thus, thermophilic cellulose degrading bacteria andcultivation equipment, improved product recovery, their enzymes have great potential in the development ofreduced waste water output, higher product viable technologies for the production of alternative fuelsconcentration, lower capital investment, lower plant from agricultural, forestry and Municipal cellulosic wastesoperational costs [12, 13], lower energy, a simple (MCWs) [24].fermentation medium, has superior productivity and Therefore, the aim of this work is to get red ofdoes not require a rigorous control of fermentation agriculture wastes by a safety manner not aggressive toparameters [14, 15], less effluent generation, low catabolic the environment and to produce valuable economicrepression [13], higher product stability, cultivation of enzymes (cellulases and xylanase) from cheap, renewablemicroorganisms specialized for the water insoluble raw material to achieve sustainable development and alsosubstrates or mixed cultivation of various fungi and last enhancing the production of cellulases and xylanase bybut not least, lower demand on sterility due to the low optimizing the fermentation conditions.water activity used in SSF [16].

Cellulase production from bacteria can be an MATERIALS AND METHODSadvantage as the enzyme production rate is normallyhigher due to bacterial high growth rate [17]. Reports on Substrate: The lignocellulosic material rice straw (RS)bacterial hydrolytic enzymes by SSF, however, are was firstly dried and milled into small pieces (3-5mm).primarily confined to only Bacillus spp. [18-21] which This milled agriculture waste was used for SSF.could be attributed to their ability to the substrateparticles to produce filamentous cells for penetration and Bacterial Strains: Two bacterial Bacillus isolatesto their specific need for water activity. Among these, (MAM-29 and MAM-38) were isolated from agricultureBacillus licheniformis enzymes are reported to have a wastes and were identified by 16S-rRNA. The twomarginally higher thermostability [22, 23], however, there Bacillus strains were facultative thermophilic andare very few reports on xylanase production in SSF to alkaliphilic as mentioned in another study.date. Optimization of SSF conditions for production, whenattempted may still improve the overall production Culture Conditions: According to Abo-State [25], the twoeconomics and also make it an attractive technology for Bacillus isolates MAM-29 and MAM-38 were inoculatedcellulase production [13]. The fermentation profile of an with 10% v/v (2X10 CFU/ml) in Luria-Bertani (L.B) brothorganism is affected by nutritional and physiological medium (10.0 g tryptone, 5.0 g yeast extract and 5.0 g NaClfactors such as carbon source, nitrogen source, additives, in 1L of distilled water) [26] and incubated at 37°C forinoculum size, pH of the media, incubation temperature 24 h in shaking incubator. Ten gram of milled RS wasand agitation rate. The hyper production of industrial introduced in 250 ml conical flask and moistened by 15 mlenzymes by optimizing these growth parameters is of distilled water. The flasks were autoclaved at 121°C for

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20 min. The sterilized flasks were inoculated by 10.0 ml of Xylanase Assay: Xylanase assay was determinedbacterial cells suspension of each strain of the well according to Chaplin [31].One ml of the crude enzymegrown Bacillus isolated strains (MAM-29 and MAM-38). supernatant was mixed with 1 ml of 2% xylan fromThe inoculated flasks were incubated at 37°C for 48 h brichwood (product of Sigma/Aldrich, St. Louis, USA)stagnant (solid state fermentation). in sodium acetate buffer (pH 5.5) and incubated at 50°C

Enzyme Extraction: According to Abo-State [25], the DNS reagent as previously mentioned. Standard curveenzymes were extracted from the fermented flasks with was determined by xylose. One unit of xylanase is the100 ml of distilled water. The whole content was filtered micromole of xylose librated per ml of culture filtrate perand squeezed through muslin cloth. The filtered extract minute under the assay conditions.was centrifuged at 8000 rpm for 15 min. by coolingcentrifuge (4°C). The clear supernatant was used as crude Extracellular Protein Determination: Protein wasenzymes for enzyme assay and extracellular protein determined according to Lowry et al. [32]. One ml of thedetermination. crude enzyme supernatant was used and 5.0 ml reaction

Enzyme Assay kept at room temperature for 10 min. Then 0.5 ml of FolinCMCase Assay: Endoglucanase, Carboxymethyl reagent (product of Fluka, Switzerland) was added to thecellulase (CMCase) activity was determined according to previous mixture. The tubes were leaved for 20 min. atWang et al. [27]. One ml of the crude enzyme supernatant room temperature and the absorbance was measured atwas incubated with 1 ml of 1% CMC in 0.1 M sodium 720 nm by spectrophotometer.acetate buffer solution pH 5.0 for 30 min at 63°C.The resulted reducing sugars were determined according Effect of pH and Buffers: Five ml of distilled water wasto Miller [28] by dinitrosalisylic acid (DNS) reagent added to ten grams of rice straw in 250ml flask and(product of Sigma/Aldrich, USA). The resulted reducing autoclaved for 20min at 121°C. After autoclaving 20ml ofsugars were determined at 540 nm by spectrophotometer filter sterilized buffer with its specific pH was added to(LW-V-200RS UV/VIS, Germany). The concentration of each flask. Then inoculated with 5.0 ml of bacterialresulted reducing sugars was determined using glucose suspension of each isolate and incubated for 48h at 37°C.standard curve. One unit of CMCase, is the micro mole of In case of tap and distilled water 25 ml was added beforeglucose liberated per ml of culture filtrate (crude enzyme) autoclaving. In this study, the effect of pH of the moistingper minute. agent were studied by using different buffers with

FPase Assay: Total cellulase (FPase) activity was the to know the best buffer and the best pH of each buffercrude enzyme supernatant was determined as described which gave the best enzymes production. Acetate bufferby Gadgil et al. [29]. One ml of the crude enzyme pH (3.7, 4.0, 4.6, 5.0 and 5.6), Citrate buffer pH (3.2, 4.0, 4.6,supernatant was incubated with 2 ml of 0.1 M citrate 5.0 and 5.6), Phosphate buffer pH (6.0, 6.4, 7.0, 7.4 and 8.0)buffer (pH 4.8) containing 50 mg Whatman No. 1 filter and Mcllvaine buffer pH (3.6, 4.0, 4.6, 5.0, 5.6, 6.0, 6.4, 7.0,paper. After incubation for 1h at 50°C, the resulted 7.4 and 7.6). Twenty ml of each buffer with its specific pHreducing sugars were determined by DNS reagent as added to ten gram of rice straw and autoclaved for 20 min.previously mentioned. One unit of FPase is the micromole at 121°C and then inoculated with 5.0 ml of bacterial cellof glucose librated per ml of culture filtrate per minute. suspension of each isolate and incubated for 48 h at 37°C.

Avicelase Assay: Avicelase activity was determined Effect of Incubation Temperature: Ten grams of milledaccording to Li and Gao [30]. One ml of the crude enzyme rice straw in 250ml Erlenmeyer flasks were moistened withsupernatant was incubated with 1ml of 2% (w/v) Avicel 20ml distilled water and inoculated with 5.0ml of the(product of Sigma, St. Louis, USA) in 0.1 M phosphate- selected bacterial cell suspension of each isolate, thencitrate buffer (pH 6.6) at 40°C for 2h. The resulted incubated at 25°C, 30°C, 37°C, 45°C and 50°C for 48h.reducing sugars were determined by DNS reagent aspreviously mentioned. One unit of Avicelase is the Optimizationof Temperature and pH: Ten grams of milledmicromole of glucose librated per ml of culture filtrate per rice straw in 250ml Erlenmeyer flasks were moistened byminute. 20ml mcllvaine buffer (pH 5.6 for isolate MAM-38 and pH

for 30min. The released reducing sugar was determined by

mixture was added in a clean dry test tube. The tubes were

different pH values, tap water and distilled water and this


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3.6 for isolate MAM-29) and the flasks autoclaved at FPase (455 and 484 U/ml) were at pH 5.6 while avicelase121°C for 10min. Then the flasks inoculated by 5.0 ml of (77 U/ml) recorded the highest production at pH 5.0 andthe bacterial cell suspension of each isolate and incubated xylanase (353 U/ml) was highest at pH 4.0 as shown inat 50°C for 72h. Fig. 8. The highest extracellular protein (951 µg/ml) has

RESULTS AND DISCUSSION xylanase production was highest when using buffers with

Effect of pH and Different Buffers: The pH value protein was highest when using buffers with high pHinfluences the cellulases and xylanase production greatly. values. When distilled water used as moisting agent, itIn the present study, tap water, distilled water and gave higher production of CMCase, FPase, avicelase,different buffers (acetate, citrate, phosphate and xylanase and extracellular protein in case of the twomcllvaine) were used to study the effect of the pH on the isolates (MAM-29 and MAM-38) as shown in Fig. 9 andproduction of cellulases and xylanase. The results of this 10 respectively than tap water. Generally, all the enzymesstudy revealed that when using acetate buffer as moisting production was high when using buffers as moistingagent, for isolate MAM-29, the highest production of agents by comparing the results with both tap andCMCase, FPase and extracellular protein (346 U/ml, 694 distilled water. One of the regulatory parameters duringU/ml and 990µg/ml) were at pH 5.0, respectively. fermentation is pH of the medium [33]. EachAvicelase production (64 U/ml) was best at pH 4.6 but microorganism holds a range pH for its growth andxylanase (299 U/ml) was high at 3.7 as shown in Fig. 1. activity with optimum value between this range. The initialIn case of isolate MAM-38, the highest CMCase pH influences many enzymatic systems and the transport(341 U/ml) was at pH 5.6 and the best FPase and xylanase of several species of enzymes across the cell membrane.production (654 U/ml and 346 U/ml) were at pH 4.0. Most bacterial species are unable to grow at reducedHowever, the best avicelase production (39 U/ml) was at moisture level and alkaline pH [34]. Results of the presentpH 5.0 as shown in Fig. 2. The highest extracellular protein study were confirmed by the results of other investigators(1181 µg/ml) had been recorded at pH 4.6. When citrate as the following. The optimum pH for the productionbuffer used as moisting agent, in case of isolate MAM-29, of xylanase by B. pumilus was in the range of pH 8the highest CMCase (438 U/ml) was at pH 4.6. FPase and (8853 U/gds) to 9 (10 125 U/gds) [35].xylanase (80 U/ml and 373 U/ml), respectively were at pH In case of isolate MAM-29, when different buffers5.0 and avicelase (464 U/ml) was at pH 4.0. The highest (acetate, citrate and mcllvaine) with pH 5.6 used asextracellular protein (885 µg/ml) was recorded at pH 5.6 as moisting agents to know which of them gave best enzymeshown in Fig. 3 In case of isolate MAM-38, highest production. Mcllvaine buffer gave best CMCase,CMCase, FPase and avicelase and extracellular protein avicelase and xylanase (363, 86 and 234 U/ml(453, 50, 524 U/ml and 1199 µg/ml) respectively were at respectively). However, acetate buffer gave best FPasepH 5.0 and the best xylanase was at pH 3.2 as shown in (564 U/ml when used as moisting agent. The highestFig. 4. protein production (885 µg/ml) was by citrate buffer as

When phosphate buffer used as moisting agent, in indicated in Fig. 11. Comparison between mcllvaine andcase of isolate MAM-29, the best CMCase and xylanase phosphate buffer with pH 7.4 as moisting agents, revealed(460 and 240 U/ml) were at pH 6.4 and the best FPase that phosphate buffer gave highest CMCase, FPase,(34 U/ml ) was at 8.0, while the best avicelase and xylanase and extracellular protein (446, 31,181 U/ml andextracellular protein were (294 U/ml and 1135 µg/ml) at pH 1135 µg/ml respectively) but mcllvaine gave highest7.4 as shown in Fig. 5. In case of isolate MAM-38, the avicelase (354 U/ml) as indicated in Fig. 12. In case ofbest CMCase, FPase and avicelase (387, 24 and 26 U/ml, isolate MAM-38, when different buffers (acetate, citraterespectively) were at pH 7.4. While the highest xylanase and mcllvaine) with pH 5.6 used as moisting agents,production (238 U/ml) was found at pH 6.4 and the results revealed that the mcllvaine buffer gave higherhighest extracellular protein was found at pH 8.0 as CMCase, avicelase and xylanase (455, 72 and 246 U/ml,indicated in Fig. 6. When mcllvaine buffer used as respectively) production. Also, acetate buffer gavemoisting agent, in case of isolate MAM-29, CMCase, highest FPase (60 U/ml) but the best protein (737 µg/ml)FPase, avicelase and xylanase (440, 48, 93, 296 U/ml, production was by citrate buffer when used as moistingrespectively) were highest at pH 3.6 as indicated in Fig. 7. agent as showed in Fig. 13. When mcllvaine andBut in case of isolate MAM-38, the highest CMCase, phosphate buffers with pH 7.4 were used as moisting

been recorded at pH 7.0. These results revealed that the

lower pH as moisting agent. In contrast, the extracellular


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Fig. 1: Effect of different pH values of acetate buffer on cellulases and xylanase production by Bacillus sp. MAM-29using SSF

Fig. 2: Effect of different pH values of acetate buffer on cellulases and xylanase production by Bacillus sp. MAM-38using SSF

Fig. 3: Effect of different pH values of citrate buffer on cellulases and xylanase production by Bacillus sp. MAM-29using SSF

Fig. 4: Effect of different pH values of citrate buffer on cellulases and xylanase production by Bacillus sp. MAM-38using SSF


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Fig. 5: Effect of different pH values of phosphate buffer on cellulases and xylanase production by Bacillus sp. MAM-29using SSF

Fig. 6: Effect of different pH values of phosphate buffer on cellulases and xylanase production by Bacillus sp. MAM-38using SSF

Fig. 7: Effect of different pH values of mcllvaine buffer on cellulases and xylanase production by Bacillus sp. MAM-29using SSF

Fig. 8: Effect of different pH values of mcllvaine buffer on cellulases and xylanase production by Bacillus sp. MAM-38using SSF


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Fig. 9: Effect of using tap and distilled water as moisting agents on cellulases and xylanase production by Bacillus sp.MAM-29 using SSF

Fig. 10: Effect of using tap and distilled water as moisting agents on cellulases and xylanase production by Bacillus sp.MAM-38 using SSF

Fig. 11: Effect of using different buffers (Acetate, Citrate and Mcllvaine) as moisting agents on cellulases and xylanaseproduction by Bacillus sp. MAM-29

agents, the results revealed that phosphate buffer gave buffer give the highest lacasse activity [36]. Similar resultsbest CMCase, xylanase and extracellular protein (387, 188 had been recorded by Ters et al. [37]. They determinedU/ml and 972µg/ml) but the highest FPase and avicelase the most stable carboxylic acid based buffer at desired(26 and 40 U/ml) was when mcllvaine buffer used as pH values and buffer strength. For example citrate buffermoisting agent as illustrated in Fig. 14. appears to be ideal choice for assaying laccase at pH

By comparing the three buffers (acetate, citrate and 4.0 and blew irrespective of the chosen buffermcllvaine) used in laccase assay, it is clear that citrate concentration. However succinate buffer at concentration


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Fig. 12: Effect of using different buffers (Phosphate and Mcllvaine) as moisting agents on cellulases and xylanaseproduction by Bacillus sp. MAM-29

Fig. 13: Effect of using different buffers (Acetate, Citrate and Mcllvaine) as moisting agents on cellulases and xylanaseproduction by Bacillus sp. MAM-38

Fig. 14: Effect of using different buffers (Phosphate and Mcllvaine) as moisting agents on cellulases and xylanaseproduction by Bacillus sp. MAM-38

Fig. 15: Effect of incubation temperature on cellulases and xylanase production by Bacillus sp. MAM-29 using SSF


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Fig. 16: Effect of incubation temperature on cellulases and xylanase production by Bacillus sp. MAM-38 using SSF

Fig. 17: Effect of optimization of the fermentation conditions on the production of cellulases and xylanase by isolateMAM-29

Fig. 18: Effect of optimization of all the fermentation conditions on the production of cellulases and xylanase by isolateMAM-38

up to 100 mM may be preferable at pH 5.0. manganese The effect of temperature on cellulases and xylanaseperoxidase (MnP) produced by P. sajor-caju on medium production in case of the two isolates had been shown inI and assayed by mcllvaine buffer pH (5.0) proved that Fig. 15 and 16. These results revealed that the bestit is the best organic acid than both acetate and citrate temperature for the production of CMCase, FPase,buffers. But MnP produced on medium II proved that Avicelase and xylanase was 50°C for the two isolates.acetate is the best buffer [38]. These results were confirmed with the results of Archana

Effect of Temperature: The incubation temperature is B. licheniformis A99 was maximized at 50°C, followed byyet another factor regulating the enzyme synthesis [39]. that at 45°C. Any temperature beyond this range was not

and Satyanarayana [40]. The production of xylanase by


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