Research ArticleIsolation Screening and Identification of CellulolyticBacteria from Natural Reserves in the Subtropical Region ofChina and Optimization of Cellulase Production byPaenibacillus terrae ME27-1
Yan-Ling Liang Zheng Zhang Min Wu Yuan Wu and Jia-Xun Feng
State Key Laboratory for Conservation and Utilization of Subtropical Agro-BioresourcesKey Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering College of Life Science and TechnologyGuangxi University 100 Daxue Road Nanning Guangxi 530004 China
Correspondence should be addressed to Jia-Xun Feng jiaxunfengsohucom
Received 11 February 2014 Accepted 8 May 2014 Published 19 June 2014
Academic Editor Encarnacion Ruiz
Copyright copy 2014 Yan-Ling Liang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
From different natural reserves in the subtropical region of China a total of 245 aerobic bacterial strains were isolated on agarplates containing sugarcane bagasse pulp as the sole carbon source Of the 245 strains 22 showed hydrolyzing zones on agar platescontaining carboxymethyl cellulose after Congo-red staining Molecular identification showed that the 22 strains belonged to 10different genera with the Burkholderia genus exhibiting the highest strain diversity and accounting for 3636 of all the 22 strainsThree isolates among the 22 strains showed higher carboxymethyl cellulase (CMCase) activity and isolate ME27-1 exhibited thehighest CMCase activity in liquid culture The strain ME27-1 was identified as Paenibacillus terrae on the basis of 16S rRNA genesequence analysis as well as physiological and biochemical properties The optimum pH and temperature for CMCase activityproduced by the strain ME27-1 were 55 and 50∘C respectively and the enzyme was stable at a wide pH range of 50ndash95 A 12-foldimprovement in the CMCase activity (208UmL) ofME27-1 was obtained under optimal conditions for CMCase productionThusthis study provided further information about the diversity of cellulose-degrading bacteria in the subtropical region of China andfound P terraeME27-1 to be highly cellulolytic
1 Introduction
With decades of studies on cellulose bioconversion cellu-lases have been playing an important role in producingfermentable sugars from lignocellulosic biomass Usuallycellulases are mainly composed of three types of synergisticenzymes endoglucanases (EC 3214) that hydrolyze theexposed cellulose chains of the cellulose polymer exoglu-canases (cellobiohydrolases EC 32191) that act to releasecellobiose from the reducing and nonreducing ends and 120573-glucosidases (EC 32121) that help to cleave the cellobioseand short-chain cello-oligosaccharide into glucose [1]
Numerousmicroorganisms that are able to degrade cellu-lose have been isolated and identified However many studieshave put more emphasis on fungi because the cellulases thatthey produce are abundant and easy to extract and some of
the fungal cellulases have been used as commercial cellulase[2] Although fungi such as Trichoderma Aspergillus Penicil-liumPhanerochaete and Fomitopsis have beenwidely studiedin recent years researchers have also been paying attentionto various bacteria that produce cellulases because of theirfast growth expression of multienzyme complexes andresistance to extreme environments [3ndash8] Bacteria belongingto the genera Clostridium Cellulomonas CellulosimicrobiumThermomonospora Bacillus Ruminococcus Erwinia Bacte-riodes Acetovibrio Streptomyces Microbispora Fibrobacterand Paenibacillus have been observed to produce differentkinds of cellulase when incubated under anaerobic or aerobicconditions [4 9 10]
Several studies have been carried out to investigate thecarboxymethyl cellulase (CMCase) activity of aerobic bacte-ria For instance a maximum CMCase activity (048UmL)
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 512497 13 pageshttpdxdoiorg1011552014512497
2 BioMed Research International
(a) (b) (c)
(d) (e)
CK
(f)
Figure 1 Hydrolyzing zones produced by bacterial strains on agar plates containing CMC after Congo-red staining (a) Strain BS16-3 (b)strain FCD1-3 (c) strain FCD2-1 (d) strain FCD3-5 (e) strain FCD7-2 (f) strain SK3-4 and (CK) Escherichia coli DH5120572
of Acinetobacter anitratus was observed in the late logarithmphase [11] Rastogi et al reported that a maximum CMCaseactivity of 002 and 0058UmLwas exhibited byBrevibacillussp DUSELG12 and Geobacillus sp DUSELR7 on days 10 and7 respectively [12] Furthermore Gupta et al isolated severalcellulose-degrading bacteria exhibiting CMCase activities inthe range of 0162ndash0400UmL [13]
With regard to studies on optimization of cellulase pro-duction by aerobic bacteria Deka et al used response surfacemethodology and found that Bacillus subtilis AS3 exhibited amaximum CMCase activity of 043UmL [14] Furthermoreusing response surface methodology and orthogonal experi-ment design for medium optimization Da Vinha et al andSheng et al observed a maximum CMCase activity of 20and 1432UmLby Streptomyces viridobrunneus SCPE-09 andPseudomonas sp HP207 respectively [15 16] Thus isolationof aerobic bacterial strains producing higher cellulase activityis gaining increasing interest
In this study diverse aerobic bacteria capable of hydrolyz-ing cellulose were isolated from the subtropical region ofChina with Burkholderia sp being the most ubiquitousFurthermore a bacterial strain ME27-1 producing CMCase
at 208UmL after optimization of culture conditions wasisolated and identified
2 Materials and Methods
21 Collection of Soil Samples The soil samples used in thisstudy were collected from Maoer Mountain (Guilin City)Longgang (Chongzuo City) Dawang Ridge (Baise City)Huaping (Guilin City) Shankou Halodrymium (Beihai City)Natural Reserves a starch factory in Fangchenggang Citya bagasse compost at the experimental farm of GuangxiUniversity (Nanning City) in Guangxi Zhuang AutonomousRegion China and Baima Snow Mountain Natural Reservein Yunnan Province China The samples were taken fromorganic-rich soil
22 Strain Isolation and Screening The soil sample suspen-sions were inoculated on Czapekrsquos medium [17] containingsugarcane bagasse pulp (in gL NaNO
3 2 MgSO
4sdot7H2O 05
NaCl 05 FeSO4sdot7H2O 001 KH
2PO4 10 yeast extract 04
pulp 5 (containing 80 water) and agar 150 pH 50) andincubated at 28∘C Subsequently single colonies were picked
BioMed Research International 3
Table 1 Cellulose-degrading bacteria isolated from different natural reserves of subtropical region in China
ldquo119863119889rdquo hydrolyzed zone diametercolony diameter on agar media containing CMC as sole carbon source ldquoNDrdquo no detectable activity
using an inoculating needle and inoculated onto Mandelsand Reese medium [18] containing carboxymethyl cellulosesodium salt (CMC-Na in gL KH
2PO4 20 (NH
4)2SO4 14
MgSO4sdot7H2O 03 CaCl
203 yeast extract 04 FeSO
4sdot7H2O
0005MnSO4 00016 ZnCl
2 00017 CoCl
2 0002 CMC-Na
50 and agar 150 pH 50) After incubation at 28∘C for 48 hall the plates were stained with 1 (wv) Congo-red solutionfor 15min and discolored with 1M NaCl for 15min [19] Thedegradation zones were visible around the bacteria showingthat the strains could hydrolyze CMC
The modified Mandels medium (also called basal medi-um) used for CMCase production by the isolates con-tained the following components (in gL KH
2PO4 15
Na2HPO4sdot7H2O 25 (NH
4)2SO4 15 MgSO
4sdot7H2O 03
CaCl2 01 FeSO
4sdot7H2O 0005 MnSO
4 00016 ZnCl
2
00017 and CoCl2 0002 pH 70) The bacterial isolates were
precultured overnight in general bacteria medium (in gLbeef extract 2 yeast extract 2 sucrose 6 and peptone 5)at 28∘C and 180 rpm Subsequently 2mL of the culture wasinoculated into 250mL conical flask containing 50mL ofbasal medium with 10 gL of CMC-Na as the sole carbonsource and incubated at 28∘C and 180 rpm for 60 h
23 Enzyme Assay Enzyme production during cultivationwas assayed at 12 h intervals up to 3 days The cultures werecentrifuged at 12000 rpm for 10min at 4∘CThe supernatantswere collected as crude enzyme for enzyme assay CMCaseAvicel cellulase (Avicelase) and filter-paper cellulase (FPase)activities were determined using the 35-dinitrosalicylic acid(DNS) method [20] The reaction systems were preparedas follows 250 120583L of crude enzyme (appropriately diluted)mixed with 250120583L of 2 (wv) CMC for determining theCMCase activity 500120583L of enzymemixedwith 1mLofAvicel(1 wv) for determining the Avicelase activity and 500120583Lof enzyme mixed with 50mg of Whatman number 1 filterpaper (10 times 60 cm) in 1mL of buffer for determining theFPase activityThe buffer used for dissolving or resuspendingthe substrates was 100mMsodium citrate buffer (pH 55)Themixtures were incubated at 50∘C for 30min for CMCase assayand for 1 h for Avicelase and FPase assay respectively Thenthe reactions were stopped by adding 1mL of DNS reagentfor CMCase assay and 3mL of DNS reagent for Avicelaseand FPase assay respectively All the mixtures were heated inboiling water for 5min for color development Subsequently200120583L of each sample was transferred to 96-well microplate
and the absorbance was measured at 540 nm [21 22] Oneunit (U) of the enzyme activity was defined as the amountof enzyme that released 1 120583mol of reducing sugars equivalentto glucose per minute during the reaction
The activity of 120573-glucosidase was measured by using p-nitrophenyl-120573-D-glucopyranoside (p-NPG) as substrateTheenzyme activity was determined by detecting the amount ofp-nitrophenol (p-NP) produced from p-NPG [23] One unit(U) of 120573-glucosidase activity was defined as the amount ofenzyme liberating 1 120583mol of p-NP per minute
24 16119878 rRNA Gene Sequencing and Phylogenetic Analysisof the CMC-Degrading Isolates TheCMC-degrading isolateswere cultivated in general bacteria medium at 28∘C for 24 hThe culturewas directly used for the amplification of bacterial16S rRNA gene by PCR [24] Two universal 16S rRNAgene primers (F27 51015840-AGAGTTTGATCCTGGCTCAG-31015840and R1492 51015840-TACGGTTACCTTGTTACGACTT-31015840) wereused [25]The 25 120583Lmixtureswere composed of 1120583Lof bacte-rial culture as template DNA 125 120583L of 2 times Taq PCR MasterMix (containing 05U Taq DNA polymerase120583L 500120583M ofeach dNTP 20mM Tris-HCl (pH 83) 100mM KCl 3mMMgCl
2 and bromophenol blue purchased from Tiangen
Biotech Beijing China) 1120583L of each primer (10 120583M) and95 120583L of double-distilledH
2OThePCR procedure employed
was as follows primary denaturation for 5min at 94∘C 30cycles of denaturation at 94∘C for 30 s annealing at 55∘Cfor 30 s and extension at 72∘C for 100 s and an additionalreaction for 10min at 72∘C The PCR products were detectedon 08 agarose gel to confirm its purity quantity and sizeThe PCR products were sent to Sangon Biotech (Shanghai)Co Ltd China for sequencing
The 16S rRNA gene sequences were compared with other16S rRNA gene sequences available in GenBank by using theBLASTN program (httpblastncbinlmnihgovBlastcgi)and aligned with similar sequences by using CLUSTX pro-gramThe phylogenetic tree was constructed by applying theneighbor-joining method usingMAGA41 program based onKimura-2 parameters with 1000 replicates of bootstrap values[26]
25 Morphological Physiological and Biochemical Identifi-cation of the Bacterial Strain ME27-1 The morphologicalproperties of the strain ME27-1 including shape size colonycharacteristics (color shape surface elevation and edge)andGram stainingwere evaluated [27]Thephysiological and
6 BioMed Research International
0
003
006
009
012
015
018
021
50 60 70 80 90 100pH
CMCa
se ac
tivity
(Um
L)
(a)
0
003
006
009
012
015
018
021
26 28 30 32 34
CMCa
se ac
tivity
(Um
L)
T (∘C)
(b)
Figure 3 Effect of initial pH and temperature on enzyme production by the strain ME27-1 (a) Initial pH (b) Temperature (119879)
biochemical characterization of the strainME27-1was carriedout by using API 50CHB microtests (bioMerieux)
26 Optimization of Cultivation Conditions for CMCase Pro-duction by the Strain ME27-1 The effect of initial pH andtemperature on CMCase production by the strain ME27-1was determined by cultivating the strain in 50mL of basalmediumcontaining 10 gL ofCMC-Na at various pH (rangingfrom 50 to 100 with an interval of 05) and temperatures (26ndash34∘C) for 60 h at 180 rpm
The effect of carbon and nitrogen sources on cellulaseproduction by the strain ME27-1 was determined by using 11different carbon sources (fructose glucose glycerol lactosesucrose maltose CMC-Na filter paper (chopped into 20mesh size) Avicel soluble starch and wheat bran whichwas chopped into 80 mesh size) and 10 different nitrogensources as below (NH
4)2SO4 NH4NO3 NaNO
3 KNO
3
NH4Cl urea soybean yeast extract tryptone and beef
extract The carbon sources were used at a concentration of10 gL instead of the carbon source in the basal mediumFurthermore different concentrations (10ndash100 gL with aninterval of 10 gL) of optimal carbon source were examinedSimilarly the effect of nitrogen sources was also studied withan initial concentration of 15 gL
The effect of different inoculum sizes (2 4 6 8and 10) on enzyme production was tested All media werein pH 80 All the flasks were incubated at 28∘CThe CMCaseactivity was detected at an interval of 12 h
27 Properties of CMCase Produced by the Bacterial StrainME27-1 To determine the optimal pH 250 120583L of crudeCMCase supernatant was incubated with 250 120583L of CMC-Na(2 wv) at 50∘C and different pH (30ndash110 with an intervalof 05) respectively To observe the effect of temperatureCMCase was incubated with 2 CMC-Na at a pH of 55 andtemperature ranging from 30 to 75∘C with an interval of 5∘C
Themaximum CMCase activity obtained at different pH andtemperatures was considered to be 100
The effect of pH on the stability of CMCase was studiedby mixing the crude enzyme with different buffers (1 9 vv)with pH ranging from 30 to 100 The CMCase activity ofthe crude enzyme after incubating at 4∘C for 24 h at differentpHwas detected To study the thermostability of the CMCaseproduced by the strain ME27-1 the crude enzyme was prein-cubated at different temperatures (varying from 30 to 75∘Cwith an interval of 5∘C) for 1 h The residual CMCase activitywas detectedThemaximumCMCase activity obtained at pH30ndash100 or temperature 30ndash75∘C was considered to be 100All the enzyme assays were carried out in triplicate
28 Nucleotide Sequence Accession Numbers All the DNAsequences of the partial 16S rRNA genes of the 22 strainsreported in this study have been deposited into the GenBankdatabase under the accession numbers from KF536877 toKF536898
3 Results and Discussion
31 Isolation and Screening of Cellulose-Degrading Bacteria Atotal of 245 cellulose-degrading aerobic bacterial strains wereisolated from different natural reserves in the subtropicalregion of China which were cultured in agar mediumcontaining sugarcane bagasse pulp as the sole carbon sourceOut of these strains 22 isolates showed hydrolyzing zoneson agar plates containing CMC-Na after Congo-red staining(Figure 1) The hydrolyzing zone diameter and colony diam-eter are listed in Table 1
Among the 22 isolates only three isolates (ME27-1 FCD1-3 and SK3-4) were found to produce measurable CMCaseafter liquid cultivation and isolate ME27-1 showed the high-est CMCase activity (017UmL) after incubation for 60 h inbasal liquid medium containing 10 gL of CMC-Na (Table 1)The CMCase activity of the other 19 strains was undetectable
BioMed Research International 7
0
009
018
027
036
045
1 2 3 4 5 6 7 8 9
CMCa
se ac
tivity
(Um
L)
Carbon sources
(a)
0
03
06
09
12
15
10 30 50 70 90
CMCa
se ac
tivity
(Um
L)
Concentration of wheat bran (gL)
(b)
0
045
09
135
18
a b c d e f g h i j
CMCa
se ac
tivity
(Um
L)
Nitrogen sources
(c)
0
03
06
09
12
15
18
00 15 30 45 60 75 90
CMCa
se ac
tivity
(Um
L)
Concentration of NH4Cl (gL)
(d)
Figure 4 Effect of carbon and nitrogen sources on CMCase production by the strainME27-1 (a) Different carbon sources 1 sim 9 representedglycerol lactose sucrose maltose CMC-Na filter paper Avicel soluble starch and wheat bran respectively (b) The concentration of wheatbran (c) Different nitrogen sources a sim j represented (NH
4
)2
SO4
NH4
NO3
NaNO3
KNO3
NH4
Cl urea soybean yeast extract tryptoneand beef extract respectively (d) The concentration of NH
4
Cl
after cultivating in various liquid media for up to 6 days andthe Avicelase FPase and 120573-glucosidase activities of all the 22bacterial strains were also undetectable
Congo-red staining has been widely used inmany studiesfor screening cellulose-degradingmicroorganisms AlthoughTeather and Wood described the relationship between thediameter of hydrolyzing zone and log enzyme concentrationthis correlation could not represent the enzyme-producingability of the microorganisms [19] In the present studyalthough some strains presented large and clear hydrolyzingzones the activities of CMCase and other cellulases producedby themwere undetectable in various liquidmedia containingCMCand other cellulosicmaterials suggesting that either theconcentration of the enzyme produced by these strains wasvery low to be detected after cultivation in liquid medium orthe ability of the strains to secrete CMCase was weak Sadhu
and Maiti also reported that the diameter of the hydrolyzingzonemay not accurately reflect the real cellulase activity [28]
In general aerobic bacteria produce low levels of Avice-lase FPase and 120573-glucosidase In a study carried out byRastogi et al Brevibacillus sp DUSELG12 andGeobacillus spDUSELR7 were found to produce a maximum FPase activityof 0027 and 0043UmL on days 7 and 8 respectively [12]Recently Soares et al found that only 91 of bacterial strainswere able to degrade Avicel on agar plates [7]
32 Identification of Cellulose-Degrading Bacteria The DNAfragments containing partial 16S rRNAgenes of the 22 isolateswere amplified and sequenced The sequences obtained werematched with those available in GenBank which revealedmaximum identity of these isolates and allowed identificationof these cellulose-degrading bacterial strains (Table 1)
8 BioMed Research International
0
03
06
09
12
15
12 24 36 48 60 72
CMCa
se ac
tivity
(Um
L)
Incubation time (h)
Figure 5 Effect of inoculum size and incubation period onCMCaseproduction by the strain ME27-1 2 (empty triangle) 4 (filledtriangle) 6 (filled circle) 8 (filled square) and 10 (empty square)Error bars show the standard deviation of experimental point (119899 =3)
It was found that the 22 aerobic bacterial strains thatcould hydrolyze cellulose belonged to 10 different gen-era Burkholderia (3636) Bacillus (1365) Citrobacter(1365) Arthrobacter (910) Enterobacter (454) Chry-seobacterium (454) Pandoraea (454) Paenibacillus(454) Dyella (454) and Pseudomonas (454) Thephylogenetic tree of the 22 strains was constructed by usingMAGA41 program (Figure 2)
Various cellulose-degrading bacteria have been foundin different environments The genus Burkholderia wasobserved to be the main cellulose-hydrolyzing bacteria andwas considered to play an important role in cellulose degra-dation in the subtropical region of China in this studyIn addition bacteria belonging to the genera ArthrobacterChryseobacterium Pandoraea and Dyella were also foundto be cellulolytic in the present study which have beenrarely reported as cellulose-degrading bacteria In a previousstudy Lo et al reported that the cellulase-producing bacterialstrains isolated from a rice field in southern Taiwan mainlybelonged to the genus Cellulomonas [9] On the other handBacilluswas reported to be the dominant cellulose-degradingbacteria in samples collected from paper mill sludges andorganic fertilizers from Red Rock Canada as well as inthose from soil compost and animal waste slurry from JejuIsland [29 30] Similarly Burkholderia was found to be themain genus of cellulase-producing bacteria in the subtropicalrainforest in Okinawa Island Japan [31]
The strain ME27-1 with higher CMCase activity wasthoroughly examined The partial 16S rRNA gene (1309 bp)from the strain ME27-1 showed a maximum identity of 99with that ofPaenibacillus terraeAM141T (T type strain)Mor-phological tests revealed that the cells of the strain ME27-1
were rod-shaped endospore-forming Gram-positive and08 times 19ndash32 120583m in size The appearance of the colonyon the TSA medium was cream-colored moist irregularswollen and pigment-free The biochemical properties ofthe strain ME27-1 are listed in Table 2 The morphologicalphysiological and biochemical properties of the strainME27-1 were found to be mostly similar to those of P terrae [27]Thus the strain ME27-1 was identified as P terrae
To our knowledge till date no study has reported aboutCMCase production by P terrae although other species ofPaenibacillus have been found to produce cellulase SomeCMCase genes cloned from Paenibacillus polymyxa GS01Paenibacillus barcinonensis Paenibacillus xylanilyticus KJ-03 and Paenibacillus cookii SS-24 have been expressed inEscherichia coli and Saccharomyces cerevisiae [32ndash35] On theother hand CMCases from Paenibacillus curdlanolyticus B-6Paenibacillus campinasensis BL11 Paenibacillus sp B39 and Ppolymyxa have been purified [36ndash39]
33 Effect of Initial pH Temperature Carbon and NitrogenSources Inoculum Size and Incubation Time on CMCase Pro-duction by P terrae ME27-1 The best incubation conditionswere pH 80 and 28∘C (Figures 3(a) and 3(b)) The CMCaseactivity declined when the initial pH and incubation tem-perature were not optimal There have been diverse reportson the optimal initial pH and temperature for cellulolyticenzyme production by Paenibacillus sp In a previous studyP curdlanolyticus B-6 was cultivated for enzyme productionat pH 70 and 37∘C [5] Furthermore Kumar et al reportedthat the optimal initial pH and temperature for CMCase pro-duction by P polymyxa were 55 and 37∘C respectively [39]Yoon et al accounted that the optimal growth temperaturefor P terrae was 30∘C which is similar to that observed foroptimal CMCase production by the strain ME27-1 [27]
Various cellulosic materials have been used to inducemicroorganisms to produce cellulaseWhen fructose and glu-cose were used as the sole carbon source no CMCase activitywas detected Wheat bran induced the highest CMCaseactivity which was about 25-fold higher than that observedin the basal medium containing CMC-Na (Figure 4(a)) Theoptimal concentration of wheat bran in the medium wasfound to be 50 gL (Figure 4(b)) Da Vinha et al used steam-pretreated sugarcane bagasse (or wheat bran) as the maincarbon source and found thatwheat branwas the best inducerfor CMCase production by S viridobrunneus SCPE-09 [15]Gao et al demonstrated that rice branwas the optimal carbonsource for CMCase production by Cellulophaga lytica LBH-14 while Kumar et al reported that high CMCase productionby P polymyxa was obtained when using mango peel assubstrate [39 40] In addition wheat straw rice straw andxylan have been reported to be good carbon sources forCMCase production by Cellulomonas sp and Cellulosimicro-bium cellulans [9 41]
Furthermore maximum CMCase activity was notedwhen using NH
4Cl as the sole nitrogen source (Figure 4(c))
and the best concentration of NH4Cl in the medium was
observed to be 3 gL (Figure 4(d)) Many reports have shownthat organic nitrogen sources are better than inorganic
BioMed Research International 9
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(a)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(b)
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(c)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(d)
Figure 6 Properties of CMCase produced by the strain ME27-1 (a) Effect of pH on CMCase activity (b) Effect of temperature on CMCaseactivity (c) Effect of pH on the stability of CMCase (d) Thermostability of CMCase The different buffers used are as follows (100mM)sodium citrate buffer (empty square pH 30ndash65) Na
2
HPO4
-NaH2
PO4
buffer (filled square pH 65ndash75) Tris-HCl buffer (empty triangle pH75ndash85) and glycine-NaOH buffer (filled triangle pH 85ndash110) Error bars show the standard deviation of experimental point (119899 = 2)
nitrogen sources [15 16 42 43] In the present study theCMCase activity of the strain ME27-1 was higher wheninorganic nitrogen sources were used as the sole nitrogensource Likewise Kumar et al and Kalogeris et al alsoobserved a similar phenomenon in their studies [39 44]
In addition use of an inoculum size of 2 resulted inmaximum CMCase activity after incubation of the strainfor 60 h (Figure 5) There has been increasing interest incellulase-producing bacteria because of their ability to growfast [45] In the present study the strain ME27-1 producedthe highest CMCase activity after 60 h of incubation On theother hand in previous studies maximum CMCase activityof Pseudomonas sp HP207 and S viridobrunneus SCPE-09was observed after 24 and 48 h of incubation respectivelywhich is much earlier than that noted for the strain ME27-1 [15 16] However different results have been reported invarious studies MaximumCMCase activity of C lytica LBH-14 was obtained after 72 h of incubation whereas that of
Brevibacillus sp DUSELG12 and Geobacillus sp DUSELR7was noted after days 9 and 7 respectively [12 40]
34 Properties of CMCase Produced by P terrae ME27-1The optimum pH and temperature of CMCase produced bystrain ME27-1 were found to be 55 and 50∘C respectively(Figures 6(a) and 6(b)) The CMCase produced by the strainME27-1 was stable from pH 40 to 110 with more than 60CMCase activity being retained (Figure 6(c)) Furthermorethe enzyme maintained 65 activity after incubation at 4∘Cand pH 110 for 24 h The temperature profiles demonstratedthat more than 95 CMCase activity was retained at 30ndash45∘C for 1 h (Figure 6(d)) However the enzyme activity wasreduced at temperatures above 50∘C In fact approximately77 residual activity was maintained after preincubating theenzyme at 50∘C for 1 h
Similar results were observed for cellulases produced by Sviridobrunneus SCPE-09 and P cookii SS-24 with an optimal
10 BioMed Research International
Table 3 Comparison of CMCase production by Paenibacillus terraeME27-1 with other bacterial and fungal strains
pH of 50 and 51 and an optimal temperature of 50∘ and 55∘Crespectively [15 35] However maximum CMCase activity ofbacteria at pH lower than 60 has been rarely observed andthe maximum CMCase activities of P campinasensis BL11 Ppolymyxa GS01 Paenibacillus sp B39 and Bacillus mycoidesS122C were observed at neutral or alkaline conditions [3738 46 47] In the present study the CMCase producedby the strain ME27-1 was stable at pH 50ndash95 and almost85 residual activity was retained Only a few studies havereported that CMCase was stable at such a wide pH range forexample Da Vinha et al reported the 60 CMCase activitywas retained within the pH range of 30ndash80 [15]
35 Comparison of CMCase Production by P terrae ME27-1and Other Microorganisms When measured at the optimalpH and temperature of CMCase P terrae ME27-1 producedCMCase activity of 208UmL under the optimized cul-tivation conditions which was a 12-fold improvement in
the CMCase production This yield of CMCase productionwas higher than most of the aerobic bacterial strains but lessthan someof aerobic bacterial strains that have been exploitedpreviously (Table 3) However the CMCase production by Pterrae ME27-1 was lower than that by several anaerobic bac-terial strains for example Clostridium papyrosolvens CFR-703 C thermocellum YM4 C thermocopriae JT3-3 (Table 3)Some anaerobic bacteria can degrade lignocellulosic sub-strates efficiently by producingmultienzyme complex termedcellulosome [36] The carbohydrate binding modules anddifferent proteins in the cellulosome allow the whole enzymecomplex to bind to the substrates which avoids the wastefulexpenditure of energy of bacteria releasing large amounts ofindividual enzymes andmakes lots of advantages over single-enzyme system [4 48]
Furthermore the CMCase produced by P terrae ME27-1 was lower than that by most aerobic fungal strains whileit was higher than that by anaerobic fungal strains (Table 3)
BioMed Research International 11
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
Figure 1 Hydrolyzing zones produced by bacterial strains on agar plates containing CMC after Congo-red staining (a) Strain BS16-3 (b)strain FCD1-3 (c) strain FCD2-1 (d) strain FCD3-5 (e) strain FCD7-2 (f) strain SK3-4 and (CK) Escherichia coli DH5120572
of Acinetobacter anitratus was observed in the late logarithmphase [11] Rastogi et al reported that a maximum CMCaseactivity of 002 and 0058UmLwas exhibited byBrevibacillussp DUSELG12 and Geobacillus sp DUSELR7 on days 10 and7 respectively [12] Furthermore Gupta et al isolated severalcellulose-degrading bacteria exhibiting CMCase activities inthe range of 0162ndash0400UmL [13]
With regard to studies on optimization of cellulase pro-duction by aerobic bacteria Deka et al used response surfacemethodology and found that Bacillus subtilis AS3 exhibited amaximum CMCase activity of 043UmL [14] Furthermoreusing response surface methodology and orthogonal experi-ment design for medium optimization Da Vinha et al andSheng et al observed a maximum CMCase activity of 20and 1432UmLby Streptomyces viridobrunneus SCPE-09 andPseudomonas sp HP207 respectively [15 16] Thus isolationof aerobic bacterial strains producing higher cellulase activityis gaining increasing interest
In this study diverse aerobic bacteria capable of hydrolyz-ing cellulose were isolated from the subtropical region ofChina with Burkholderia sp being the most ubiquitousFurthermore a bacterial strain ME27-1 producing CMCase
at 208UmL after optimization of culture conditions wasisolated and identified
2 Materials and Methods
21 Collection of Soil Samples The soil samples used in thisstudy were collected from Maoer Mountain (Guilin City)Longgang (Chongzuo City) Dawang Ridge (Baise City)Huaping (Guilin City) Shankou Halodrymium (Beihai City)Natural Reserves a starch factory in Fangchenggang Citya bagasse compost at the experimental farm of GuangxiUniversity (Nanning City) in Guangxi Zhuang AutonomousRegion China and Baima Snow Mountain Natural Reservein Yunnan Province China The samples were taken fromorganic-rich soil
22 Strain Isolation and Screening The soil sample suspen-sions were inoculated on Czapekrsquos medium [17] containingsugarcane bagasse pulp (in gL NaNO
3 2 MgSO
4sdot7H2O 05
NaCl 05 FeSO4sdot7H2O 001 KH
2PO4 10 yeast extract 04
pulp 5 (containing 80 water) and agar 150 pH 50) andincubated at 28∘C Subsequently single colonies were picked
BioMed Research International 3
Table 1 Cellulose-degrading bacteria isolated from different natural reserves of subtropical region in China
ldquo119863119889rdquo hydrolyzed zone diametercolony diameter on agar media containing CMC as sole carbon source ldquoNDrdquo no detectable activity
using an inoculating needle and inoculated onto Mandelsand Reese medium [18] containing carboxymethyl cellulosesodium salt (CMC-Na in gL KH
2PO4 20 (NH
4)2SO4 14
MgSO4sdot7H2O 03 CaCl
203 yeast extract 04 FeSO
4sdot7H2O
0005MnSO4 00016 ZnCl
2 00017 CoCl
2 0002 CMC-Na
50 and agar 150 pH 50) After incubation at 28∘C for 48 hall the plates were stained with 1 (wv) Congo-red solutionfor 15min and discolored with 1M NaCl for 15min [19] Thedegradation zones were visible around the bacteria showingthat the strains could hydrolyze CMC
The modified Mandels medium (also called basal medi-um) used for CMCase production by the isolates con-tained the following components (in gL KH
2PO4 15
Na2HPO4sdot7H2O 25 (NH
4)2SO4 15 MgSO
4sdot7H2O 03
CaCl2 01 FeSO
4sdot7H2O 0005 MnSO
4 00016 ZnCl
2
00017 and CoCl2 0002 pH 70) The bacterial isolates were
precultured overnight in general bacteria medium (in gLbeef extract 2 yeast extract 2 sucrose 6 and peptone 5)at 28∘C and 180 rpm Subsequently 2mL of the culture wasinoculated into 250mL conical flask containing 50mL ofbasal medium with 10 gL of CMC-Na as the sole carbonsource and incubated at 28∘C and 180 rpm for 60 h
23 Enzyme Assay Enzyme production during cultivationwas assayed at 12 h intervals up to 3 days The cultures werecentrifuged at 12000 rpm for 10min at 4∘CThe supernatantswere collected as crude enzyme for enzyme assay CMCaseAvicel cellulase (Avicelase) and filter-paper cellulase (FPase)activities were determined using the 35-dinitrosalicylic acid(DNS) method [20] The reaction systems were preparedas follows 250 120583L of crude enzyme (appropriately diluted)mixed with 250120583L of 2 (wv) CMC for determining theCMCase activity 500120583L of enzymemixedwith 1mLofAvicel(1 wv) for determining the Avicelase activity and 500120583Lof enzyme mixed with 50mg of Whatman number 1 filterpaper (10 times 60 cm) in 1mL of buffer for determining theFPase activityThe buffer used for dissolving or resuspendingthe substrates was 100mMsodium citrate buffer (pH 55)Themixtures were incubated at 50∘C for 30min for CMCase assayand for 1 h for Avicelase and FPase assay respectively Thenthe reactions were stopped by adding 1mL of DNS reagentfor CMCase assay and 3mL of DNS reagent for Avicelaseand FPase assay respectively All the mixtures were heated inboiling water for 5min for color development Subsequently200120583L of each sample was transferred to 96-well microplate
and the absorbance was measured at 540 nm [21 22] Oneunit (U) of the enzyme activity was defined as the amountof enzyme that released 1 120583mol of reducing sugars equivalentto glucose per minute during the reaction
The activity of 120573-glucosidase was measured by using p-nitrophenyl-120573-D-glucopyranoside (p-NPG) as substrateTheenzyme activity was determined by detecting the amount ofp-nitrophenol (p-NP) produced from p-NPG [23] One unit(U) of 120573-glucosidase activity was defined as the amount ofenzyme liberating 1 120583mol of p-NP per minute
24 16119878 rRNA Gene Sequencing and Phylogenetic Analysisof the CMC-Degrading Isolates TheCMC-degrading isolateswere cultivated in general bacteria medium at 28∘C for 24 hThe culturewas directly used for the amplification of bacterial16S rRNA gene by PCR [24] Two universal 16S rRNAgene primers (F27 51015840-AGAGTTTGATCCTGGCTCAG-31015840and R1492 51015840-TACGGTTACCTTGTTACGACTT-31015840) wereused [25]The 25 120583Lmixtureswere composed of 1120583Lof bacte-rial culture as template DNA 125 120583L of 2 times Taq PCR MasterMix (containing 05U Taq DNA polymerase120583L 500120583M ofeach dNTP 20mM Tris-HCl (pH 83) 100mM KCl 3mMMgCl
2 and bromophenol blue purchased from Tiangen
Biotech Beijing China) 1120583L of each primer (10 120583M) and95 120583L of double-distilledH
2OThePCR procedure employed
was as follows primary denaturation for 5min at 94∘C 30cycles of denaturation at 94∘C for 30 s annealing at 55∘Cfor 30 s and extension at 72∘C for 100 s and an additionalreaction for 10min at 72∘C The PCR products were detectedon 08 agarose gel to confirm its purity quantity and sizeThe PCR products were sent to Sangon Biotech (Shanghai)Co Ltd China for sequencing
The 16S rRNA gene sequences were compared with other16S rRNA gene sequences available in GenBank by using theBLASTN program (httpblastncbinlmnihgovBlastcgi)and aligned with similar sequences by using CLUSTX pro-gramThe phylogenetic tree was constructed by applying theneighbor-joining method usingMAGA41 program based onKimura-2 parameters with 1000 replicates of bootstrap values[26]
25 Morphological Physiological and Biochemical Identifi-cation of the Bacterial Strain ME27-1 The morphologicalproperties of the strain ME27-1 including shape size colonycharacteristics (color shape surface elevation and edge)andGram stainingwere evaluated [27]Thephysiological and
6 BioMed Research International
0
003
006
009
012
015
018
021
50 60 70 80 90 100pH
CMCa
se ac
tivity
(Um
L)
(a)
0
003
006
009
012
015
018
021
26 28 30 32 34
CMCa
se ac
tivity
(Um
L)
T (∘C)
(b)
Figure 3 Effect of initial pH and temperature on enzyme production by the strain ME27-1 (a) Initial pH (b) Temperature (119879)
biochemical characterization of the strainME27-1was carriedout by using API 50CHB microtests (bioMerieux)
26 Optimization of Cultivation Conditions for CMCase Pro-duction by the Strain ME27-1 The effect of initial pH andtemperature on CMCase production by the strain ME27-1was determined by cultivating the strain in 50mL of basalmediumcontaining 10 gL ofCMC-Na at various pH (rangingfrom 50 to 100 with an interval of 05) and temperatures (26ndash34∘C) for 60 h at 180 rpm
The effect of carbon and nitrogen sources on cellulaseproduction by the strain ME27-1 was determined by using 11different carbon sources (fructose glucose glycerol lactosesucrose maltose CMC-Na filter paper (chopped into 20mesh size) Avicel soluble starch and wheat bran whichwas chopped into 80 mesh size) and 10 different nitrogensources as below (NH
4)2SO4 NH4NO3 NaNO
3 KNO
3
NH4Cl urea soybean yeast extract tryptone and beef
extract The carbon sources were used at a concentration of10 gL instead of the carbon source in the basal mediumFurthermore different concentrations (10ndash100 gL with aninterval of 10 gL) of optimal carbon source were examinedSimilarly the effect of nitrogen sources was also studied withan initial concentration of 15 gL
The effect of different inoculum sizes (2 4 6 8and 10) on enzyme production was tested All media werein pH 80 All the flasks were incubated at 28∘CThe CMCaseactivity was detected at an interval of 12 h
27 Properties of CMCase Produced by the Bacterial StrainME27-1 To determine the optimal pH 250 120583L of crudeCMCase supernatant was incubated with 250 120583L of CMC-Na(2 wv) at 50∘C and different pH (30ndash110 with an intervalof 05) respectively To observe the effect of temperatureCMCase was incubated with 2 CMC-Na at a pH of 55 andtemperature ranging from 30 to 75∘C with an interval of 5∘C
Themaximum CMCase activity obtained at different pH andtemperatures was considered to be 100
The effect of pH on the stability of CMCase was studiedby mixing the crude enzyme with different buffers (1 9 vv)with pH ranging from 30 to 100 The CMCase activity ofthe crude enzyme after incubating at 4∘C for 24 h at differentpHwas detected To study the thermostability of the CMCaseproduced by the strain ME27-1 the crude enzyme was prein-cubated at different temperatures (varying from 30 to 75∘Cwith an interval of 5∘C) for 1 h The residual CMCase activitywas detectedThemaximumCMCase activity obtained at pH30ndash100 or temperature 30ndash75∘C was considered to be 100All the enzyme assays were carried out in triplicate
28 Nucleotide Sequence Accession Numbers All the DNAsequences of the partial 16S rRNA genes of the 22 strainsreported in this study have been deposited into the GenBankdatabase under the accession numbers from KF536877 toKF536898
3 Results and Discussion
31 Isolation and Screening of Cellulose-Degrading Bacteria Atotal of 245 cellulose-degrading aerobic bacterial strains wereisolated from different natural reserves in the subtropicalregion of China which were cultured in agar mediumcontaining sugarcane bagasse pulp as the sole carbon sourceOut of these strains 22 isolates showed hydrolyzing zoneson agar plates containing CMC-Na after Congo-red staining(Figure 1) The hydrolyzing zone diameter and colony diam-eter are listed in Table 1
Among the 22 isolates only three isolates (ME27-1 FCD1-3 and SK3-4) were found to produce measurable CMCaseafter liquid cultivation and isolate ME27-1 showed the high-est CMCase activity (017UmL) after incubation for 60 h inbasal liquid medium containing 10 gL of CMC-Na (Table 1)The CMCase activity of the other 19 strains was undetectable
BioMed Research International 7
0
009
018
027
036
045
1 2 3 4 5 6 7 8 9
CMCa
se ac
tivity
(Um
L)
Carbon sources
(a)
0
03
06
09
12
15
10 30 50 70 90
CMCa
se ac
tivity
(Um
L)
Concentration of wheat bran (gL)
(b)
0
045
09
135
18
a b c d e f g h i j
CMCa
se ac
tivity
(Um
L)
Nitrogen sources
(c)
0
03
06
09
12
15
18
00 15 30 45 60 75 90
CMCa
se ac
tivity
(Um
L)
Concentration of NH4Cl (gL)
(d)
Figure 4 Effect of carbon and nitrogen sources on CMCase production by the strainME27-1 (a) Different carbon sources 1 sim 9 representedglycerol lactose sucrose maltose CMC-Na filter paper Avicel soluble starch and wheat bran respectively (b) The concentration of wheatbran (c) Different nitrogen sources a sim j represented (NH
4
)2
SO4
NH4
NO3
NaNO3
KNO3
NH4
Cl urea soybean yeast extract tryptoneand beef extract respectively (d) The concentration of NH
4
Cl
after cultivating in various liquid media for up to 6 days andthe Avicelase FPase and 120573-glucosidase activities of all the 22bacterial strains were also undetectable
Congo-red staining has been widely used inmany studiesfor screening cellulose-degradingmicroorganisms AlthoughTeather and Wood described the relationship between thediameter of hydrolyzing zone and log enzyme concentrationthis correlation could not represent the enzyme-producingability of the microorganisms [19] In the present studyalthough some strains presented large and clear hydrolyzingzones the activities of CMCase and other cellulases producedby themwere undetectable in various liquidmedia containingCMCand other cellulosicmaterials suggesting that either theconcentration of the enzyme produced by these strains wasvery low to be detected after cultivation in liquid medium orthe ability of the strains to secrete CMCase was weak Sadhu
and Maiti also reported that the diameter of the hydrolyzingzonemay not accurately reflect the real cellulase activity [28]
In general aerobic bacteria produce low levels of Avice-lase FPase and 120573-glucosidase In a study carried out byRastogi et al Brevibacillus sp DUSELG12 andGeobacillus spDUSELR7 were found to produce a maximum FPase activityof 0027 and 0043UmL on days 7 and 8 respectively [12]Recently Soares et al found that only 91 of bacterial strainswere able to degrade Avicel on agar plates [7]
32 Identification of Cellulose-Degrading Bacteria The DNAfragments containing partial 16S rRNAgenes of the 22 isolateswere amplified and sequenced The sequences obtained werematched with those available in GenBank which revealedmaximum identity of these isolates and allowed identificationof these cellulose-degrading bacterial strains (Table 1)
8 BioMed Research International
0
03
06
09
12
15
12 24 36 48 60 72
CMCa
se ac
tivity
(Um
L)
Incubation time (h)
Figure 5 Effect of inoculum size and incubation period onCMCaseproduction by the strain ME27-1 2 (empty triangle) 4 (filledtriangle) 6 (filled circle) 8 (filled square) and 10 (empty square)Error bars show the standard deviation of experimental point (119899 =3)
It was found that the 22 aerobic bacterial strains thatcould hydrolyze cellulose belonged to 10 different gen-era Burkholderia (3636) Bacillus (1365) Citrobacter(1365) Arthrobacter (910) Enterobacter (454) Chry-seobacterium (454) Pandoraea (454) Paenibacillus(454) Dyella (454) and Pseudomonas (454) Thephylogenetic tree of the 22 strains was constructed by usingMAGA41 program (Figure 2)
Various cellulose-degrading bacteria have been foundin different environments The genus Burkholderia wasobserved to be the main cellulose-hydrolyzing bacteria andwas considered to play an important role in cellulose degra-dation in the subtropical region of China in this studyIn addition bacteria belonging to the genera ArthrobacterChryseobacterium Pandoraea and Dyella were also foundto be cellulolytic in the present study which have beenrarely reported as cellulose-degrading bacteria In a previousstudy Lo et al reported that the cellulase-producing bacterialstrains isolated from a rice field in southern Taiwan mainlybelonged to the genus Cellulomonas [9] On the other handBacilluswas reported to be the dominant cellulose-degradingbacteria in samples collected from paper mill sludges andorganic fertilizers from Red Rock Canada as well as inthose from soil compost and animal waste slurry from JejuIsland [29 30] Similarly Burkholderia was found to be themain genus of cellulase-producing bacteria in the subtropicalrainforest in Okinawa Island Japan [31]
The strain ME27-1 with higher CMCase activity wasthoroughly examined The partial 16S rRNA gene (1309 bp)from the strain ME27-1 showed a maximum identity of 99with that ofPaenibacillus terraeAM141T (T type strain)Mor-phological tests revealed that the cells of the strain ME27-1
were rod-shaped endospore-forming Gram-positive and08 times 19ndash32 120583m in size The appearance of the colonyon the TSA medium was cream-colored moist irregularswollen and pigment-free The biochemical properties ofthe strain ME27-1 are listed in Table 2 The morphologicalphysiological and biochemical properties of the strainME27-1 were found to be mostly similar to those of P terrae [27]Thus the strain ME27-1 was identified as P terrae
To our knowledge till date no study has reported aboutCMCase production by P terrae although other species ofPaenibacillus have been found to produce cellulase SomeCMCase genes cloned from Paenibacillus polymyxa GS01Paenibacillus barcinonensis Paenibacillus xylanilyticus KJ-03 and Paenibacillus cookii SS-24 have been expressed inEscherichia coli and Saccharomyces cerevisiae [32ndash35] On theother hand CMCases from Paenibacillus curdlanolyticus B-6Paenibacillus campinasensis BL11 Paenibacillus sp B39 and Ppolymyxa have been purified [36ndash39]
33 Effect of Initial pH Temperature Carbon and NitrogenSources Inoculum Size and Incubation Time on CMCase Pro-duction by P terrae ME27-1 The best incubation conditionswere pH 80 and 28∘C (Figures 3(a) and 3(b)) The CMCaseactivity declined when the initial pH and incubation tem-perature were not optimal There have been diverse reportson the optimal initial pH and temperature for cellulolyticenzyme production by Paenibacillus sp In a previous studyP curdlanolyticus B-6 was cultivated for enzyme productionat pH 70 and 37∘C [5] Furthermore Kumar et al reportedthat the optimal initial pH and temperature for CMCase pro-duction by P polymyxa were 55 and 37∘C respectively [39]Yoon et al accounted that the optimal growth temperaturefor P terrae was 30∘C which is similar to that observed foroptimal CMCase production by the strain ME27-1 [27]
Various cellulosic materials have been used to inducemicroorganisms to produce cellulaseWhen fructose and glu-cose were used as the sole carbon source no CMCase activitywas detected Wheat bran induced the highest CMCaseactivity which was about 25-fold higher than that observedin the basal medium containing CMC-Na (Figure 4(a)) Theoptimal concentration of wheat bran in the medium wasfound to be 50 gL (Figure 4(b)) Da Vinha et al used steam-pretreated sugarcane bagasse (or wheat bran) as the maincarbon source and found thatwheat branwas the best inducerfor CMCase production by S viridobrunneus SCPE-09 [15]Gao et al demonstrated that rice branwas the optimal carbonsource for CMCase production by Cellulophaga lytica LBH-14 while Kumar et al reported that high CMCase productionby P polymyxa was obtained when using mango peel assubstrate [39 40] In addition wheat straw rice straw andxylan have been reported to be good carbon sources forCMCase production by Cellulomonas sp and Cellulosimicro-bium cellulans [9 41]
Furthermore maximum CMCase activity was notedwhen using NH
4Cl as the sole nitrogen source (Figure 4(c))
and the best concentration of NH4Cl in the medium was
observed to be 3 gL (Figure 4(d)) Many reports have shownthat organic nitrogen sources are better than inorganic
BioMed Research International 9
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(a)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(b)
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(c)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(d)
Figure 6 Properties of CMCase produced by the strain ME27-1 (a) Effect of pH on CMCase activity (b) Effect of temperature on CMCaseactivity (c) Effect of pH on the stability of CMCase (d) Thermostability of CMCase The different buffers used are as follows (100mM)sodium citrate buffer (empty square pH 30ndash65) Na
2
HPO4
-NaH2
PO4
buffer (filled square pH 65ndash75) Tris-HCl buffer (empty triangle pH75ndash85) and glycine-NaOH buffer (filled triangle pH 85ndash110) Error bars show the standard deviation of experimental point (119899 = 2)
nitrogen sources [15 16 42 43] In the present study theCMCase activity of the strain ME27-1 was higher wheninorganic nitrogen sources were used as the sole nitrogensource Likewise Kumar et al and Kalogeris et al alsoobserved a similar phenomenon in their studies [39 44]
In addition use of an inoculum size of 2 resulted inmaximum CMCase activity after incubation of the strainfor 60 h (Figure 5) There has been increasing interest incellulase-producing bacteria because of their ability to growfast [45] In the present study the strain ME27-1 producedthe highest CMCase activity after 60 h of incubation On theother hand in previous studies maximum CMCase activityof Pseudomonas sp HP207 and S viridobrunneus SCPE-09was observed after 24 and 48 h of incubation respectivelywhich is much earlier than that noted for the strain ME27-1 [15 16] However different results have been reported invarious studies MaximumCMCase activity of C lytica LBH-14 was obtained after 72 h of incubation whereas that of
Brevibacillus sp DUSELG12 and Geobacillus sp DUSELR7was noted after days 9 and 7 respectively [12 40]
34 Properties of CMCase Produced by P terrae ME27-1The optimum pH and temperature of CMCase produced bystrain ME27-1 were found to be 55 and 50∘C respectively(Figures 6(a) and 6(b)) The CMCase produced by the strainME27-1 was stable from pH 40 to 110 with more than 60CMCase activity being retained (Figure 6(c)) Furthermorethe enzyme maintained 65 activity after incubation at 4∘Cand pH 110 for 24 h The temperature profiles demonstratedthat more than 95 CMCase activity was retained at 30ndash45∘C for 1 h (Figure 6(d)) However the enzyme activity wasreduced at temperatures above 50∘C In fact approximately77 residual activity was maintained after preincubating theenzyme at 50∘C for 1 h
Similar results were observed for cellulases produced by Sviridobrunneus SCPE-09 and P cookii SS-24 with an optimal
10 BioMed Research International
Table 3 Comparison of CMCase production by Paenibacillus terraeME27-1 with other bacterial and fungal strains
pH of 50 and 51 and an optimal temperature of 50∘ and 55∘Crespectively [15 35] However maximum CMCase activity ofbacteria at pH lower than 60 has been rarely observed andthe maximum CMCase activities of P campinasensis BL11 Ppolymyxa GS01 Paenibacillus sp B39 and Bacillus mycoidesS122C were observed at neutral or alkaline conditions [3738 46 47] In the present study the CMCase producedby the strain ME27-1 was stable at pH 50ndash95 and almost85 residual activity was retained Only a few studies havereported that CMCase was stable at such a wide pH range forexample Da Vinha et al reported the 60 CMCase activitywas retained within the pH range of 30ndash80 [15]
35 Comparison of CMCase Production by P terrae ME27-1and Other Microorganisms When measured at the optimalpH and temperature of CMCase P terrae ME27-1 producedCMCase activity of 208UmL under the optimized cul-tivation conditions which was a 12-fold improvement in
the CMCase production This yield of CMCase productionwas higher than most of the aerobic bacterial strains but lessthan someof aerobic bacterial strains that have been exploitedpreviously (Table 3) However the CMCase production by Pterrae ME27-1 was lower than that by several anaerobic bac-terial strains for example Clostridium papyrosolvens CFR-703 C thermocellum YM4 C thermocopriae JT3-3 (Table 3)Some anaerobic bacteria can degrade lignocellulosic sub-strates efficiently by producingmultienzyme complex termedcellulosome [36] The carbohydrate binding modules anddifferent proteins in the cellulosome allow the whole enzymecomplex to bind to the substrates which avoids the wastefulexpenditure of energy of bacteria releasing large amounts ofindividual enzymes andmakes lots of advantages over single-enzyme system [4 48]
Furthermore the CMCase produced by P terrae ME27-1 was lower than that by most aerobic fungal strains whileit was higher than that by anaerobic fungal strains (Table 3)
BioMed Research International 11
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
ldquo119863119889rdquo hydrolyzed zone diametercolony diameter on agar media containing CMC as sole carbon source ldquoNDrdquo no detectable activity
using an inoculating needle and inoculated onto Mandelsand Reese medium [18] containing carboxymethyl cellulosesodium salt (CMC-Na in gL KH
2PO4 20 (NH
4)2SO4 14
MgSO4sdot7H2O 03 CaCl
203 yeast extract 04 FeSO
4sdot7H2O
0005MnSO4 00016 ZnCl
2 00017 CoCl
2 0002 CMC-Na
50 and agar 150 pH 50) After incubation at 28∘C for 48 hall the plates were stained with 1 (wv) Congo-red solutionfor 15min and discolored with 1M NaCl for 15min [19] Thedegradation zones were visible around the bacteria showingthat the strains could hydrolyze CMC
The modified Mandels medium (also called basal medi-um) used for CMCase production by the isolates con-tained the following components (in gL KH
2PO4 15
Na2HPO4sdot7H2O 25 (NH
4)2SO4 15 MgSO
4sdot7H2O 03
CaCl2 01 FeSO
4sdot7H2O 0005 MnSO
4 00016 ZnCl
2
00017 and CoCl2 0002 pH 70) The bacterial isolates were
precultured overnight in general bacteria medium (in gLbeef extract 2 yeast extract 2 sucrose 6 and peptone 5)at 28∘C and 180 rpm Subsequently 2mL of the culture wasinoculated into 250mL conical flask containing 50mL ofbasal medium with 10 gL of CMC-Na as the sole carbonsource and incubated at 28∘C and 180 rpm for 60 h
23 Enzyme Assay Enzyme production during cultivationwas assayed at 12 h intervals up to 3 days The cultures werecentrifuged at 12000 rpm for 10min at 4∘CThe supernatantswere collected as crude enzyme for enzyme assay CMCaseAvicel cellulase (Avicelase) and filter-paper cellulase (FPase)activities were determined using the 35-dinitrosalicylic acid(DNS) method [20] The reaction systems were preparedas follows 250 120583L of crude enzyme (appropriately diluted)mixed with 250120583L of 2 (wv) CMC for determining theCMCase activity 500120583L of enzymemixedwith 1mLofAvicel(1 wv) for determining the Avicelase activity and 500120583Lof enzyme mixed with 50mg of Whatman number 1 filterpaper (10 times 60 cm) in 1mL of buffer for determining theFPase activityThe buffer used for dissolving or resuspendingthe substrates was 100mMsodium citrate buffer (pH 55)Themixtures were incubated at 50∘C for 30min for CMCase assayand for 1 h for Avicelase and FPase assay respectively Thenthe reactions were stopped by adding 1mL of DNS reagentfor CMCase assay and 3mL of DNS reagent for Avicelaseand FPase assay respectively All the mixtures were heated inboiling water for 5min for color development Subsequently200120583L of each sample was transferred to 96-well microplate
and the absorbance was measured at 540 nm [21 22] Oneunit (U) of the enzyme activity was defined as the amountof enzyme that released 1 120583mol of reducing sugars equivalentto glucose per minute during the reaction
The activity of 120573-glucosidase was measured by using p-nitrophenyl-120573-D-glucopyranoside (p-NPG) as substrateTheenzyme activity was determined by detecting the amount ofp-nitrophenol (p-NP) produced from p-NPG [23] One unit(U) of 120573-glucosidase activity was defined as the amount ofenzyme liberating 1 120583mol of p-NP per minute
24 16119878 rRNA Gene Sequencing and Phylogenetic Analysisof the CMC-Degrading Isolates TheCMC-degrading isolateswere cultivated in general bacteria medium at 28∘C for 24 hThe culturewas directly used for the amplification of bacterial16S rRNA gene by PCR [24] Two universal 16S rRNAgene primers (F27 51015840-AGAGTTTGATCCTGGCTCAG-31015840and R1492 51015840-TACGGTTACCTTGTTACGACTT-31015840) wereused [25]The 25 120583Lmixtureswere composed of 1120583Lof bacte-rial culture as template DNA 125 120583L of 2 times Taq PCR MasterMix (containing 05U Taq DNA polymerase120583L 500120583M ofeach dNTP 20mM Tris-HCl (pH 83) 100mM KCl 3mMMgCl
2 and bromophenol blue purchased from Tiangen
Biotech Beijing China) 1120583L of each primer (10 120583M) and95 120583L of double-distilledH
2OThePCR procedure employed
was as follows primary denaturation for 5min at 94∘C 30cycles of denaturation at 94∘C for 30 s annealing at 55∘Cfor 30 s and extension at 72∘C for 100 s and an additionalreaction for 10min at 72∘C The PCR products were detectedon 08 agarose gel to confirm its purity quantity and sizeThe PCR products were sent to Sangon Biotech (Shanghai)Co Ltd China for sequencing
The 16S rRNA gene sequences were compared with other16S rRNA gene sequences available in GenBank by using theBLASTN program (httpblastncbinlmnihgovBlastcgi)and aligned with similar sequences by using CLUSTX pro-gramThe phylogenetic tree was constructed by applying theneighbor-joining method usingMAGA41 program based onKimura-2 parameters with 1000 replicates of bootstrap values[26]
25 Morphological Physiological and Biochemical Identifi-cation of the Bacterial Strain ME27-1 The morphologicalproperties of the strain ME27-1 including shape size colonycharacteristics (color shape surface elevation and edge)andGram stainingwere evaluated [27]Thephysiological and
6 BioMed Research International
0
003
006
009
012
015
018
021
50 60 70 80 90 100pH
CMCa
se ac
tivity
(Um
L)
(a)
0
003
006
009
012
015
018
021
26 28 30 32 34
CMCa
se ac
tivity
(Um
L)
T (∘C)
(b)
Figure 3 Effect of initial pH and temperature on enzyme production by the strain ME27-1 (a) Initial pH (b) Temperature (119879)
biochemical characterization of the strainME27-1was carriedout by using API 50CHB microtests (bioMerieux)
26 Optimization of Cultivation Conditions for CMCase Pro-duction by the Strain ME27-1 The effect of initial pH andtemperature on CMCase production by the strain ME27-1was determined by cultivating the strain in 50mL of basalmediumcontaining 10 gL ofCMC-Na at various pH (rangingfrom 50 to 100 with an interval of 05) and temperatures (26ndash34∘C) for 60 h at 180 rpm
The effect of carbon and nitrogen sources on cellulaseproduction by the strain ME27-1 was determined by using 11different carbon sources (fructose glucose glycerol lactosesucrose maltose CMC-Na filter paper (chopped into 20mesh size) Avicel soluble starch and wheat bran whichwas chopped into 80 mesh size) and 10 different nitrogensources as below (NH
4)2SO4 NH4NO3 NaNO
3 KNO
3
NH4Cl urea soybean yeast extract tryptone and beef
extract The carbon sources were used at a concentration of10 gL instead of the carbon source in the basal mediumFurthermore different concentrations (10ndash100 gL with aninterval of 10 gL) of optimal carbon source were examinedSimilarly the effect of nitrogen sources was also studied withan initial concentration of 15 gL
The effect of different inoculum sizes (2 4 6 8and 10) on enzyme production was tested All media werein pH 80 All the flasks were incubated at 28∘CThe CMCaseactivity was detected at an interval of 12 h
27 Properties of CMCase Produced by the Bacterial StrainME27-1 To determine the optimal pH 250 120583L of crudeCMCase supernatant was incubated with 250 120583L of CMC-Na(2 wv) at 50∘C and different pH (30ndash110 with an intervalof 05) respectively To observe the effect of temperatureCMCase was incubated with 2 CMC-Na at a pH of 55 andtemperature ranging from 30 to 75∘C with an interval of 5∘C
Themaximum CMCase activity obtained at different pH andtemperatures was considered to be 100
The effect of pH on the stability of CMCase was studiedby mixing the crude enzyme with different buffers (1 9 vv)with pH ranging from 30 to 100 The CMCase activity ofthe crude enzyme after incubating at 4∘C for 24 h at differentpHwas detected To study the thermostability of the CMCaseproduced by the strain ME27-1 the crude enzyme was prein-cubated at different temperatures (varying from 30 to 75∘Cwith an interval of 5∘C) for 1 h The residual CMCase activitywas detectedThemaximumCMCase activity obtained at pH30ndash100 or temperature 30ndash75∘C was considered to be 100All the enzyme assays were carried out in triplicate
28 Nucleotide Sequence Accession Numbers All the DNAsequences of the partial 16S rRNA genes of the 22 strainsreported in this study have been deposited into the GenBankdatabase under the accession numbers from KF536877 toKF536898
3 Results and Discussion
31 Isolation and Screening of Cellulose-Degrading Bacteria Atotal of 245 cellulose-degrading aerobic bacterial strains wereisolated from different natural reserves in the subtropicalregion of China which were cultured in agar mediumcontaining sugarcane bagasse pulp as the sole carbon sourceOut of these strains 22 isolates showed hydrolyzing zoneson agar plates containing CMC-Na after Congo-red staining(Figure 1) The hydrolyzing zone diameter and colony diam-eter are listed in Table 1
Among the 22 isolates only three isolates (ME27-1 FCD1-3 and SK3-4) were found to produce measurable CMCaseafter liquid cultivation and isolate ME27-1 showed the high-est CMCase activity (017UmL) after incubation for 60 h inbasal liquid medium containing 10 gL of CMC-Na (Table 1)The CMCase activity of the other 19 strains was undetectable
BioMed Research International 7
0
009
018
027
036
045
1 2 3 4 5 6 7 8 9
CMCa
se ac
tivity
(Um
L)
Carbon sources
(a)
0
03
06
09
12
15
10 30 50 70 90
CMCa
se ac
tivity
(Um
L)
Concentration of wheat bran (gL)
(b)
0
045
09
135
18
a b c d e f g h i j
CMCa
se ac
tivity
(Um
L)
Nitrogen sources
(c)
0
03
06
09
12
15
18
00 15 30 45 60 75 90
CMCa
se ac
tivity
(Um
L)
Concentration of NH4Cl (gL)
(d)
Figure 4 Effect of carbon and nitrogen sources on CMCase production by the strainME27-1 (a) Different carbon sources 1 sim 9 representedglycerol lactose sucrose maltose CMC-Na filter paper Avicel soluble starch and wheat bran respectively (b) The concentration of wheatbran (c) Different nitrogen sources a sim j represented (NH
4
)2
SO4
NH4
NO3
NaNO3
KNO3
NH4
Cl urea soybean yeast extract tryptoneand beef extract respectively (d) The concentration of NH
4
Cl
after cultivating in various liquid media for up to 6 days andthe Avicelase FPase and 120573-glucosidase activities of all the 22bacterial strains were also undetectable
Congo-red staining has been widely used inmany studiesfor screening cellulose-degradingmicroorganisms AlthoughTeather and Wood described the relationship between thediameter of hydrolyzing zone and log enzyme concentrationthis correlation could not represent the enzyme-producingability of the microorganisms [19] In the present studyalthough some strains presented large and clear hydrolyzingzones the activities of CMCase and other cellulases producedby themwere undetectable in various liquidmedia containingCMCand other cellulosicmaterials suggesting that either theconcentration of the enzyme produced by these strains wasvery low to be detected after cultivation in liquid medium orthe ability of the strains to secrete CMCase was weak Sadhu
and Maiti also reported that the diameter of the hydrolyzingzonemay not accurately reflect the real cellulase activity [28]
In general aerobic bacteria produce low levels of Avice-lase FPase and 120573-glucosidase In a study carried out byRastogi et al Brevibacillus sp DUSELG12 andGeobacillus spDUSELR7 were found to produce a maximum FPase activityof 0027 and 0043UmL on days 7 and 8 respectively [12]Recently Soares et al found that only 91 of bacterial strainswere able to degrade Avicel on agar plates [7]
32 Identification of Cellulose-Degrading Bacteria The DNAfragments containing partial 16S rRNAgenes of the 22 isolateswere amplified and sequenced The sequences obtained werematched with those available in GenBank which revealedmaximum identity of these isolates and allowed identificationof these cellulose-degrading bacterial strains (Table 1)
8 BioMed Research International
0
03
06
09
12
15
12 24 36 48 60 72
CMCa
se ac
tivity
(Um
L)
Incubation time (h)
Figure 5 Effect of inoculum size and incubation period onCMCaseproduction by the strain ME27-1 2 (empty triangle) 4 (filledtriangle) 6 (filled circle) 8 (filled square) and 10 (empty square)Error bars show the standard deviation of experimental point (119899 =3)
It was found that the 22 aerobic bacterial strains thatcould hydrolyze cellulose belonged to 10 different gen-era Burkholderia (3636) Bacillus (1365) Citrobacter(1365) Arthrobacter (910) Enterobacter (454) Chry-seobacterium (454) Pandoraea (454) Paenibacillus(454) Dyella (454) and Pseudomonas (454) Thephylogenetic tree of the 22 strains was constructed by usingMAGA41 program (Figure 2)
Various cellulose-degrading bacteria have been foundin different environments The genus Burkholderia wasobserved to be the main cellulose-hydrolyzing bacteria andwas considered to play an important role in cellulose degra-dation in the subtropical region of China in this studyIn addition bacteria belonging to the genera ArthrobacterChryseobacterium Pandoraea and Dyella were also foundto be cellulolytic in the present study which have beenrarely reported as cellulose-degrading bacteria In a previousstudy Lo et al reported that the cellulase-producing bacterialstrains isolated from a rice field in southern Taiwan mainlybelonged to the genus Cellulomonas [9] On the other handBacilluswas reported to be the dominant cellulose-degradingbacteria in samples collected from paper mill sludges andorganic fertilizers from Red Rock Canada as well as inthose from soil compost and animal waste slurry from JejuIsland [29 30] Similarly Burkholderia was found to be themain genus of cellulase-producing bacteria in the subtropicalrainforest in Okinawa Island Japan [31]
The strain ME27-1 with higher CMCase activity wasthoroughly examined The partial 16S rRNA gene (1309 bp)from the strain ME27-1 showed a maximum identity of 99with that ofPaenibacillus terraeAM141T (T type strain)Mor-phological tests revealed that the cells of the strain ME27-1
were rod-shaped endospore-forming Gram-positive and08 times 19ndash32 120583m in size The appearance of the colonyon the TSA medium was cream-colored moist irregularswollen and pigment-free The biochemical properties ofthe strain ME27-1 are listed in Table 2 The morphologicalphysiological and biochemical properties of the strainME27-1 were found to be mostly similar to those of P terrae [27]Thus the strain ME27-1 was identified as P terrae
To our knowledge till date no study has reported aboutCMCase production by P terrae although other species ofPaenibacillus have been found to produce cellulase SomeCMCase genes cloned from Paenibacillus polymyxa GS01Paenibacillus barcinonensis Paenibacillus xylanilyticus KJ-03 and Paenibacillus cookii SS-24 have been expressed inEscherichia coli and Saccharomyces cerevisiae [32ndash35] On theother hand CMCases from Paenibacillus curdlanolyticus B-6Paenibacillus campinasensis BL11 Paenibacillus sp B39 and Ppolymyxa have been purified [36ndash39]
33 Effect of Initial pH Temperature Carbon and NitrogenSources Inoculum Size and Incubation Time on CMCase Pro-duction by P terrae ME27-1 The best incubation conditionswere pH 80 and 28∘C (Figures 3(a) and 3(b)) The CMCaseactivity declined when the initial pH and incubation tem-perature were not optimal There have been diverse reportson the optimal initial pH and temperature for cellulolyticenzyme production by Paenibacillus sp In a previous studyP curdlanolyticus B-6 was cultivated for enzyme productionat pH 70 and 37∘C [5] Furthermore Kumar et al reportedthat the optimal initial pH and temperature for CMCase pro-duction by P polymyxa were 55 and 37∘C respectively [39]Yoon et al accounted that the optimal growth temperaturefor P terrae was 30∘C which is similar to that observed foroptimal CMCase production by the strain ME27-1 [27]
Various cellulosic materials have been used to inducemicroorganisms to produce cellulaseWhen fructose and glu-cose were used as the sole carbon source no CMCase activitywas detected Wheat bran induced the highest CMCaseactivity which was about 25-fold higher than that observedin the basal medium containing CMC-Na (Figure 4(a)) Theoptimal concentration of wheat bran in the medium wasfound to be 50 gL (Figure 4(b)) Da Vinha et al used steam-pretreated sugarcane bagasse (or wheat bran) as the maincarbon source and found thatwheat branwas the best inducerfor CMCase production by S viridobrunneus SCPE-09 [15]Gao et al demonstrated that rice branwas the optimal carbonsource for CMCase production by Cellulophaga lytica LBH-14 while Kumar et al reported that high CMCase productionby P polymyxa was obtained when using mango peel assubstrate [39 40] In addition wheat straw rice straw andxylan have been reported to be good carbon sources forCMCase production by Cellulomonas sp and Cellulosimicro-bium cellulans [9 41]
Furthermore maximum CMCase activity was notedwhen using NH
4Cl as the sole nitrogen source (Figure 4(c))
and the best concentration of NH4Cl in the medium was
observed to be 3 gL (Figure 4(d)) Many reports have shownthat organic nitrogen sources are better than inorganic
BioMed Research International 9
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(a)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(b)
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(c)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(d)
Figure 6 Properties of CMCase produced by the strain ME27-1 (a) Effect of pH on CMCase activity (b) Effect of temperature on CMCaseactivity (c) Effect of pH on the stability of CMCase (d) Thermostability of CMCase The different buffers used are as follows (100mM)sodium citrate buffer (empty square pH 30ndash65) Na
2
HPO4
-NaH2
PO4
buffer (filled square pH 65ndash75) Tris-HCl buffer (empty triangle pH75ndash85) and glycine-NaOH buffer (filled triangle pH 85ndash110) Error bars show the standard deviation of experimental point (119899 = 2)
nitrogen sources [15 16 42 43] In the present study theCMCase activity of the strain ME27-1 was higher wheninorganic nitrogen sources were used as the sole nitrogensource Likewise Kumar et al and Kalogeris et al alsoobserved a similar phenomenon in their studies [39 44]
In addition use of an inoculum size of 2 resulted inmaximum CMCase activity after incubation of the strainfor 60 h (Figure 5) There has been increasing interest incellulase-producing bacteria because of their ability to growfast [45] In the present study the strain ME27-1 producedthe highest CMCase activity after 60 h of incubation On theother hand in previous studies maximum CMCase activityof Pseudomonas sp HP207 and S viridobrunneus SCPE-09was observed after 24 and 48 h of incubation respectivelywhich is much earlier than that noted for the strain ME27-1 [15 16] However different results have been reported invarious studies MaximumCMCase activity of C lytica LBH-14 was obtained after 72 h of incubation whereas that of
Brevibacillus sp DUSELG12 and Geobacillus sp DUSELR7was noted after days 9 and 7 respectively [12 40]
34 Properties of CMCase Produced by P terrae ME27-1The optimum pH and temperature of CMCase produced bystrain ME27-1 were found to be 55 and 50∘C respectively(Figures 6(a) and 6(b)) The CMCase produced by the strainME27-1 was stable from pH 40 to 110 with more than 60CMCase activity being retained (Figure 6(c)) Furthermorethe enzyme maintained 65 activity after incubation at 4∘Cand pH 110 for 24 h The temperature profiles demonstratedthat more than 95 CMCase activity was retained at 30ndash45∘C for 1 h (Figure 6(d)) However the enzyme activity wasreduced at temperatures above 50∘C In fact approximately77 residual activity was maintained after preincubating theenzyme at 50∘C for 1 h
Similar results were observed for cellulases produced by Sviridobrunneus SCPE-09 and P cookii SS-24 with an optimal
10 BioMed Research International
Table 3 Comparison of CMCase production by Paenibacillus terraeME27-1 with other bacterial and fungal strains
pH of 50 and 51 and an optimal temperature of 50∘ and 55∘Crespectively [15 35] However maximum CMCase activity ofbacteria at pH lower than 60 has been rarely observed andthe maximum CMCase activities of P campinasensis BL11 Ppolymyxa GS01 Paenibacillus sp B39 and Bacillus mycoidesS122C were observed at neutral or alkaline conditions [3738 46 47] In the present study the CMCase producedby the strain ME27-1 was stable at pH 50ndash95 and almost85 residual activity was retained Only a few studies havereported that CMCase was stable at such a wide pH range forexample Da Vinha et al reported the 60 CMCase activitywas retained within the pH range of 30ndash80 [15]
35 Comparison of CMCase Production by P terrae ME27-1and Other Microorganisms When measured at the optimalpH and temperature of CMCase P terrae ME27-1 producedCMCase activity of 208UmL under the optimized cul-tivation conditions which was a 12-fold improvement in
the CMCase production This yield of CMCase productionwas higher than most of the aerobic bacterial strains but lessthan someof aerobic bacterial strains that have been exploitedpreviously (Table 3) However the CMCase production by Pterrae ME27-1 was lower than that by several anaerobic bac-terial strains for example Clostridium papyrosolvens CFR-703 C thermocellum YM4 C thermocopriae JT3-3 (Table 3)Some anaerobic bacteria can degrade lignocellulosic sub-strates efficiently by producingmultienzyme complex termedcellulosome [36] The carbohydrate binding modules anddifferent proteins in the cellulosome allow the whole enzymecomplex to bind to the substrates which avoids the wastefulexpenditure of energy of bacteria releasing large amounts ofindividual enzymes andmakes lots of advantages over single-enzyme system [4 48]
Furthermore the CMCase produced by P terrae ME27-1 was lower than that by most aerobic fungal strains whileit was higher than that by anaerobic fungal strains (Table 3)
BioMed Research International 11
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
and the absorbance was measured at 540 nm [21 22] Oneunit (U) of the enzyme activity was defined as the amountof enzyme that released 1 120583mol of reducing sugars equivalentto glucose per minute during the reaction
The activity of 120573-glucosidase was measured by using p-nitrophenyl-120573-D-glucopyranoside (p-NPG) as substrateTheenzyme activity was determined by detecting the amount ofp-nitrophenol (p-NP) produced from p-NPG [23] One unit(U) of 120573-glucosidase activity was defined as the amount ofenzyme liberating 1 120583mol of p-NP per minute
24 16119878 rRNA Gene Sequencing and Phylogenetic Analysisof the CMC-Degrading Isolates TheCMC-degrading isolateswere cultivated in general bacteria medium at 28∘C for 24 hThe culturewas directly used for the amplification of bacterial16S rRNA gene by PCR [24] Two universal 16S rRNAgene primers (F27 51015840-AGAGTTTGATCCTGGCTCAG-31015840and R1492 51015840-TACGGTTACCTTGTTACGACTT-31015840) wereused [25]The 25 120583Lmixtureswere composed of 1120583Lof bacte-rial culture as template DNA 125 120583L of 2 times Taq PCR MasterMix (containing 05U Taq DNA polymerase120583L 500120583M ofeach dNTP 20mM Tris-HCl (pH 83) 100mM KCl 3mMMgCl
2 and bromophenol blue purchased from Tiangen
Biotech Beijing China) 1120583L of each primer (10 120583M) and95 120583L of double-distilledH
2OThePCR procedure employed
was as follows primary denaturation for 5min at 94∘C 30cycles of denaturation at 94∘C for 30 s annealing at 55∘Cfor 30 s and extension at 72∘C for 100 s and an additionalreaction for 10min at 72∘C The PCR products were detectedon 08 agarose gel to confirm its purity quantity and sizeThe PCR products were sent to Sangon Biotech (Shanghai)Co Ltd China for sequencing
The 16S rRNA gene sequences were compared with other16S rRNA gene sequences available in GenBank by using theBLASTN program (httpblastncbinlmnihgovBlastcgi)and aligned with similar sequences by using CLUSTX pro-gramThe phylogenetic tree was constructed by applying theneighbor-joining method usingMAGA41 program based onKimura-2 parameters with 1000 replicates of bootstrap values[26]
25 Morphological Physiological and Biochemical Identifi-cation of the Bacterial Strain ME27-1 The morphologicalproperties of the strain ME27-1 including shape size colonycharacteristics (color shape surface elevation and edge)andGram stainingwere evaluated [27]Thephysiological and
6 BioMed Research International
0
003
006
009
012
015
018
021
50 60 70 80 90 100pH
CMCa
se ac
tivity
(Um
L)
(a)
0
003
006
009
012
015
018
021
26 28 30 32 34
CMCa
se ac
tivity
(Um
L)
T (∘C)
(b)
Figure 3 Effect of initial pH and temperature on enzyme production by the strain ME27-1 (a) Initial pH (b) Temperature (119879)
biochemical characterization of the strainME27-1was carriedout by using API 50CHB microtests (bioMerieux)
26 Optimization of Cultivation Conditions for CMCase Pro-duction by the Strain ME27-1 The effect of initial pH andtemperature on CMCase production by the strain ME27-1was determined by cultivating the strain in 50mL of basalmediumcontaining 10 gL ofCMC-Na at various pH (rangingfrom 50 to 100 with an interval of 05) and temperatures (26ndash34∘C) for 60 h at 180 rpm
The effect of carbon and nitrogen sources on cellulaseproduction by the strain ME27-1 was determined by using 11different carbon sources (fructose glucose glycerol lactosesucrose maltose CMC-Na filter paper (chopped into 20mesh size) Avicel soluble starch and wheat bran whichwas chopped into 80 mesh size) and 10 different nitrogensources as below (NH
4)2SO4 NH4NO3 NaNO
3 KNO
3
NH4Cl urea soybean yeast extract tryptone and beef
extract The carbon sources were used at a concentration of10 gL instead of the carbon source in the basal mediumFurthermore different concentrations (10ndash100 gL with aninterval of 10 gL) of optimal carbon source were examinedSimilarly the effect of nitrogen sources was also studied withan initial concentration of 15 gL
The effect of different inoculum sizes (2 4 6 8and 10) on enzyme production was tested All media werein pH 80 All the flasks were incubated at 28∘CThe CMCaseactivity was detected at an interval of 12 h
27 Properties of CMCase Produced by the Bacterial StrainME27-1 To determine the optimal pH 250 120583L of crudeCMCase supernatant was incubated with 250 120583L of CMC-Na(2 wv) at 50∘C and different pH (30ndash110 with an intervalof 05) respectively To observe the effect of temperatureCMCase was incubated with 2 CMC-Na at a pH of 55 andtemperature ranging from 30 to 75∘C with an interval of 5∘C
Themaximum CMCase activity obtained at different pH andtemperatures was considered to be 100
The effect of pH on the stability of CMCase was studiedby mixing the crude enzyme with different buffers (1 9 vv)with pH ranging from 30 to 100 The CMCase activity ofthe crude enzyme after incubating at 4∘C for 24 h at differentpHwas detected To study the thermostability of the CMCaseproduced by the strain ME27-1 the crude enzyme was prein-cubated at different temperatures (varying from 30 to 75∘Cwith an interval of 5∘C) for 1 h The residual CMCase activitywas detectedThemaximumCMCase activity obtained at pH30ndash100 or temperature 30ndash75∘C was considered to be 100All the enzyme assays were carried out in triplicate
28 Nucleotide Sequence Accession Numbers All the DNAsequences of the partial 16S rRNA genes of the 22 strainsreported in this study have been deposited into the GenBankdatabase under the accession numbers from KF536877 toKF536898
3 Results and Discussion
31 Isolation and Screening of Cellulose-Degrading Bacteria Atotal of 245 cellulose-degrading aerobic bacterial strains wereisolated from different natural reserves in the subtropicalregion of China which were cultured in agar mediumcontaining sugarcane bagasse pulp as the sole carbon sourceOut of these strains 22 isolates showed hydrolyzing zoneson agar plates containing CMC-Na after Congo-red staining(Figure 1) The hydrolyzing zone diameter and colony diam-eter are listed in Table 1
Among the 22 isolates only three isolates (ME27-1 FCD1-3 and SK3-4) were found to produce measurable CMCaseafter liquid cultivation and isolate ME27-1 showed the high-est CMCase activity (017UmL) after incubation for 60 h inbasal liquid medium containing 10 gL of CMC-Na (Table 1)The CMCase activity of the other 19 strains was undetectable
BioMed Research International 7
0
009
018
027
036
045
1 2 3 4 5 6 7 8 9
CMCa
se ac
tivity
(Um
L)
Carbon sources
(a)
0
03
06
09
12
15
10 30 50 70 90
CMCa
se ac
tivity
(Um
L)
Concentration of wheat bran (gL)
(b)
0
045
09
135
18
a b c d e f g h i j
CMCa
se ac
tivity
(Um
L)
Nitrogen sources
(c)
0
03
06
09
12
15
18
00 15 30 45 60 75 90
CMCa
se ac
tivity
(Um
L)
Concentration of NH4Cl (gL)
(d)
Figure 4 Effect of carbon and nitrogen sources on CMCase production by the strainME27-1 (a) Different carbon sources 1 sim 9 representedglycerol lactose sucrose maltose CMC-Na filter paper Avicel soluble starch and wheat bran respectively (b) The concentration of wheatbran (c) Different nitrogen sources a sim j represented (NH
4
)2
SO4
NH4
NO3
NaNO3
KNO3
NH4
Cl urea soybean yeast extract tryptoneand beef extract respectively (d) The concentration of NH
4
Cl
after cultivating in various liquid media for up to 6 days andthe Avicelase FPase and 120573-glucosidase activities of all the 22bacterial strains were also undetectable
Congo-red staining has been widely used inmany studiesfor screening cellulose-degradingmicroorganisms AlthoughTeather and Wood described the relationship between thediameter of hydrolyzing zone and log enzyme concentrationthis correlation could not represent the enzyme-producingability of the microorganisms [19] In the present studyalthough some strains presented large and clear hydrolyzingzones the activities of CMCase and other cellulases producedby themwere undetectable in various liquidmedia containingCMCand other cellulosicmaterials suggesting that either theconcentration of the enzyme produced by these strains wasvery low to be detected after cultivation in liquid medium orthe ability of the strains to secrete CMCase was weak Sadhu
and Maiti also reported that the diameter of the hydrolyzingzonemay not accurately reflect the real cellulase activity [28]
In general aerobic bacteria produce low levels of Avice-lase FPase and 120573-glucosidase In a study carried out byRastogi et al Brevibacillus sp DUSELG12 andGeobacillus spDUSELR7 were found to produce a maximum FPase activityof 0027 and 0043UmL on days 7 and 8 respectively [12]Recently Soares et al found that only 91 of bacterial strainswere able to degrade Avicel on agar plates [7]
32 Identification of Cellulose-Degrading Bacteria The DNAfragments containing partial 16S rRNAgenes of the 22 isolateswere amplified and sequenced The sequences obtained werematched with those available in GenBank which revealedmaximum identity of these isolates and allowed identificationof these cellulose-degrading bacterial strains (Table 1)
8 BioMed Research International
0
03
06
09
12
15
12 24 36 48 60 72
CMCa
se ac
tivity
(Um
L)
Incubation time (h)
Figure 5 Effect of inoculum size and incubation period onCMCaseproduction by the strain ME27-1 2 (empty triangle) 4 (filledtriangle) 6 (filled circle) 8 (filled square) and 10 (empty square)Error bars show the standard deviation of experimental point (119899 =3)
It was found that the 22 aerobic bacterial strains thatcould hydrolyze cellulose belonged to 10 different gen-era Burkholderia (3636) Bacillus (1365) Citrobacter(1365) Arthrobacter (910) Enterobacter (454) Chry-seobacterium (454) Pandoraea (454) Paenibacillus(454) Dyella (454) and Pseudomonas (454) Thephylogenetic tree of the 22 strains was constructed by usingMAGA41 program (Figure 2)
Various cellulose-degrading bacteria have been foundin different environments The genus Burkholderia wasobserved to be the main cellulose-hydrolyzing bacteria andwas considered to play an important role in cellulose degra-dation in the subtropical region of China in this studyIn addition bacteria belonging to the genera ArthrobacterChryseobacterium Pandoraea and Dyella were also foundto be cellulolytic in the present study which have beenrarely reported as cellulose-degrading bacteria In a previousstudy Lo et al reported that the cellulase-producing bacterialstrains isolated from a rice field in southern Taiwan mainlybelonged to the genus Cellulomonas [9] On the other handBacilluswas reported to be the dominant cellulose-degradingbacteria in samples collected from paper mill sludges andorganic fertilizers from Red Rock Canada as well as inthose from soil compost and animal waste slurry from JejuIsland [29 30] Similarly Burkholderia was found to be themain genus of cellulase-producing bacteria in the subtropicalrainforest in Okinawa Island Japan [31]
The strain ME27-1 with higher CMCase activity wasthoroughly examined The partial 16S rRNA gene (1309 bp)from the strain ME27-1 showed a maximum identity of 99with that ofPaenibacillus terraeAM141T (T type strain)Mor-phological tests revealed that the cells of the strain ME27-1
were rod-shaped endospore-forming Gram-positive and08 times 19ndash32 120583m in size The appearance of the colonyon the TSA medium was cream-colored moist irregularswollen and pigment-free The biochemical properties ofthe strain ME27-1 are listed in Table 2 The morphologicalphysiological and biochemical properties of the strainME27-1 were found to be mostly similar to those of P terrae [27]Thus the strain ME27-1 was identified as P terrae
To our knowledge till date no study has reported aboutCMCase production by P terrae although other species ofPaenibacillus have been found to produce cellulase SomeCMCase genes cloned from Paenibacillus polymyxa GS01Paenibacillus barcinonensis Paenibacillus xylanilyticus KJ-03 and Paenibacillus cookii SS-24 have been expressed inEscherichia coli and Saccharomyces cerevisiae [32ndash35] On theother hand CMCases from Paenibacillus curdlanolyticus B-6Paenibacillus campinasensis BL11 Paenibacillus sp B39 and Ppolymyxa have been purified [36ndash39]
33 Effect of Initial pH Temperature Carbon and NitrogenSources Inoculum Size and Incubation Time on CMCase Pro-duction by P terrae ME27-1 The best incubation conditionswere pH 80 and 28∘C (Figures 3(a) and 3(b)) The CMCaseactivity declined when the initial pH and incubation tem-perature were not optimal There have been diverse reportson the optimal initial pH and temperature for cellulolyticenzyme production by Paenibacillus sp In a previous studyP curdlanolyticus B-6 was cultivated for enzyme productionat pH 70 and 37∘C [5] Furthermore Kumar et al reportedthat the optimal initial pH and temperature for CMCase pro-duction by P polymyxa were 55 and 37∘C respectively [39]Yoon et al accounted that the optimal growth temperaturefor P terrae was 30∘C which is similar to that observed foroptimal CMCase production by the strain ME27-1 [27]
Various cellulosic materials have been used to inducemicroorganisms to produce cellulaseWhen fructose and glu-cose were used as the sole carbon source no CMCase activitywas detected Wheat bran induced the highest CMCaseactivity which was about 25-fold higher than that observedin the basal medium containing CMC-Na (Figure 4(a)) Theoptimal concentration of wheat bran in the medium wasfound to be 50 gL (Figure 4(b)) Da Vinha et al used steam-pretreated sugarcane bagasse (or wheat bran) as the maincarbon source and found thatwheat branwas the best inducerfor CMCase production by S viridobrunneus SCPE-09 [15]Gao et al demonstrated that rice branwas the optimal carbonsource for CMCase production by Cellulophaga lytica LBH-14 while Kumar et al reported that high CMCase productionby P polymyxa was obtained when using mango peel assubstrate [39 40] In addition wheat straw rice straw andxylan have been reported to be good carbon sources forCMCase production by Cellulomonas sp and Cellulosimicro-bium cellulans [9 41]
Furthermore maximum CMCase activity was notedwhen using NH
4Cl as the sole nitrogen source (Figure 4(c))
and the best concentration of NH4Cl in the medium was
observed to be 3 gL (Figure 4(d)) Many reports have shownthat organic nitrogen sources are better than inorganic
BioMed Research International 9
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(a)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(b)
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(c)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(d)
Figure 6 Properties of CMCase produced by the strain ME27-1 (a) Effect of pH on CMCase activity (b) Effect of temperature on CMCaseactivity (c) Effect of pH on the stability of CMCase (d) Thermostability of CMCase The different buffers used are as follows (100mM)sodium citrate buffer (empty square pH 30ndash65) Na
2
HPO4
-NaH2
PO4
buffer (filled square pH 65ndash75) Tris-HCl buffer (empty triangle pH75ndash85) and glycine-NaOH buffer (filled triangle pH 85ndash110) Error bars show the standard deviation of experimental point (119899 = 2)
nitrogen sources [15 16 42 43] In the present study theCMCase activity of the strain ME27-1 was higher wheninorganic nitrogen sources were used as the sole nitrogensource Likewise Kumar et al and Kalogeris et al alsoobserved a similar phenomenon in their studies [39 44]
In addition use of an inoculum size of 2 resulted inmaximum CMCase activity after incubation of the strainfor 60 h (Figure 5) There has been increasing interest incellulase-producing bacteria because of their ability to growfast [45] In the present study the strain ME27-1 producedthe highest CMCase activity after 60 h of incubation On theother hand in previous studies maximum CMCase activityof Pseudomonas sp HP207 and S viridobrunneus SCPE-09was observed after 24 and 48 h of incubation respectivelywhich is much earlier than that noted for the strain ME27-1 [15 16] However different results have been reported invarious studies MaximumCMCase activity of C lytica LBH-14 was obtained after 72 h of incubation whereas that of
Brevibacillus sp DUSELG12 and Geobacillus sp DUSELR7was noted after days 9 and 7 respectively [12 40]
34 Properties of CMCase Produced by P terrae ME27-1The optimum pH and temperature of CMCase produced bystrain ME27-1 were found to be 55 and 50∘C respectively(Figures 6(a) and 6(b)) The CMCase produced by the strainME27-1 was stable from pH 40 to 110 with more than 60CMCase activity being retained (Figure 6(c)) Furthermorethe enzyme maintained 65 activity after incubation at 4∘Cand pH 110 for 24 h The temperature profiles demonstratedthat more than 95 CMCase activity was retained at 30ndash45∘C for 1 h (Figure 6(d)) However the enzyme activity wasreduced at temperatures above 50∘C In fact approximately77 residual activity was maintained after preincubating theenzyme at 50∘C for 1 h
Similar results were observed for cellulases produced by Sviridobrunneus SCPE-09 and P cookii SS-24 with an optimal
10 BioMed Research International
Table 3 Comparison of CMCase production by Paenibacillus terraeME27-1 with other bacterial and fungal strains
pH of 50 and 51 and an optimal temperature of 50∘ and 55∘Crespectively [15 35] However maximum CMCase activity ofbacteria at pH lower than 60 has been rarely observed andthe maximum CMCase activities of P campinasensis BL11 Ppolymyxa GS01 Paenibacillus sp B39 and Bacillus mycoidesS122C were observed at neutral or alkaline conditions [3738 46 47] In the present study the CMCase producedby the strain ME27-1 was stable at pH 50ndash95 and almost85 residual activity was retained Only a few studies havereported that CMCase was stable at such a wide pH range forexample Da Vinha et al reported the 60 CMCase activitywas retained within the pH range of 30ndash80 [15]
35 Comparison of CMCase Production by P terrae ME27-1and Other Microorganisms When measured at the optimalpH and temperature of CMCase P terrae ME27-1 producedCMCase activity of 208UmL under the optimized cul-tivation conditions which was a 12-fold improvement in
the CMCase production This yield of CMCase productionwas higher than most of the aerobic bacterial strains but lessthan someof aerobic bacterial strains that have been exploitedpreviously (Table 3) However the CMCase production by Pterrae ME27-1 was lower than that by several anaerobic bac-terial strains for example Clostridium papyrosolvens CFR-703 C thermocellum YM4 C thermocopriae JT3-3 (Table 3)Some anaerobic bacteria can degrade lignocellulosic sub-strates efficiently by producingmultienzyme complex termedcellulosome [36] The carbohydrate binding modules anddifferent proteins in the cellulosome allow the whole enzymecomplex to bind to the substrates which avoids the wastefulexpenditure of energy of bacteria releasing large amounts ofindividual enzymes andmakes lots of advantages over single-enzyme system [4 48]
Furthermore the CMCase produced by P terrae ME27-1 was lower than that by most aerobic fungal strains whileit was higher than that by anaerobic fungal strains (Table 3)
BioMed Research International 11
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
and the absorbance was measured at 540 nm [21 22] Oneunit (U) of the enzyme activity was defined as the amountof enzyme that released 1 120583mol of reducing sugars equivalentto glucose per minute during the reaction
The activity of 120573-glucosidase was measured by using p-nitrophenyl-120573-D-glucopyranoside (p-NPG) as substrateTheenzyme activity was determined by detecting the amount ofp-nitrophenol (p-NP) produced from p-NPG [23] One unit(U) of 120573-glucosidase activity was defined as the amount ofenzyme liberating 1 120583mol of p-NP per minute
24 16119878 rRNA Gene Sequencing and Phylogenetic Analysisof the CMC-Degrading Isolates TheCMC-degrading isolateswere cultivated in general bacteria medium at 28∘C for 24 hThe culturewas directly used for the amplification of bacterial16S rRNA gene by PCR [24] Two universal 16S rRNAgene primers (F27 51015840-AGAGTTTGATCCTGGCTCAG-31015840and R1492 51015840-TACGGTTACCTTGTTACGACTT-31015840) wereused [25]The 25 120583Lmixtureswere composed of 1120583Lof bacte-rial culture as template DNA 125 120583L of 2 times Taq PCR MasterMix (containing 05U Taq DNA polymerase120583L 500120583M ofeach dNTP 20mM Tris-HCl (pH 83) 100mM KCl 3mMMgCl
2 and bromophenol blue purchased from Tiangen
Biotech Beijing China) 1120583L of each primer (10 120583M) and95 120583L of double-distilledH
2OThePCR procedure employed
was as follows primary denaturation for 5min at 94∘C 30cycles of denaturation at 94∘C for 30 s annealing at 55∘Cfor 30 s and extension at 72∘C for 100 s and an additionalreaction for 10min at 72∘C The PCR products were detectedon 08 agarose gel to confirm its purity quantity and sizeThe PCR products were sent to Sangon Biotech (Shanghai)Co Ltd China for sequencing
The 16S rRNA gene sequences were compared with other16S rRNA gene sequences available in GenBank by using theBLASTN program (httpblastncbinlmnihgovBlastcgi)and aligned with similar sequences by using CLUSTX pro-gramThe phylogenetic tree was constructed by applying theneighbor-joining method usingMAGA41 program based onKimura-2 parameters with 1000 replicates of bootstrap values[26]
25 Morphological Physiological and Biochemical Identifi-cation of the Bacterial Strain ME27-1 The morphologicalproperties of the strain ME27-1 including shape size colonycharacteristics (color shape surface elevation and edge)andGram stainingwere evaluated [27]Thephysiological and
6 BioMed Research International
0
003
006
009
012
015
018
021
50 60 70 80 90 100pH
CMCa
se ac
tivity
(Um
L)
(a)
0
003
006
009
012
015
018
021
26 28 30 32 34
CMCa
se ac
tivity
(Um
L)
T (∘C)
(b)
Figure 3 Effect of initial pH and temperature on enzyme production by the strain ME27-1 (a) Initial pH (b) Temperature (119879)
biochemical characterization of the strainME27-1was carriedout by using API 50CHB microtests (bioMerieux)
26 Optimization of Cultivation Conditions for CMCase Pro-duction by the Strain ME27-1 The effect of initial pH andtemperature on CMCase production by the strain ME27-1was determined by cultivating the strain in 50mL of basalmediumcontaining 10 gL ofCMC-Na at various pH (rangingfrom 50 to 100 with an interval of 05) and temperatures (26ndash34∘C) for 60 h at 180 rpm
The effect of carbon and nitrogen sources on cellulaseproduction by the strain ME27-1 was determined by using 11different carbon sources (fructose glucose glycerol lactosesucrose maltose CMC-Na filter paper (chopped into 20mesh size) Avicel soluble starch and wheat bran whichwas chopped into 80 mesh size) and 10 different nitrogensources as below (NH
4)2SO4 NH4NO3 NaNO
3 KNO
3
NH4Cl urea soybean yeast extract tryptone and beef
extract The carbon sources were used at a concentration of10 gL instead of the carbon source in the basal mediumFurthermore different concentrations (10ndash100 gL with aninterval of 10 gL) of optimal carbon source were examinedSimilarly the effect of nitrogen sources was also studied withan initial concentration of 15 gL
The effect of different inoculum sizes (2 4 6 8and 10) on enzyme production was tested All media werein pH 80 All the flasks were incubated at 28∘CThe CMCaseactivity was detected at an interval of 12 h
27 Properties of CMCase Produced by the Bacterial StrainME27-1 To determine the optimal pH 250 120583L of crudeCMCase supernatant was incubated with 250 120583L of CMC-Na(2 wv) at 50∘C and different pH (30ndash110 with an intervalof 05) respectively To observe the effect of temperatureCMCase was incubated with 2 CMC-Na at a pH of 55 andtemperature ranging from 30 to 75∘C with an interval of 5∘C
Themaximum CMCase activity obtained at different pH andtemperatures was considered to be 100
The effect of pH on the stability of CMCase was studiedby mixing the crude enzyme with different buffers (1 9 vv)with pH ranging from 30 to 100 The CMCase activity ofthe crude enzyme after incubating at 4∘C for 24 h at differentpHwas detected To study the thermostability of the CMCaseproduced by the strain ME27-1 the crude enzyme was prein-cubated at different temperatures (varying from 30 to 75∘Cwith an interval of 5∘C) for 1 h The residual CMCase activitywas detectedThemaximumCMCase activity obtained at pH30ndash100 or temperature 30ndash75∘C was considered to be 100All the enzyme assays were carried out in triplicate
28 Nucleotide Sequence Accession Numbers All the DNAsequences of the partial 16S rRNA genes of the 22 strainsreported in this study have been deposited into the GenBankdatabase under the accession numbers from KF536877 toKF536898
3 Results and Discussion
31 Isolation and Screening of Cellulose-Degrading Bacteria Atotal of 245 cellulose-degrading aerobic bacterial strains wereisolated from different natural reserves in the subtropicalregion of China which were cultured in agar mediumcontaining sugarcane bagasse pulp as the sole carbon sourceOut of these strains 22 isolates showed hydrolyzing zoneson agar plates containing CMC-Na after Congo-red staining(Figure 1) The hydrolyzing zone diameter and colony diam-eter are listed in Table 1
Among the 22 isolates only three isolates (ME27-1 FCD1-3 and SK3-4) were found to produce measurable CMCaseafter liquid cultivation and isolate ME27-1 showed the high-est CMCase activity (017UmL) after incubation for 60 h inbasal liquid medium containing 10 gL of CMC-Na (Table 1)The CMCase activity of the other 19 strains was undetectable
BioMed Research International 7
0
009
018
027
036
045
1 2 3 4 5 6 7 8 9
CMCa
se ac
tivity
(Um
L)
Carbon sources
(a)
0
03
06
09
12
15
10 30 50 70 90
CMCa
se ac
tivity
(Um
L)
Concentration of wheat bran (gL)
(b)
0
045
09
135
18
a b c d e f g h i j
CMCa
se ac
tivity
(Um
L)
Nitrogen sources
(c)
0
03
06
09
12
15
18
00 15 30 45 60 75 90
CMCa
se ac
tivity
(Um
L)
Concentration of NH4Cl (gL)
(d)
Figure 4 Effect of carbon and nitrogen sources on CMCase production by the strainME27-1 (a) Different carbon sources 1 sim 9 representedglycerol lactose sucrose maltose CMC-Na filter paper Avicel soluble starch and wheat bran respectively (b) The concentration of wheatbran (c) Different nitrogen sources a sim j represented (NH
4
)2
SO4
NH4
NO3
NaNO3
KNO3
NH4
Cl urea soybean yeast extract tryptoneand beef extract respectively (d) The concentration of NH
4
Cl
after cultivating in various liquid media for up to 6 days andthe Avicelase FPase and 120573-glucosidase activities of all the 22bacterial strains were also undetectable
Congo-red staining has been widely used inmany studiesfor screening cellulose-degradingmicroorganisms AlthoughTeather and Wood described the relationship between thediameter of hydrolyzing zone and log enzyme concentrationthis correlation could not represent the enzyme-producingability of the microorganisms [19] In the present studyalthough some strains presented large and clear hydrolyzingzones the activities of CMCase and other cellulases producedby themwere undetectable in various liquidmedia containingCMCand other cellulosicmaterials suggesting that either theconcentration of the enzyme produced by these strains wasvery low to be detected after cultivation in liquid medium orthe ability of the strains to secrete CMCase was weak Sadhu
and Maiti also reported that the diameter of the hydrolyzingzonemay not accurately reflect the real cellulase activity [28]
In general aerobic bacteria produce low levels of Avice-lase FPase and 120573-glucosidase In a study carried out byRastogi et al Brevibacillus sp DUSELG12 andGeobacillus spDUSELR7 were found to produce a maximum FPase activityof 0027 and 0043UmL on days 7 and 8 respectively [12]Recently Soares et al found that only 91 of bacterial strainswere able to degrade Avicel on agar plates [7]
32 Identification of Cellulose-Degrading Bacteria The DNAfragments containing partial 16S rRNAgenes of the 22 isolateswere amplified and sequenced The sequences obtained werematched with those available in GenBank which revealedmaximum identity of these isolates and allowed identificationof these cellulose-degrading bacterial strains (Table 1)
8 BioMed Research International
0
03
06
09
12
15
12 24 36 48 60 72
CMCa
se ac
tivity
(Um
L)
Incubation time (h)
Figure 5 Effect of inoculum size and incubation period onCMCaseproduction by the strain ME27-1 2 (empty triangle) 4 (filledtriangle) 6 (filled circle) 8 (filled square) and 10 (empty square)Error bars show the standard deviation of experimental point (119899 =3)
It was found that the 22 aerobic bacterial strains thatcould hydrolyze cellulose belonged to 10 different gen-era Burkholderia (3636) Bacillus (1365) Citrobacter(1365) Arthrobacter (910) Enterobacter (454) Chry-seobacterium (454) Pandoraea (454) Paenibacillus(454) Dyella (454) and Pseudomonas (454) Thephylogenetic tree of the 22 strains was constructed by usingMAGA41 program (Figure 2)
Various cellulose-degrading bacteria have been foundin different environments The genus Burkholderia wasobserved to be the main cellulose-hydrolyzing bacteria andwas considered to play an important role in cellulose degra-dation in the subtropical region of China in this studyIn addition bacteria belonging to the genera ArthrobacterChryseobacterium Pandoraea and Dyella were also foundto be cellulolytic in the present study which have beenrarely reported as cellulose-degrading bacteria In a previousstudy Lo et al reported that the cellulase-producing bacterialstrains isolated from a rice field in southern Taiwan mainlybelonged to the genus Cellulomonas [9] On the other handBacilluswas reported to be the dominant cellulose-degradingbacteria in samples collected from paper mill sludges andorganic fertilizers from Red Rock Canada as well as inthose from soil compost and animal waste slurry from JejuIsland [29 30] Similarly Burkholderia was found to be themain genus of cellulase-producing bacteria in the subtropicalrainforest in Okinawa Island Japan [31]
The strain ME27-1 with higher CMCase activity wasthoroughly examined The partial 16S rRNA gene (1309 bp)from the strain ME27-1 showed a maximum identity of 99with that ofPaenibacillus terraeAM141T (T type strain)Mor-phological tests revealed that the cells of the strain ME27-1
were rod-shaped endospore-forming Gram-positive and08 times 19ndash32 120583m in size The appearance of the colonyon the TSA medium was cream-colored moist irregularswollen and pigment-free The biochemical properties ofthe strain ME27-1 are listed in Table 2 The morphologicalphysiological and biochemical properties of the strainME27-1 were found to be mostly similar to those of P terrae [27]Thus the strain ME27-1 was identified as P terrae
To our knowledge till date no study has reported aboutCMCase production by P terrae although other species ofPaenibacillus have been found to produce cellulase SomeCMCase genes cloned from Paenibacillus polymyxa GS01Paenibacillus barcinonensis Paenibacillus xylanilyticus KJ-03 and Paenibacillus cookii SS-24 have been expressed inEscherichia coli and Saccharomyces cerevisiae [32ndash35] On theother hand CMCases from Paenibacillus curdlanolyticus B-6Paenibacillus campinasensis BL11 Paenibacillus sp B39 and Ppolymyxa have been purified [36ndash39]
33 Effect of Initial pH Temperature Carbon and NitrogenSources Inoculum Size and Incubation Time on CMCase Pro-duction by P terrae ME27-1 The best incubation conditionswere pH 80 and 28∘C (Figures 3(a) and 3(b)) The CMCaseactivity declined when the initial pH and incubation tem-perature were not optimal There have been diverse reportson the optimal initial pH and temperature for cellulolyticenzyme production by Paenibacillus sp In a previous studyP curdlanolyticus B-6 was cultivated for enzyme productionat pH 70 and 37∘C [5] Furthermore Kumar et al reportedthat the optimal initial pH and temperature for CMCase pro-duction by P polymyxa were 55 and 37∘C respectively [39]Yoon et al accounted that the optimal growth temperaturefor P terrae was 30∘C which is similar to that observed foroptimal CMCase production by the strain ME27-1 [27]
Various cellulosic materials have been used to inducemicroorganisms to produce cellulaseWhen fructose and glu-cose were used as the sole carbon source no CMCase activitywas detected Wheat bran induced the highest CMCaseactivity which was about 25-fold higher than that observedin the basal medium containing CMC-Na (Figure 4(a)) Theoptimal concentration of wheat bran in the medium wasfound to be 50 gL (Figure 4(b)) Da Vinha et al used steam-pretreated sugarcane bagasse (or wheat bran) as the maincarbon source and found thatwheat branwas the best inducerfor CMCase production by S viridobrunneus SCPE-09 [15]Gao et al demonstrated that rice branwas the optimal carbonsource for CMCase production by Cellulophaga lytica LBH-14 while Kumar et al reported that high CMCase productionby P polymyxa was obtained when using mango peel assubstrate [39 40] In addition wheat straw rice straw andxylan have been reported to be good carbon sources forCMCase production by Cellulomonas sp and Cellulosimicro-bium cellulans [9 41]
Furthermore maximum CMCase activity was notedwhen using NH
4Cl as the sole nitrogen source (Figure 4(c))
and the best concentration of NH4Cl in the medium was
observed to be 3 gL (Figure 4(d)) Many reports have shownthat organic nitrogen sources are better than inorganic
BioMed Research International 9
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(a)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(b)
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(c)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(d)
Figure 6 Properties of CMCase produced by the strain ME27-1 (a) Effect of pH on CMCase activity (b) Effect of temperature on CMCaseactivity (c) Effect of pH on the stability of CMCase (d) Thermostability of CMCase The different buffers used are as follows (100mM)sodium citrate buffer (empty square pH 30ndash65) Na
2
HPO4
-NaH2
PO4
buffer (filled square pH 65ndash75) Tris-HCl buffer (empty triangle pH75ndash85) and glycine-NaOH buffer (filled triangle pH 85ndash110) Error bars show the standard deviation of experimental point (119899 = 2)
nitrogen sources [15 16 42 43] In the present study theCMCase activity of the strain ME27-1 was higher wheninorganic nitrogen sources were used as the sole nitrogensource Likewise Kumar et al and Kalogeris et al alsoobserved a similar phenomenon in their studies [39 44]
In addition use of an inoculum size of 2 resulted inmaximum CMCase activity after incubation of the strainfor 60 h (Figure 5) There has been increasing interest incellulase-producing bacteria because of their ability to growfast [45] In the present study the strain ME27-1 producedthe highest CMCase activity after 60 h of incubation On theother hand in previous studies maximum CMCase activityof Pseudomonas sp HP207 and S viridobrunneus SCPE-09was observed after 24 and 48 h of incubation respectivelywhich is much earlier than that noted for the strain ME27-1 [15 16] However different results have been reported invarious studies MaximumCMCase activity of C lytica LBH-14 was obtained after 72 h of incubation whereas that of
Brevibacillus sp DUSELG12 and Geobacillus sp DUSELR7was noted after days 9 and 7 respectively [12 40]
34 Properties of CMCase Produced by P terrae ME27-1The optimum pH and temperature of CMCase produced bystrain ME27-1 were found to be 55 and 50∘C respectively(Figures 6(a) and 6(b)) The CMCase produced by the strainME27-1 was stable from pH 40 to 110 with more than 60CMCase activity being retained (Figure 6(c)) Furthermorethe enzyme maintained 65 activity after incubation at 4∘Cand pH 110 for 24 h The temperature profiles demonstratedthat more than 95 CMCase activity was retained at 30ndash45∘C for 1 h (Figure 6(d)) However the enzyme activity wasreduced at temperatures above 50∘C In fact approximately77 residual activity was maintained after preincubating theenzyme at 50∘C for 1 h
Similar results were observed for cellulases produced by Sviridobrunneus SCPE-09 and P cookii SS-24 with an optimal
10 BioMed Research International
Table 3 Comparison of CMCase production by Paenibacillus terraeME27-1 with other bacterial and fungal strains
pH of 50 and 51 and an optimal temperature of 50∘ and 55∘Crespectively [15 35] However maximum CMCase activity ofbacteria at pH lower than 60 has been rarely observed andthe maximum CMCase activities of P campinasensis BL11 Ppolymyxa GS01 Paenibacillus sp B39 and Bacillus mycoidesS122C were observed at neutral or alkaline conditions [3738 46 47] In the present study the CMCase producedby the strain ME27-1 was stable at pH 50ndash95 and almost85 residual activity was retained Only a few studies havereported that CMCase was stable at such a wide pH range forexample Da Vinha et al reported the 60 CMCase activitywas retained within the pH range of 30ndash80 [15]
35 Comparison of CMCase Production by P terrae ME27-1and Other Microorganisms When measured at the optimalpH and temperature of CMCase P terrae ME27-1 producedCMCase activity of 208UmL under the optimized cul-tivation conditions which was a 12-fold improvement in
the CMCase production This yield of CMCase productionwas higher than most of the aerobic bacterial strains but lessthan someof aerobic bacterial strains that have been exploitedpreviously (Table 3) However the CMCase production by Pterrae ME27-1 was lower than that by several anaerobic bac-terial strains for example Clostridium papyrosolvens CFR-703 C thermocellum YM4 C thermocopriae JT3-3 (Table 3)Some anaerobic bacteria can degrade lignocellulosic sub-strates efficiently by producingmultienzyme complex termedcellulosome [36] The carbohydrate binding modules anddifferent proteins in the cellulosome allow the whole enzymecomplex to bind to the substrates which avoids the wastefulexpenditure of energy of bacteria releasing large amounts ofindividual enzymes andmakes lots of advantages over single-enzyme system [4 48]
Furthermore the CMCase produced by P terrae ME27-1 was lower than that by most aerobic fungal strains whileit was higher than that by anaerobic fungal strains (Table 3)
BioMed Research International 11
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
Figure 3 Effect of initial pH and temperature on enzyme production by the strain ME27-1 (a) Initial pH (b) Temperature (119879)
biochemical characterization of the strainME27-1was carriedout by using API 50CHB microtests (bioMerieux)
26 Optimization of Cultivation Conditions for CMCase Pro-duction by the Strain ME27-1 The effect of initial pH andtemperature on CMCase production by the strain ME27-1was determined by cultivating the strain in 50mL of basalmediumcontaining 10 gL ofCMC-Na at various pH (rangingfrom 50 to 100 with an interval of 05) and temperatures (26ndash34∘C) for 60 h at 180 rpm
The effect of carbon and nitrogen sources on cellulaseproduction by the strain ME27-1 was determined by using 11different carbon sources (fructose glucose glycerol lactosesucrose maltose CMC-Na filter paper (chopped into 20mesh size) Avicel soluble starch and wheat bran whichwas chopped into 80 mesh size) and 10 different nitrogensources as below (NH
4)2SO4 NH4NO3 NaNO
3 KNO
3
NH4Cl urea soybean yeast extract tryptone and beef
extract The carbon sources were used at a concentration of10 gL instead of the carbon source in the basal mediumFurthermore different concentrations (10ndash100 gL with aninterval of 10 gL) of optimal carbon source were examinedSimilarly the effect of nitrogen sources was also studied withan initial concentration of 15 gL
The effect of different inoculum sizes (2 4 6 8and 10) on enzyme production was tested All media werein pH 80 All the flasks were incubated at 28∘CThe CMCaseactivity was detected at an interval of 12 h
27 Properties of CMCase Produced by the Bacterial StrainME27-1 To determine the optimal pH 250 120583L of crudeCMCase supernatant was incubated with 250 120583L of CMC-Na(2 wv) at 50∘C and different pH (30ndash110 with an intervalof 05) respectively To observe the effect of temperatureCMCase was incubated with 2 CMC-Na at a pH of 55 andtemperature ranging from 30 to 75∘C with an interval of 5∘C
Themaximum CMCase activity obtained at different pH andtemperatures was considered to be 100
The effect of pH on the stability of CMCase was studiedby mixing the crude enzyme with different buffers (1 9 vv)with pH ranging from 30 to 100 The CMCase activity ofthe crude enzyme after incubating at 4∘C for 24 h at differentpHwas detected To study the thermostability of the CMCaseproduced by the strain ME27-1 the crude enzyme was prein-cubated at different temperatures (varying from 30 to 75∘Cwith an interval of 5∘C) for 1 h The residual CMCase activitywas detectedThemaximumCMCase activity obtained at pH30ndash100 or temperature 30ndash75∘C was considered to be 100All the enzyme assays were carried out in triplicate
28 Nucleotide Sequence Accession Numbers All the DNAsequences of the partial 16S rRNA genes of the 22 strainsreported in this study have been deposited into the GenBankdatabase under the accession numbers from KF536877 toKF536898
3 Results and Discussion
31 Isolation and Screening of Cellulose-Degrading Bacteria Atotal of 245 cellulose-degrading aerobic bacterial strains wereisolated from different natural reserves in the subtropicalregion of China which were cultured in agar mediumcontaining sugarcane bagasse pulp as the sole carbon sourceOut of these strains 22 isolates showed hydrolyzing zoneson agar plates containing CMC-Na after Congo-red staining(Figure 1) The hydrolyzing zone diameter and colony diam-eter are listed in Table 1
Among the 22 isolates only three isolates (ME27-1 FCD1-3 and SK3-4) were found to produce measurable CMCaseafter liquid cultivation and isolate ME27-1 showed the high-est CMCase activity (017UmL) after incubation for 60 h inbasal liquid medium containing 10 gL of CMC-Na (Table 1)The CMCase activity of the other 19 strains was undetectable
BioMed Research International 7
0
009
018
027
036
045
1 2 3 4 5 6 7 8 9
CMCa
se ac
tivity
(Um
L)
Carbon sources
(a)
0
03
06
09
12
15
10 30 50 70 90
CMCa
se ac
tivity
(Um
L)
Concentration of wheat bran (gL)
(b)
0
045
09
135
18
a b c d e f g h i j
CMCa
se ac
tivity
(Um
L)
Nitrogen sources
(c)
0
03
06
09
12
15
18
00 15 30 45 60 75 90
CMCa
se ac
tivity
(Um
L)
Concentration of NH4Cl (gL)
(d)
Figure 4 Effect of carbon and nitrogen sources on CMCase production by the strainME27-1 (a) Different carbon sources 1 sim 9 representedglycerol lactose sucrose maltose CMC-Na filter paper Avicel soluble starch and wheat bran respectively (b) The concentration of wheatbran (c) Different nitrogen sources a sim j represented (NH
4
)2
SO4
NH4
NO3
NaNO3
KNO3
NH4
Cl urea soybean yeast extract tryptoneand beef extract respectively (d) The concentration of NH
4
Cl
after cultivating in various liquid media for up to 6 days andthe Avicelase FPase and 120573-glucosidase activities of all the 22bacterial strains were also undetectable
Congo-red staining has been widely used inmany studiesfor screening cellulose-degradingmicroorganisms AlthoughTeather and Wood described the relationship between thediameter of hydrolyzing zone and log enzyme concentrationthis correlation could not represent the enzyme-producingability of the microorganisms [19] In the present studyalthough some strains presented large and clear hydrolyzingzones the activities of CMCase and other cellulases producedby themwere undetectable in various liquidmedia containingCMCand other cellulosicmaterials suggesting that either theconcentration of the enzyme produced by these strains wasvery low to be detected after cultivation in liquid medium orthe ability of the strains to secrete CMCase was weak Sadhu
and Maiti also reported that the diameter of the hydrolyzingzonemay not accurately reflect the real cellulase activity [28]
In general aerobic bacteria produce low levels of Avice-lase FPase and 120573-glucosidase In a study carried out byRastogi et al Brevibacillus sp DUSELG12 andGeobacillus spDUSELR7 were found to produce a maximum FPase activityof 0027 and 0043UmL on days 7 and 8 respectively [12]Recently Soares et al found that only 91 of bacterial strainswere able to degrade Avicel on agar plates [7]
32 Identification of Cellulose-Degrading Bacteria The DNAfragments containing partial 16S rRNAgenes of the 22 isolateswere amplified and sequenced The sequences obtained werematched with those available in GenBank which revealedmaximum identity of these isolates and allowed identificationof these cellulose-degrading bacterial strains (Table 1)
8 BioMed Research International
0
03
06
09
12
15
12 24 36 48 60 72
CMCa
se ac
tivity
(Um
L)
Incubation time (h)
Figure 5 Effect of inoculum size and incubation period onCMCaseproduction by the strain ME27-1 2 (empty triangle) 4 (filledtriangle) 6 (filled circle) 8 (filled square) and 10 (empty square)Error bars show the standard deviation of experimental point (119899 =3)
It was found that the 22 aerobic bacterial strains thatcould hydrolyze cellulose belonged to 10 different gen-era Burkholderia (3636) Bacillus (1365) Citrobacter(1365) Arthrobacter (910) Enterobacter (454) Chry-seobacterium (454) Pandoraea (454) Paenibacillus(454) Dyella (454) and Pseudomonas (454) Thephylogenetic tree of the 22 strains was constructed by usingMAGA41 program (Figure 2)
Various cellulose-degrading bacteria have been foundin different environments The genus Burkholderia wasobserved to be the main cellulose-hydrolyzing bacteria andwas considered to play an important role in cellulose degra-dation in the subtropical region of China in this studyIn addition bacteria belonging to the genera ArthrobacterChryseobacterium Pandoraea and Dyella were also foundto be cellulolytic in the present study which have beenrarely reported as cellulose-degrading bacteria In a previousstudy Lo et al reported that the cellulase-producing bacterialstrains isolated from a rice field in southern Taiwan mainlybelonged to the genus Cellulomonas [9] On the other handBacilluswas reported to be the dominant cellulose-degradingbacteria in samples collected from paper mill sludges andorganic fertilizers from Red Rock Canada as well as inthose from soil compost and animal waste slurry from JejuIsland [29 30] Similarly Burkholderia was found to be themain genus of cellulase-producing bacteria in the subtropicalrainforest in Okinawa Island Japan [31]
The strain ME27-1 with higher CMCase activity wasthoroughly examined The partial 16S rRNA gene (1309 bp)from the strain ME27-1 showed a maximum identity of 99with that ofPaenibacillus terraeAM141T (T type strain)Mor-phological tests revealed that the cells of the strain ME27-1
were rod-shaped endospore-forming Gram-positive and08 times 19ndash32 120583m in size The appearance of the colonyon the TSA medium was cream-colored moist irregularswollen and pigment-free The biochemical properties ofthe strain ME27-1 are listed in Table 2 The morphologicalphysiological and biochemical properties of the strainME27-1 were found to be mostly similar to those of P terrae [27]Thus the strain ME27-1 was identified as P terrae
To our knowledge till date no study has reported aboutCMCase production by P terrae although other species ofPaenibacillus have been found to produce cellulase SomeCMCase genes cloned from Paenibacillus polymyxa GS01Paenibacillus barcinonensis Paenibacillus xylanilyticus KJ-03 and Paenibacillus cookii SS-24 have been expressed inEscherichia coli and Saccharomyces cerevisiae [32ndash35] On theother hand CMCases from Paenibacillus curdlanolyticus B-6Paenibacillus campinasensis BL11 Paenibacillus sp B39 and Ppolymyxa have been purified [36ndash39]
33 Effect of Initial pH Temperature Carbon and NitrogenSources Inoculum Size and Incubation Time on CMCase Pro-duction by P terrae ME27-1 The best incubation conditionswere pH 80 and 28∘C (Figures 3(a) and 3(b)) The CMCaseactivity declined when the initial pH and incubation tem-perature were not optimal There have been diverse reportson the optimal initial pH and temperature for cellulolyticenzyme production by Paenibacillus sp In a previous studyP curdlanolyticus B-6 was cultivated for enzyme productionat pH 70 and 37∘C [5] Furthermore Kumar et al reportedthat the optimal initial pH and temperature for CMCase pro-duction by P polymyxa were 55 and 37∘C respectively [39]Yoon et al accounted that the optimal growth temperaturefor P terrae was 30∘C which is similar to that observed foroptimal CMCase production by the strain ME27-1 [27]
Various cellulosic materials have been used to inducemicroorganisms to produce cellulaseWhen fructose and glu-cose were used as the sole carbon source no CMCase activitywas detected Wheat bran induced the highest CMCaseactivity which was about 25-fold higher than that observedin the basal medium containing CMC-Na (Figure 4(a)) Theoptimal concentration of wheat bran in the medium wasfound to be 50 gL (Figure 4(b)) Da Vinha et al used steam-pretreated sugarcane bagasse (or wheat bran) as the maincarbon source and found thatwheat branwas the best inducerfor CMCase production by S viridobrunneus SCPE-09 [15]Gao et al demonstrated that rice branwas the optimal carbonsource for CMCase production by Cellulophaga lytica LBH-14 while Kumar et al reported that high CMCase productionby P polymyxa was obtained when using mango peel assubstrate [39 40] In addition wheat straw rice straw andxylan have been reported to be good carbon sources forCMCase production by Cellulomonas sp and Cellulosimicro-bium cellulans [9 41]
Furthermore maximum CMCase activity was notedwhen using NH
4Cl as the sole nitrogen source (Figure 4(c))
and the best concentration of NH4Cl in the medium was
observed to be 3 gL (Figure 4(d)) Many reports have shownthat organic nitrogen sources are better than inorganic
BioMed Research International 9
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(a)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(b)
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(c)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(d)
Figure 6 Properties of CMCase produced by the strain ME27-1 (a) Effect of pH on CMCase activity (b) Effect of temperature on CMCaseactivity (c) Effect of pH on the stability of CMCase (d) Thermostability of CMCase The different buffers used are as follows (100mM)sodium citrate buffer (empty square pH 30ndash65) Na
2
HPO4
-NaH2
PO4
buffer (filled square pH 65ndash75) Tris-HCl buffer (empty triangle pH75ndash85) and glycine-NaOH buffer (filled triangle pH 85ndash110) Error bars show the standard deviation of experimental point (119899 = 2)
nitrogen sources [15 16 42 43] In the present study theCMCase activity of the strain ME27-1 was higher wheninorganic nitrogen sources were used as the sole nitrogensource Likewise Kumar et al and Kalogeris et al alsoobserved a similar phenomenon in their studies [39 44]
In addition use of an inoculum size of 2 resulted inmaximum CMCase activity after incubation of the strainfor 60 h (Figure 5) There has been increasing interest incellulase-producing bacteria because of their ability to growfast [45] In the present study the strain ME27-1 producedthe highest CMCase activity after 60 h of incubation On theother hand in previous studies maximum CMCase activityof Pseudomonas sp HP207 and S viridobrunneus SCPE-09was observed after 24 and 48 h of incubation respectivelywhich is much earlier than that noted for the strain ME27-1 [15 16] However different results have been reported invarious studies MaximumCMCase activity of C lytica LBH-14 was obtained after 72 h of incubation whereas that of
Brevibacillus sp DUSELG12 and Geobacillus sp DUSELR7was noted after days 9 and 7 respectively [12 40]
34 Properties of CMCase Produced by P terrae ME27-1The optimum pH and temperature of CMCase produced bystrain ME27-1 were found to be 55 and 50∘C respectively(Figures 6(a) and 6(b)) The CMCase produced by the strainME27-1 was stable from pH 40 to 110 with more than 60CMCase activity being retained (Figure 6(c)) Furthermorethe enzyme maintained 65 activity after incubation at 4∘Cand pH 110 for 24 h The temperature profiles demonstratedthat more than 95 CMCase activity was retained at 30ndash45∘C for 1 h (Figure 6(d)) However the enzyme activity wasreduced at temperatures above 50∘C In fact approximately77 residual activity was maintained after preincubating theenzyme at 50∘C for 1 h
Similar results were observed for cellulases produced by Sviridobrunneus SCPE-09 and P cookii SS-24 with an optimal
10 BioMed Research International
Table 3 Comparison of CMCase production by Paenibacillus terraeME27-1 with other bacterial and fungal strains
pH of 50 and 51 and an optimal temperature of 50∘ and 55∘Crespectively [15 35] However maximum CMCase activity ofbacteria at pH lower than 60 has been rarely observed andthe maximum CMCase activities of P campinasensis BL11 Ppolymyxa GS01 Paenibacillus sp B39 and Bacillus mycoidesS122C were observed at neutral or alkaline conditions [3738 46 47] In the present study the CMCase producedby the strain ME27-1 was stable at pH 50ndash95 and almost85 residual activity was retained Only a few studies havereported that CMCase was stable at such a wide pH range forexample Da Vinha et al reported the 60 CMCase activitywas retained within the pH range of 30ndash80 [15]
35 Comparison of CMCase Production by P terrae ME27-1and Other Microorganisms When measured at the optimalpH and temperature of CMCase P terrae ME27-1 producedCMCase activity of 208UmL under the optimized cul-tivation conditions which was a 12-fold improvement in
the CMCase production This yield of CMCase productionwas higher than most of the aerobic bacterial strains but lessthan someof aerobic bacterial strains that have been exploitedpreviously (Table 3) However the CMCase production by Pterrae ME27-1 was lower than that by several anaerobic bac-terial strains for example Clostridium papyrosolvens CFR-703 C thermocellum YM4 C thermocopriae JT3-3 (Table 3)Some anaerobic bacteria can degrade lignocellulosic sub-strates efficiently by producingmultienzyme complex termedcellulosome [36] The carbohydrate binding modules anddifferent proteins in the cellulosome allow the whole enzymecomplex to bind to the substrates which avoids the wastefulexpenditure of energy of bacteria releasing large amounts ofindividual enzymes andmakes lots of advantages over single-enzyme system [4 48]
Furthermore the CMCase produced by P terrae ME27-1 was lower than that by most aerobic fungal strains whileit was higher than that by anaerobic fungal strains (Table 3)
BioMed Research International 11
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
Figure 4 Effect of carbon and nitrogen sources on CMCase production by the strainME27-1 (a) Different carbon sources 1 sim 9 representedglycerol lactose sucrose maltose CMC-Na filter paper Avicel soluble starch and wheat bran respectively (b) The concentration of wheatbran (c) Different nitrogen sources a sim j represented (NH
4
)2
SO4
NH4
NO3
NaNO3
KNO3
NH4
Cl urea soybean yeast extract tryptoneand beef extract respectively (d) The concentration of NH
4
Cl
after cultivating in various liquid media for up to 6 days andthe Avicelase FPase and 120573-glucosidase activities of all the 22bacterial strains were also undetectable
Congo-red staining has been widely used inmany studiesfor screening cellulose-degradingmicroorganisms AlthoughTeather and Wood described the relationship between thediameter of hydrolyzing zone and log enzyme concentrationthis correlation could not represent the enzyme-producingability of the microorganisms [19] In the present studyalthough some strains presented large and clear hydrolyzingzones the activities of CMCase and other cellulases producedby themwere undetectable in various liquidmedia containingCMCand other cellulosicmaterials suggesting that either theconcentration of the enzyme produced by these strains wasvery low to be detected after cultivation in liquid medium orthe ability of the strains to secrete CMCase was weak Sadhu
and Maiti also reported that the diameter of the hydrolyzingzonemay not accurately reflect the real cellulase activity [28]
In general aerobic bacteria produce low levels of Avice-lase FPase and 120573-glucosidase In a study carried out byRastogi et al Brevibacillus sp DUSELG12 andGeobacillus spDUSELR7 were found to produce a maximum FPase activityof 0027 and 0043UmL on days 7 and 8 respectively [12]Recently Soares et al found that only 91 of bacterial strainswere able to degrade Avicel on agar plates [7]
32 Identification of Cellulose-Degrading Bacteria The DNAfragments containing partial 16S rRNAgenes of the 22 isolateswere amplified and sequenced The sequences obtained werematched with those available in GenBank which revealedmaximum identity of these isolates and allowed identificationof these cellulose-degrading bacterial strains (Table 1)
8 BioMed Research International
0
03
06
09
12
15
12 24 36 48 60 72
CMCa
se ac
tivity
(Um
L)
Incubation time (h)
Figure 5 Effect of inoculum size and incubation period onCMCaseproduction by the strain ME27-1 2 (empty triangle) 4 (filledtriangle) 6 (filled circle) 8 (filled square) and 10 (empty square)Error bars show the standard deviation of experimental point (119899 =3)
It was found that the 22 aerobic bacterial strains thatcould hydrolyze cellulose belonged to 10 different gen-era Burkholderia (3636) Bacillus (1365) Citrobacter(1365) Arthrobacter (910) Enterobacter (454) Chry-seobacterium (454) Pandoraea (454) Paenibacillus(454) Dyella (454) and Pseudomonas (454) Thephylogenetic tree of the 22 strains was constructed by usingMAGA41 program (Figure 2)
Various cellulose-degrading bacteria have been foundin different environments The genus Burkholderia wasobserved to be the main cellulose-hydrolyzing bacteria andwas considered to play an important role in cellulose degra-dation in the subtropical region of China in this studyIn addition bacteria belonging to the genera ArthrobacterChryseobacterium Pandoraea and Dyella were also foundto be cellulolytic in the present study which have beenrarely reported as cellulose-degrading bacteria In a previousstudy Lo et al reported that the cellulase-producing bacterialstrains isolated from a rice field in southern Taiwan mainlybelonged to the genus Cellulomonas [9] On the other handBacilluswas reported to be the dominant cellulose-degradingbacteria in samples collected from paper mill sludges andorganic fertilizers from Red Rock Canada as well as inthose from soil compost and animal waste slurry from JejuIsland [29 30] Similarly Burkholderia was found to be themain genus of cellulase-producing bacteria in the subtropicalrainforest in Okinawa Island Japan [31]
The strain ME27-1 with higher CMCase activity wasthoroughly examined The partial 16S rRNA gene (1309 bp)from the strain ME27-1 showed a maximum identity of 99with that ofPaenibacillus terraeAM141T (T type strain)Mor-phological tests revealed that the cells of the strain ME27-1
were rod-shaped endospore-forming Gram-positive and08 times 19ndash32 120583m in size The appearance of the colonyon the TSA medium was cream-colored moist irregularswollen and pigment-free The biochemical properties ofthe strain ME27-1 are listed in Table 2 The morphologicalphysiological and biochemical properties of the strainME27-1 were found to be mostly similar to those of P terrae [27]Thus the strain ME27-1 was identified as P terrae
To our knowledge till date no study has reported aboutCMCase production by P terrae although other species ofPaenibacillus have been found to produce cellulase SomeCMCase genes cloned from Paenibacillus polymyxa GS01Paenibacillus barcinonensis Paenibacillus xylanilyticus KJ-03 and Paenibacillus cookii SS-24 have been expressed inEscherichia coli and Saccharomyces cerevisiae [32ndash35] On theother hand CMCases from Paenibacillus curdlanolyticus B-6Paenibacillus campinasensis BL11 Paenibacillus sp B39 and Ppolymyxa have been purified [36ndash39]
33 Effect of Initial pH Temperature Carbon and NitrogenSources Inoculum Size and Incubation Time on CMCase Pro-duction by P terrae ME27-1 The best incubation conditionswere pH 80 and 28∘C (Figures 3(a) and 3(b)) The CMCaseactivity declined when the initial pH and incubation tem-perature were not optimal There have been diverse reportson the optimal initial pH and temperature for cellulolyticenzyme production by Paenibacillus sp In a previous studyP curdlanolyticus B-6 was cultivated for enzyme productionat pH 70 and 37∘C [5] Furthermore Kumar et al reportedthat the optimal initial pH and temperature for CMCase pro-duction by P polymyxa were 55 and 37∘C respectively [39]Yoon et al accounted that the optimal growth temperaturefor P terrae was 30∘C which is similar to that observed foroptimal CMCase production by the strain ME27-1 [27]
Various cellulosic materials have been used to inducemicroorganisms to produce cellulaseWhen fructose and glu-cose were used as the sole carbon source no CMCase activitywas detected Wheat bran induced the highest CMCaseactivity which was about 25-fold higher than that observedin the basal medium containing CMC-Na (Figure 4(a)) Theoptimal concentration of wheat bran in the medium wasfound to be 50 gL (Figure 4(b)) Da Vinha et al used steam-pretreated sugarcane bagasse (or wheat bran) as the maincarbon source and found thatwheat branwas the best inducerfor CMCase production by S viridobrunneus SCPE-09 [15]Gao et al demonstrated that rice branwas the optimal carbonsource for CMCase production by Cellulophaga lytica LBH-14 while Kumar et al reported that high CMCase productionby P polymyxa was obtained when using mango peel assubstrate [39 40] In addition wheat straw rice straw andxylan have been reported to be good carbon sources forCMCase production by Cellulomonas sp and Cellulosimicro-bium cellulans [9 41]
Furthermore maximum CMCase activity was notedwhen using NH
4Cl as the sole nitrogen source (Figure 4(c))
and the best concentration of NH4Cl in the medium was
observed to be 3 gL (Figure 4(d)) Many reports have shownthat organic nitrogen sources are better than inorganic
BioMed Research International 9
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(a)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(b)
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(c)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(d)
Figure 6 Properties of CMCase produced by the strain ME27-1 (a) Effect of pH on CMCase activity (b) Effect of temperature on CMCaseactivity (c) Effect of pH on the stability of CMCase (d) Thermostability of CMCase The different buffers used are as follows (100mM)sodium citrate buffer (empty square pH 30ndash65) Na
2
HPO4
-NaH2
PO4
buffer (filled square pH 65ndash75) Tris-HCl buffer (empty triangle pH75ndash85) and glycine-NaOH buffer (filled triangle pH 85ndash110) Error bars show the standard deviation of experimental point (119899 = 2)
nitrogen sources [15 16 42 43] In the present study theCMCase activity of the strain ME27-1 was higher wheninorganic nitrogen sources were used as the sole nitrogensource Likewise Kumar et al and Kalogeris et al alsoobserved a similar phenomenon in their studies [39 44]
In addition use of an inoculum size of 2 resulted inmaximum CMCase activity after incubation of the strainfor 60 h (Figure 5) There has been increasing interest incellulase-producing bacteria because of their ability to growfast [45] In the present study the strain ME27-1 producedthe highest CMCase activity after 60 h of incubation On theother hand in previous studies maximum CMCase activityof Pseudomonas sp HP207 and S viridobrunneus SCPE-09was observed after 24 and 48 h of incubation respectivelywhich is much earlier than that noted for the strain ME27-1 [15 16] However different results have been reported invarious studies MaximumCMCase activity of C lytica LBH-14 was obtained after 72 h of incubation whereas that of
Brevibacillus sp DUSELG12 and Geobacillus sp DUSELR7was noted after days 9 and 7 respectively [12 40]
34 Properties of CMCase Produced by P terrae ME27-1The optimum pH and temperature of CMCase produced bystrain ME27-1 were found to be 55 and 50∘C respectively(Figures 6(a) and 6(b)) The CMCase produced by the strainME27-1 was stable from pH 40 to 110 with more than 60CMCase activity being retained (Figure 6(c)) Furthermorethe enzyme maintained 65 activity after incubation at 4∘Cand pH 110 for 24 h The temperature profiles demonstratedthat more than 95 CMCase activity was retained at 30ndash45∘C for 1 h (Figure 6(d)) However the enzyme activity wasreduced at temperatures above 50∘C In fact approximately77 residual activity was maintained after preincubating theenzyme at 50∘C for 1 h
Similar results were observed for cellulases produced by Sviridobrunneus SCPE-09 and P cookii SS-24 with an optimal
10 BioMed Research International
Table 3 Comparison of CMCase production by Paenibacillus terraeME27-1 with other bacterial and fungal strains
pH of 50 and 51 and an optimal temperature of 50∘ and 55∘Crespectively [15 35] However maximum CMCase activity ofbacteria at pH lower than 60 has been rarely observed andthe maximum CMCase activities of P campinasensis BL11 Ppolymyxa GS01 Paenibacillus sp B39 and Bacillus mycoidesS122C were observed at neutral or alkaline conditions [3738 46 47] In the present study the CMCase producedby the strain ME27-1 was stable at pH 50ndash95 and almost85 residual activity was retained Only a few studies havereported that CMCase was stable at such a wide pH range forexample Da Vinha et al reported the 60 CMCase activitywas retained within the pH range of 30ndash80 [15]
35 Comparison of CMCase Production by P terrae ME27-1and Other Microorganisms When measured at the optimalpH and temperature of CMCase P terrae ME27-1 producedCMCase activity of 208UmL under the optimized cul-tivation conditions which was a 12-fold improvement in
the CMCase production This yield of CMCase productionwas higher than most of the aerobic bacterial strains but lessthan someof aerobic bacterial strains that have been exploitedpreviously (Table 3) However the CMCase production by Pterrae ME27-1 was lower than that by several anaerobic bac-terial strains for example Clostridium papyrosolvens CFR-703 C thermocellum YM4 C thermocopriae JT3-3 (Table 3)Some anaerobic bacteria can degrade lignocellulosic sub-strates efficiently by producingmultienzyme complex termedcellulosome [36] The carbohydrate binding modules anddifferent proteins in the cellulosome allow the whole enzymecomplex to bind to the substrates which avoids the wastefulexpenditure of energy of bacteria releasing large amounts ofindividual enzymes andmakes lots of advantages over single-enzyme system [4 48]
Furthermore the CMCase produced by P terrae ME27-1 was lower than that by most aerobic fungal strains whileit was higher than that by anaerobic fungal strains (Table 3)
BioMed Research International 11
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
Figure 5 Effect of inoculum size and incubation period onCMCaseproduction by the strain ME27-1 2 (empty triangle) 4 (filledtriangle) 6 (filled circle) 8 (filled square) and 10 (empty square)Error bars show the standard deviation of experimental point (119899 =3)
It was found that the 22 aerobic bacterial strains thatcould hydrolyze cellulose belonged to 10 different gen-era Burkholderia (3636) Bacillus (1365) Citrobacter(1365) Arthrobacter (910) Enterobacter (454) Chry-seobacterium (454) Pandoraea (454) Paenibacillus(454) Dyella (454) and Pseudomonas (454) Thephylogenetic tree of the 22 strains was constructed by usingMAGA41 program (Figure 2)
Various cellulose-degrading bacteria have been foundin different environments The genus Burkholderia wasobserved to be the main cellulose-hydrolyzing bacteria andwas considered to play an important role in cellulose degra-dation in the subtropical region of China in this studyIn addition bacteria belonging to the genera ArthrobacterChryseobacterium Pandoraea and Dyella were also foundto be cellulolytic in the present study which have beenrarely reported as cellulose-degrading bacteria In a previousstudy Lo et al reported that the cellulase-producing bacterialstrains isolated from a rice field in southern Taiwan mainlybelonged to the genus Cellulomonas [9] On the other handBacilluswas reported to be the dominant cellulose-degradingbacteria in samples collected from paper mill sludges andorganic fertilizers from Red Rock Canada as well as inthose from soil compost and animal waste slurry from JejuIsland [29 30] Similarly Burkholderia was found to be themain genus of cellulase-producing bacteria in the subtropicalrainforest in Okinawa Island Japan [31]
The strain ME27-1 with higher CMCase activity wasthoroughly examined The partial 16S rRNA gene (1309 bp)from the strain ME27-1 showed a maximum identity of 99with that ofPaenibacillus terraeAM141T (T type strain)Mor-phological tests revealed that the cells of the strain ME27-1
were rod-shaped endospore-forming Gram-positive and08 times 19ndash32 120583m in size The appearance of the colonyon the TSA medium was cream-colored moist irregularswollen and pigment-free The biochemical properties ofthe strain ME27-1 are listed in Table 2 The morphologicalphysiological and biochemical properties of the strainME27-1 were found to be mostly similar to those of P terrae [27]Thus the strain ME27-1 was identified as P terrae
To our knowledge till date no study has reported aboutCMCase production by P terrae although other species ofPaenibacillus have been found to produce cellulase SomeCMCase genes cloned from Paenibacillus polymyxa GS01Paenibacillus barcinonensis Paenibacillus xylanilyticus KJ-03 and Paenibacillus cookii SS-24 have been expressed inEscherichia coli and Saccharomyces cerevisiae [32ndash35] On theother hand CMCases from Paenibacillus curdlanolyticus B-6Paenibacillus campinasensis BL11 Paenibacillus sp B39 and Ppolymyxa have been purified [36ndash39]
33 Effect of Initial pH Temperature Carbon and NitrogenSources Inoculum Size and Incubation Time on CMCase Pro-duction by P terrae ME27-1 The best incubation conditionswere pH 80 and 28∘C (Figures 3(a) and 3(b)) The CMCaseactivity declined when the initial pH and incubation tem-perature were not optimal There have been diverse reportson the optimal initial pH and temperature for cellulolyticenzyme production by Paenibacillus sp In a previous studyP curdlanolyticus B-6 was cultivated for enzyme productionat pH 70 and 37∘C [5] Furthermore Kumar et al reportedthat the optimal initial pH and temperature for CMCase pro-duction by P polymyxa were 55 and 37∘C respectively [39]Yoon et al accounted that the optimal growth temperaturefor P terrae was 30∘C which is similar to that observed foroptimal CMCase production by the strain ME27-1 [27]
Various cellulosic materials have been used to inducemicroorganisms to produce cellulaseWhen fructose and glu-cose were used as the sole carbon source no CMCase activitywas detected Wheat bran induced the highest CMCaseactivity which was about 25-fold higher than that observedin the basal medium containing CMC-Na (Figure 4(a)) Theoptimal concentration of wheat bran in the medium wasfound to be 50 gL (Figure 4(b)) Da Vinha et al used steam-pretreated sugarcane bagasse (or wheat bran) as the maincarbon source and found thatwheat branwas the best inducerfor CMCase production by S viridobrunneus SCPE-09 [15]Gao et al demonstrated that rice branwas the optimal carbonsource for CMCase production by Cellulophaga lytica LBH-14 while Kumar et al reported that high CMCase productionby P polymyxa was obtained when using mango peel assubstrate [39 40] In addition wheat straw rice straw andxylan have been reported to be good carbon sources forCMCase production by Cellulomonas sp and Cellulosimicro-bium cellulans [9 41]
Furthermore maximum CMCase activity was notedwhen using NH
4Cl as the sole nitrogen source (Figure 4(c))
and the best concentration of NH4Cl in the medium was
observed to be 3 gL (Figure 4(d)) Many reports have shownthat organic nitrogen sources are better than inorganic
BioMed Research International 9
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(a)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(b)
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100 110
Rela
tive a
ctiv
ity (
)
pH
(c)
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75
Rela
tive a
ctiv
ity (
)
T (∘C)
(d)
Figure 6 Properties of CMCase produced by the strain ME27-1 (a) Effect of pH on CMCase activity (b) Effect of temperature on CMCaseactivity (c) Effect of pH on the stability of CMCase (d) Thermostability of CMCase The different buffers used are as follows (100mM)sodium citrate buffer (empty square pH 30ndash65) Na
2
HPO4
-NaH2
PO4
buffer (filled square pH 65ndash75) Tris-HCl buffer (empty triangle pH75ndash85) and glycine-NaOH buffer (filled triangle pH 85ndash110) Error bars show the standard deviation of experimental point (119899 = 2)
nitrogen sources [15 16 42 43] In the present study theCMCase activity of the strain ME27-1 was higher wheninorganic nitrogen sources were used as the sole nitrogensource Likewise Kumar et al and Kalogeris et al alsoobserved a similar phenomenon in their studies [39 44]
In addition use of an inoculum size of 2 resulted inmaximum CMCase activity after incubation of the strainfor 60 h (Figure 5) There has been increasing interest incellulase-producing bacteria because of their ability to growfast [45] In the present study the strain ME27-1 producedthe highest CMCase activity after 60 h of incubation On theother hand in previous studies maximum CMCase activityof Pseudomonas sp HP207 and S viridobrunneus SCPE-09was observed after 24 and 48 h of incubation respectivelywhich is much earlier than that noted for the strain ME27-1 [15 16] However different results have been reported invarious studies MaximumCMCase activity of C lytica LBH-14 was obtained after 72 h of incubation whereas that of
Brevibacillus sp DUSELG12 and Geobacillus sp DUSELR7was noted after days 9 and 7 respectively [12 40]
34 Properties of CMCase Produced by P terrae ME27-1The optimum pH and temperature of CMCase produced bystrain ME27-1 were found to be 55 and 50∘C respectively(Figures 6(a) and 6(b)) The CMCase produced by the strainME27-1 was stable from pH 40 to 110 with more than 60CMCase activity being retained (Figure 6(c)) Furthermorethe enzyme maintained 65 activity after incubation at 4∘Cand pH 110 for 24 h The temperature profiles demonstratedthat more than 95 CMCase activity was retained at 30ndash45∘C for 1 h (Figure 6(d)) However the enzyme activity wasreduced at temperatures above 50∘C In fact approximately77 residual activity was maintained after preincubating theenzyme at 50∘C for 1 h
Similar results were observed for cellulases produced by Sviridobrunneus SCPE-09 and P cookii SS-24 with an optimal
10 BioMed Research International
Table 3 Comparison of CMCase production by Paenibacillus terraeME27-1 with other bacterial and fungal strains
pH of 50 and 51 and an optimal temperature of 50∘ and 55∘Crespectively [15 35] However maximum CMCase activity ofbacteria at pH lower than 60 has been rarely observed andthe maximum CMCase activities of P campinasensis BL11 Ppolymyxa GS01 Paenibacillus sp B39 and Bacillus mycoidesS122C were observed at neutral or alkaline conditions [3738 46 47] In the present study the CMCase producedby the strain ME27-1 was stable at pH 50ndash95 and almost85 residual activity was retained Only a few studies havereported that CMCase was stable at such a wide pH range forexample Da Vinha et al reported the 60 CMCase activitywas retained within the pH range of 30ndash80 [15]
35 Comparison of CMCase Production by P terrae ME27-1and Other Microorganisms When measured at the optimalpH and temperature of CMCase P terrae ME27-1 producedCMCase activity of 208UmL under the optimized cul-tivation conditions which was a 12-fold improvement in
the CMCase production This yield of CMCase productionwas higher than most of the aerobic bacterial strains but lessthan someof aerobic bacterial strains that have been exploitedpreviously (Table 3) However the CMCase production by Pterrae ME27-1 was lower than that by several anaerobic bac-terial strains for example Clostridium papyrosolvens CFR-703 C thermocellum YM4 C thermocopriae JT3-3 (Table 3)Some anaerobic bacteria can degrade lignocellulosic sub-strates efficiently by producingmultienzyme complex termedcellulosome [36] The carbohydrate binding modules anddifferent proteins in the cellulosome allow the whole enzymecomplex to bind to the substrates which avoids the wastefulexpenditure of energy of bacteria releasing large amounts ofindividual enzymes andmakes lots of advantages over single-enzyme system [4 48]
Furthermore the CMCase produced by P terrae ME27-1 was lower than that by most aerobic fungal strains whileit was higher than that by anaerobic fungal strains (Table 3)
BioMed Research International 11
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
Figure 6 Properties of CMCase produced by the strain ME27-1 (a) Effect of pH on CMCase activity (b) Effect of temperature on CMCaseactivity (c) Effect of pH on the stability of CMCase (d) Thermostability of CMCase The different buffers used are as follows (100mM)sodium citrate buffer (empty square pH 30ndash65) Na
2
HPO4
-NaH2
PO4
buffer (filled square pH 65ndash75) Tris-HCl buffer (empty triangle pH75ndash85) and glycine-NaOH buffer (filled triangle pH 85ndash110) Error bars show the standard deviation of experimental point (119899 = 2)
nitrogen sources [15 16 42 43] In the present study theCMCase activity of the strain ME27-1 was higher wheninorganic nitrogen sources were used as the sole nitrogensource Likewise Kumar et al and Kalogeris et al alsoobserved a similar phenomenon in their studies [39 44]
In addition use of an inoculum size of 2 resulted inmaximum CMCase activity after incubation of the strainfor 60 h (Figure 5) There has been increasing interest incellulase-producing bacteria because of their ability to growfast [45] In the present study the strain ME27-1 producedthe highest CMCase activity after 60 h of incubation On theother hand in previous studies maximum CMCase activityof Pseudomonas sp HP207 and S viridobrunneus SCPE-09was observed after 24 and 48 h of incubation respectivelywhich is much earlier than that noted for the strain ME27-1 [15 16] However different results have been reported invarious studies MaximumCMCase activity of C lytica LBH-14 was obtained after 72 h of incubation whereas that of
Brevibacillus sp DUSELG12 and Geobacillus sp DUSELR7was noted after days 9 and 7 respectively [12 40]
34 Properties of CMCase Produced by P terrae ME27-1The optimum pH and temperature of CMCase produced bystrain ME27-1 were found to be 55 and 50∘C respectively(Figures 6(a) and 6(b)) The CMCase produced by the strainME27-1 was stable from pH 40 to 110 with more than 60CMCase activity being retained (Figure 6(c)) Furthermorethe enzyme maintained 65 activity after incubation at 4∘Cand pH 110 for 24 h The temperature profiles demonstratedthat more than 95 CMCase activity was retained at 30ndash45∘C for 1 h (Figure 6(d)) However the enzyme activity wasreduced at temperatures above 50∘C In fact approximately77 residual activity was maintained after preincubating theenzyme at 50∘C for 1 h
Similar results were observed for cellulases produced by Sviridobrunneus SCPE-09 and P cookii SS-24 with an optimal
10 BioMed Research International
Table 3 Comparison of CMCase production by Paenibacillus terraeME27-1 with other bacterial and fungal strains
pH of 50 and 51 and an optimal temperature of 50∘ and 55∘Crespectively [15 35] However maximum CMCase activity ofbacteria at pH lower than 60 has been rarely observed andthe maximum CMCase activities of P campinasensis BL11 Ppolymyxa GS01 Paenibacillus sp B39 and Bacillus mycoidesS122C were observed at neutral or alkaline conditions [3738 46 47] In the present study the CMCase producedby the strain ME27-1 was stable at pH 50ndash95 and almost85 residual activity was retained Only a few studies havereported that CMCase was stable at such a wide pH range forexample Da Vinha et al reported the 60 CMCase activitywas retained within the pH range of 30ndash80 [15]
35 Comparison of CMCase Production by P terrae ME27-1and Other Microorganisms When measured at the optimalpH and temperature of CMCase P terrae ME27-1 producedCMCase activity of 208UmL under the optimized cul-tivation conditions which was a 12-fold improvement in
the CMCase production This yield of CMCase productionwas higher than most of the aerobic bacterial strains but lessthan someof aerobic bacterial strains that have been exploitedpreviously (Table 3) However the CMCase production by Pterrae ME27-1 was lower than that by several anaerobic bac-terial strains for example Clostridium papyrosolvens CFR-703 C thermocellum YM4 C thermocopriae JT3-3 (Table 3)Some anaerobic bacteria can degrade lignocellulosic sub-strates efficiently by producingmultienzyme complex termedcellulosome [36] The carbohydrate binding modules anddifferent proteins in the cellulosome allow the whole enzymecomplex to bind to the substrates which avoids the wastefulexpenditure of energy of bacteria releasing large amounts ofindividual enzymes andmakes lots of advantages over single-enzyme system [4 48]
Furthermore the CMCase produced by P terrae ME27-1 was lower than that by most aerobic fungal strains whileit was higher than that by anaerobic fungal strains (Table 3)
BioMed Research International 11
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
pH of 50 and 51 and an optimal temperature of 50∘ and 55∘Crespectively [15 35] However maximum CMCase activity ofbacteria at pH lower than 60 has been rarely observed andthe maximum CMCase activities of P campinasensis BL11 Ppolymyxa GS01 Paenibacillus sp B39 and Bacillus mycoidesS122C were observed at neutral or alkaline conditions [3738 46 47] In the present study the CMCase producedby the strain ME27-1 was stable at pH 50ndash95 and almost85 residual activity was retained Only a few studies havereported that CMCase was stable at such a wide pH range forexample Da Vinha et al reported the 60 CMCase activitywas retained within the pH range of 30ndash80 [15]
35 Comparison of CMCase Production by P terrae ME27-1and Other Microorganisms When measured at the optimalpH and temperature of CMCase P terrae ME27-1 producedCMCase activity of 208UmL under the optimized cul-tivation conditions which was a 12-fold improvement in
the CMCase production This yield of CMCase productionwas higher than most of the aerobic bacterial strains but lessthan someof aerobic bacterial strains that have been exploitedpreviously (Table 3) However the CMCase production by Pterrae ME27-1 was lower than that by several anaerobic bac-terial strains for example Clostridium papyrosolvens CFR-703 C thermocellum YM4 C thermocopriae JT3-3 (Table 3)Some anaerobic bacteria can degrade lignocellulosic sub-strates efficiently by producingmultienzyme complex termedcellulosome [36] The carbohydrate binding modules anddifferent proteins in the cellulosome allow the whole enzymecomplex to bind to the substrates which avoids the wastefulexpenditure of energy of bacteria releasing large amounts ofindividual enzymes andmakes lots of advantages over single-enzyme system [4 48]
Furthermore the CMCase produced by P terrae ME27-1 was lower than that by most aerobic fungal strains whileit was higher than that by anaerobic fungal strains (Table 3)
BioMed Research International 11
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
The CMCase production by most bacteria was usually lowerthan that by aerobic fungal strains Genomic analysis showedthat less glycosyl hydrolases existed in aerobic bacterialstrains than aerobic fungal strains which may explain whyaerobic bacteria usually produce lower CMCase activity [48]
4 Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolatedfrom different natural reserves in the subtropical region ofChina and the genus Burkholderia was found to be the mostprevalent and predominant The strain ME27-1 identified tobe P terrae showed the highest CMCase activity among the22 strains isolated and after optimization of the cultivationconditions the enzyme activity was significantly improvedto 208UmL This bacterial species has been rarely foundto produce cellulase Thus this study revealed the diversityof cellulose-degrading bacteria in the subtropical region ofChina and found that P terrae ME27-1 was a good CMCaseproducer
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by a Grant from the NationalNatural Science Foundation of China (30960013) a Grantfrom the Guangxi Natural Science Foundation (2012GXNS-FGA060005) and the Bagui Scholar Program of Guangxi(2011A001)
References
[1] J L A Bras A Cartmell A L M Carvalho et al ldquoStructuralinsights into a unique cellulase fold andmechanism of cellulosehydrolysisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 13 pp 5237ndash5242 2011
[2] A V Gusakov and A P Sinitsyn ldquoCellulases from Penicilliumspecies for producing fuels from biomassrdquo Biofuels vol 3 no 4pp 463ndash477 2012
[3] L F Martins D Kolling M Camassola A J P Dillon andL P Ramos ldquoComparison of Penicillium echinulatum andTrichoderma reesei cellulases in relation to their activity againstvarious cellulosic substratesrdquo Bioresource Technology vol 99no 5 pp 1417ndash1424 2008
[4] M Maki K T Leung and W Qin ldquoThe prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomassrdquo International Journal of Biological Sciences vol 5 no5 pp 500ndash516 2009
[5] R Waeonukul K L Kyu K Sakka and K RatanakhanokchaildquoIsolation and characterization of a multienzyme complex(cellulosome) of the Paenibacillus curdlanolyticus B-6 grownon Avicel under aerobic conditionsrdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 610ndash614 2009
[6] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010
under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011
[7] F L Soares Jr I S Melo A C F Dias and F D AndreoteldquoCellulolytic bacteria from soils in harsh environmentsrdquoWorldJournal of Microbiology and Biotechnology vol 28 no 5 pp2195ndash2203 2012
[8] KMarjamaa K Toth P A Bromann G Szakacs andK KruusldquoNovel Penicillium cellulases for total hydrolysis of lignocellu-losicsrdquo Enzyme and Microbial Technology vol 52 no 6-7 pp358ndash369 2013
[9] Y-C LoGD SarataleW-MChenM-D Bai and J-S ChangldquoIsolation of cellulose-hydrolytic bacteria and applications ofthe cellulolytic enzymes for cellulosic biohydrogen productionrdquoEnzyme and Microbial Technology vol 44 no 6-7 pp 417ndash4252009
[10] D B Wilson ldquoMicrobial diversity of cellulose hydrolysisrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 259ndash263 2011
[11] M M Ekperigin ldquoPreliminary studies of cellulase productionby Acinetobacter anitratus and Branhamella sprdquo African Journalof Biotechnology vol 6 no 1 pp 028ndash033 2007
[12] G Rastogi G L Muppidi R N Gurram et al ldquoIsolation andcharacterization of cellulose-degrading bacteria from the deepsubsurface of the Homestake gold mine Lead South DakotaUSArdquo Journal of Industrial Microbiology and Biotechnology vol36 no 4 pp 585ndash598 2009
[13] P Gupta K Samant and A Sahu ldquoIsolation of cellulose-degrading bacteria and determination of their cellulolyticpotentialrdquo International Journal of Microbiology vol 2012Article ID 578925 5 pages 2012
[14] D Deka P Bhargavi A Sharma D Goyal M Jawed and AGoyal ldquoEnhancement of cellulase activity from a new strainof Bacillus subtilis by medium optimization and analysis withvarious cellulosic substratesrdquo Enzyme Research vol 2011 no 1Article ID 151656 2011
[15] F N M Da Vinha M P Gravina-Oliveira M N Franco et alldquoCellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substraterdquo AppliedBiochemistry and Biotechnology vol 164 no 3 pp 256ndash2672011
[16] P Sheng S Huang Q Wang A Wang and H Zhang ldquoIso-lation screening and optimization of the fermentation con-ditions of highly cellulolytic bacteria from the hindgutof Holotrichia parallela larvae (Coleoptera Scarabaeidae)rdquoApplied Biochemistry and Biotechnology vol 167 no 2 pp 270ndash284 2012
[17] F TWolf ldquoTheproduction of indole acetic acid byUstilago zeaeand its possible significance in tumor formationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 38 no 2 pp 106ndash111 1952
[18] M Mandels and E T Reese ldquoInduction of cellulase in Tri-choderma viride as influenced by carbon sources and metalsrdquoJournal of Bacteriology vol 73 no 2 pp 269ndash278 1957
[19] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982
[20] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959
[21] T K Ghose ldquoMeasurement of cellulase activityrdquo Pure andApplied Chemistry vol 59 no 2 pp 257ndash268 1987
12 BioMed Research International
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
[22] R Rawat and L Tewari ldquoPurification and characterization ofan acidothermophilic cellulase enzyme produced by Bacillussubtilis strain LFS3rdquo Extremophiles vol 16 no 4 pp 637ndash6442012
[23] Y Feng C-J DuanH Pang et al ldquoCloning and identification ofnovel cellulase genes fromunculturedmicroorganisms in rabbitcecum and characterization of the expressed cellulasesrdquoAppliedMicrobiology and Biotechnology vol 75 no 2 pp 319ndash328 2007
[24] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCRproductsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000
[25] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash175 John Wiley amp Sons Chichester UK1991
[26] S Kumar M Nei J Dudley and K Tamura ldquoMEGA abiologist-centric software for evolutionary analysis of DNA andprotein sequencesrdquo Briefings in Bioinformatics vol 9 no 4 pp299ndash306 2008
[27] J-H Yoon H-M Oh B-D Yoon K H Kang and Y-H ParkldquoPaenibacillus kribbensis sp nov and Paenibacillus terrae spnov bioflocculants for efficient harvesting of algal cellsrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 53 no 1 pp 295ndash301 2003
[28] S Sadhu and T K Maiti ldquoCellulase production by bacteria areviewrdquo British Microbiology Research Journal vol 3 no 3 pp235ndash258 2013
[29] M L Maki M Broere K T Leung andW Qin ldquoCharacteriza-tion of some efficient cellulase producing bacteria isolated frompaper mill sludges and organic fertilizersrdquo International Journalof Biochemistry andMolecular Biology vol 2 no 2 pp 146ndash1542011
[30] Y K Kim S C Lee Y Y Cho H J Oh and Y H Ko ldquoIsolationof cellulolytic Bacillus subtilis strains from agricultural environ-mentsrdquo ISRNMicrobiology vol 2012 Article ID650563 9 pages2012
[31] K Fujii A Oosugi and S Sekiuchi ldquoCellulolytic microbesin the Yanbaru a subtropical rainforest with an endemicbiota on Okinawa Island Japanrdquo Bioscience Biotechnology andBiochemistry vol 76 no 5 pp 906ndash911 2012
[32] K M Cho S Y Hong S M Lee et al ldquoA cel44C-man26A geneof endophytic Paenibacillus polymyxa GS01 has multi-glycosylhydrolases in two catalytic domainsrdquo Applied Microbiology andBiotechnology vol 73 no 3 pp 618ndash630 2006
[33] MMormeneo F J Pastor and J Zueco ldquoEfficient expression ofa Paenibacillus barcinonensis endoglucanase in Saccharomycescerevisiaerdquo Journal of IndustrialMicrobiology and Biotechnologyvol 39 no 1 pp 115ndash123 2012
[34] I-H Park J Chang Y-S Lee S-J Fang and Y-L Choi ldquoGenecloning of endoglucanase Cel5A from cellulose-degradingPaenibacillus xylanilyticus KJ-03 and purification and charac-terization of the recombinant enzymerdquo Protein Journal vol 31no 3 pp 238ndash245 2012
[35] S Shinoda S Kanamasa and M Arai ldquoCloning of an endogly-canase gene from Paenibacillus cookii and characterization ofthe recombinant enzymerdquo Biotechnology Letters vol 34 no 2pp 281ndash286 2012
[36] P Pason K L Kyu and K Ratanakhanokchai ldquoPaenibacil-lus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzymesystem that degrades insoluble polysaccharidesrdquo Applied andEnvironmentalMicrobiology vol 72 no 4 pp 2483ndash2490 2006
[37] C-H Ko W-L Chen C-H Tsai W-N Jane C-C Liu andJ Tu ldquoPaenibacillus campinasensis BL11 a wood material-utilizing bacterial strain isolated from black liquorrdquo BioresourceTechnology vol 98 no 14 pp 2727ndash2733 2007
[38] C-MWang C-L Shyu S-P Ho and S-H Chiou ldquoCharacter-ization of a novel thermophilic cellulose-degrading bacteriumPaenibacillus sp strain B39rdquo Letters in AppliedMicrobiology vol47 no 1 pp 46ndash53 2008
[39] D Kumar M Ashfaque M Muthukumar M Singh and NGarg ldquoProduction and characterization of carboxymethyl cel-lulase from Paenibacillus polymyxa using mango peel as sub-straterdquo Journal of Environmental Biology vol 33 no 1 pp 81ndash842012
[40] W Gao E-J Lee S-U Lee J Li C-H Chung and J-WLee ldquoEnhanced carboxymethylcellulase production by a newlyisolated marine bacterium Cellulophaga lytica LBH-14 usingrice branrdquo Journal of Microbiology and Biotechnology vol 22no 10 pp 1412ndash1422 2012
[41] B Wen X Yuan Y Cao Y Liu X Wang and Z Cui ldquoOpti-mization of liquid fermentation of microbial consortiumWSD-5 followed by saccharification and acidification of wheat strawrdquoBioresource Technology vol 118 pp 141ndash149 2012
[42] K Geetha and P Gunasekaran ldquoOptimization of nutrientmedium containing agricultural waste for xylanase productionbyBacillus pumilusB20rdquoBiotechnology andBioprocess Engineer-ing vol 15 no 5 pp 882ndash889 2010
[43] H-J Kim Y-J Lee W Gao C-H Chung C-W Son andJ-W Lee ldquoStatistical optimization of fermentation conditionsand comparison of their influences on production of cellulasesby a psychrophilic marine bacterium Psychrobacter aquimarisLBH-10 using orthogonal array methodrdquo Biotechnology andBioprocess Engineering vol 16 no 3 pp 542ndash548 2011
[44] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003
[45] W-J Lu H-TWang S-J Yang Z-CWang and Y-F Nie ldquoIso-lation and characterization of mesophilic cellulose-degradingbacteria from flower stalks-vegetable waste co-compostingsystemrdquo Journal of General andAppliedMicrobiology vol 51 no6 pp 353ndash360 2006
[46] M C Kye J H Sun R K Math et al ldquoCloning of two cellulasegenes from endophytic Paenibacillus polymyxa GS01 and com-parison with cel44C-man26Ardquo Journal of Basic Microbiologyvol 48 no 6 pp 464ndash472 2008
[47] N Balasubramanian D Toubarro M Teixeira and N SimosldquoPurification and biochemical characterization of a novelthermo-stable carboxymethyl cellulase from Azorean isolateBacillus mycoides S122Crdquo Applied Biochemistry and Biotechnol-ogy vol 168 no 8 pp 2191ndash2204 2012
[48] P J Brumm ldquoBacterial genomes what they teach us aboutcellulose degradationrdquo Biofuels vol 4 no 6 pp 669ndash681 2013
[49] T Shankar and L Isaiarasu ldquoCellulase production by Bacilluspumilus EWBCM1 under varying cultural conditionsrdquoMiddle-East Journal of Scientific Research vol 8 no 1 pp 40ndash45 2011
[50] O S Kotchoni O O Shonukan and W E Gachomo ldquoBacilluspumilus BpCRI 6 a promising candidate for cellulase produc-tion under conditions of catabolite repressionrdquo African Journalof Biotechnology vol 2 no 6 pp 140ndash146 2003
[51] A L Grigorevski De Lima R Pires Do Nascimento E P DaSilva Bon and R R R Coelho ldquoStreptomyces drozdowiczii
BioMed Research International 13
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
cellulase production using agro-industrial by-products and itspotential use in the detergent and textile industriesrdquo Enzymeand Microbial Technology vol 37 no 2 pp 272ndash277 2005
[52] Z Jaradat A Dawagreh Q Ababneh and I Saadoun ldquoInflu-ence of culture conditions on cellulase production by Strepto-myces sp (strain J2)rdquo Jordan Journal of Biological Science vol 1no 4 pp 141ndash146 2008
[53] E PMacedo C L O Cerqueira D A J Souza A S R Bispo RR R Coelho and R P Nascimento ldquoProduction of cellulose-degrading enzyme on sisal and other agro-industrial residuesusing a new Brazilian actinobacteria strain Streptomyces spSLBA-08rdquoBrazilian Journal of Chemical Engineering vol 30 no4 pp 729ndash735 2013
[54] F-J Chu C-W Lin Y-P I C-HWu andD-H Chen ldquoHydrol-ysis of bamboo cellulose and cellulase characteristics by Strep-tomyces griseoaurantiacus ZQBC691rdquo Journal of the TaiwanInstitute of Chemical Engineers vol 43 no 2 pp 220ndash225 2012
[55] Y Mori ldquoComparison of the cellulolytic systems of ClostridiumthermocellumYM4 and JW20rdquoBiotechnology Letters vol 14 no2 pp 131ndash136 1992
[56] F Jin and K Toda ldquoNutrient effects on cellulase production bythe new speciesClostridium thermocopriaerdquoAppliedMicrobiol-ogy and Biotechnology vol 31 no 5-6 pp 597ndash600 1989
[57] D S Rani S Thirumale and K Nand ldquoProduction of cellulasebyClostridium papyrosolvensCFR-703rdquoWorld Journal ofMicro-biology and Biotechnology vol 20 no 6 pp 629ndash632 2004
[58] R Assareh H Shahbani Zahiri K Akbari Noghabi S Amin-zadeh and G Bakhshi khaniki ldquoCharacterization of the newlyisolated Geobacillus sp T1 the efficient cellulase-producer onuntreated barley and wheat strawsrdquo Bioresource Technology vol120 pp 99ndash105 2012
[59] M S Umikalsom A B Ariff Z H Shamsuddin C C TongM A Hassan andM I A Karim ldquoProduction of cellulase by awild strain of Chaetomium globosum using delignified oil palmempty-fruit-bunch fibre as substraterdquoApplied Microbiology andBiotechnology vol 47 no 5 pp 590ndash595 1997
[60] R Lucas A Robles M T Garcıa G A De Cienfuegosand A Galvez ldquoProduction purification and properties of anendoglucanase produced by the hyphomycete Chalara (synThielaviopsis) paradoxaCH32rdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 79ndash85 2001
[61] L M F Gottschalk R A Oliveira and E P D S Bon ldquoCel-lulases xylanases 120573-glucosidase and ferulic acid esterase pro-duced by Trichoderma and Aspergillus act synergistically inthe hydrolysis of sugarcane bagasserdquo Biochemical EngineeringJournal vol 51 no 1-2 pp 72ndash78 2010
[62] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004
[63] X Sun Z Liu K Zheng X Song and Y Qu ldquoThe compositionof basal and induced cellulase systems in Penicillium decumbensunder induction or repression conditionsrdquo Enzyme and Micro-bial Technology vol 42 no 7 pp 560ndash567 2008
[64] R Singh A J Varma R Seeta Laxman andMRao ldquoHydrolysisof cellulose derived from steam exploded bagasse by Penicilliumcellulases comparison with commercial cellulaserdquo BioresourceTechnology vol 100 no 24 pp 6679ndash6681 2009
[65] S E Lowe M K Theodorou and A P J Trinci ldquoCellulasesand xylanase of an anaerobic rumen fungus grown on wheatstraw wheat straw holocellulose cellulose and xylanrdquo Appliedand Environmental Microbiology vol 53 no 6 pp 1216ndash12231987
[66] D O Mountfort and R A Asher ldquoProduction and regulationof cellulase by two strains of the rumen anaerobic fungus Neo-callimastix frontalisrdquo Applied and Environmental Microbiologyvol 49 no 5 pp 1314ndash1322 1985
[67] M D Romero J Aguado L Gonzalez and M Ladero ldquoCellu-lase production by Neurospora crassa on wheat strawrdquo Enzymeand Microbial Technology vol 25 no 3ndash5 pp 244ndash250 1999
[68] B A Gashe ldquoCellulase production and activity by Trichodermasp A-001rdquo Journal of Applied Bacteriology vol 73 no 1 pp 79ndash82 1992
[69] Y J Cai S J Chapman J A Buswell and S-T Chang ldquoPro-duction and distribution of endoglucanase cellobiohydrolaseand 120573- glucosidase components of the cellulolytic system ofVolvariella volvacea the edible straw mushroomrdquo Applied andEnvironmental Microbiology vol 65 no 2 pp 553ndash559 1999
