Research ArticleIsolation of Cellulose Degrading Fungi from Decaying BananaPseudostem and Strelitzia alba

L M Legodi D La Grange E L Jansen van Rensburg and I Ncube

University of Limpopo Department of Biochemistry Microbiology and Biotechnology Private Bag X 1106 Sovenga 0727 South Africa

Correspondence should be addressed to L M Legodi 9731824keyakaulacza

Received 24 April 2019 Accepted 7 July 2019 Published 25 July 2019

Academic Editor David Ballou

Copyright copy 2019 L M Legodi et al This 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

Cellulases are a group of hydrolytic enzymes that break down cellulose to glucose units These enzymes are used in the foodbeverage textile pulp and paper and the biofuel industries The aim of this study was to isolate fungi from natural compostand produce cellulases in submerged fermentation (SmF) Initial selection was based on the ability of the fungi to grow on agarcontaining Avicel followed by cellulase activity determination in the form of endoglucanase and total cellulase activity Ten fungalisolates obtained from the screening process showed good endoglucanase activity on carboxymethyl cellulose-Congo Red agarplates Six of the fungal isolates were selected based on high total cellulase activity and identified as belonging to the generaTrichoderma and Aspergillus In SmF of synthetic media with an initial pH of 65 at 30∘C Trichoderma longibrachiatum LMLSAUL14-1 produced total cellulase activity of 8 FPUmL and endoglucanase activity of 23 UmLwhilstTrichoderma harzianum LMLBP0713-5 produced 6 FPUmL and endoglucanase activity of 16 UmL The produced levels of both cellulases and endoglucanase byTrichoderma species were higher than the levels for the Aspergillus fumigatus strains Aspergillus fumigatus LMLPS 13-4 producedhigher 120573-glucosidase 38 UmL activity than Trichoderma species

1 Introduction

Cellulose is a simple linear organic polymer of 120573-1-4 linkedglucopyranose units with varying degrees of polymerization(DPs) In primary cell walls the DP range is 5000 ndash 7500glucopyranose units whereas the DP in secondary cell wallsis approximately 10000 and 15000 [1] This polysaccharideis abundantly available in materials such as agro-wastesmunicipal wastes and forest residues The hydrolysis ofcellulosic biomass through enzymatic reaction is a preferredmethod over chemical reaction due to the absence of sugardegradation in the enzymatic process [2]

Cellulases are enzymeswith different specificities to catal-yse the hydrolysis of glycosidic bonds within cellulose (Jecu2000 Khan et al 2016)The enzymatic hydrolysis of celluloseinvolves exoglucanases (exo-14-120573-glucanases EC 32191)endoglucanases (endo-14-120573-glucanases EC 3214) and 120573-glucosidases (120573-D-glucoside glucohydrolase EC 32121)which exhibit high specificity for the 120573-14 glycosidic linkages(Jecu 2000) Total cellulase is an activity involving thesynergistic actions of the above enzymes that is measured

using insoluble substrates such as the Whatman No 1 filterpaper cotton linter microcrystalline cellulose (Avicel) orbacterial cellulose [2]

The mechanism of action reveals that endoglucanasescleaves cellulose internally at the 120573-1-4 glycosidic linkagereleasing oligosaccharide chains of different lengths whereasexoglucanases cleaves the oligosaccharide chains from eitherthe reducing or nonreducing ends to release disaccharidestri- and tetrasaccharides 120573-Glucosidase cleaves the 120573-1-4linkage in the disaccharide (cellobiose) to release glucosemolecules and it can also hydrolyse very short chain 120573-14 oligoglucosides up to cellohexaose but the reaction ratedecreases with chain length ([3 4] Saini et al 2015)

Cellulases are increasingly demanded for use in the bio-fuel industry to produce bioethanol from cellulosic biomassTechnology for the utilization of cellulosic biomass for theproduction of bioethanol is progressing slowly due to highcost of cellulases production the recalcitrant nature of cellu-losic biomass and inefficiency of available cellulases to releasemore fermentable sugars [5] Research efforts have beenundertaken to improve the catalytic efficiency of the known

HindawiEnzyme ResearchVolume 2019 Article ID 1390890 10 pageshttpsdoiorg10115520191390890

2 Enzyme Research

enzymes identify new enzymes and optimise enzyme mixpreparations for cellulosic biomass hydrolysis [6] Improvingfungal hydrolytic activity and finding stable enzymes that aretolerant to extreme conditions have become priority [7]

Agricultural waste such as banana by-products providesan alternative to the use of food crops such as corn and wheatin ethanol production [8] During the harvest of bananawaste generated including leaves rachis and pseudostemall contain high levels of cellulose [9 10] These celluloserich materials are discarded and left to decay either in theplantation site or at a dumping site [10] The decaying ofplant material is facilitated by cellulolytic microorganismsHence this study aimed to isolate fungi that secrete cellulaseswith ability to hydrolyse cellulose in banana pseudostem tofermentable sugars for the bioethanol industry

2 Materials and Methods

21 Sample Collection and Screening Upper soil sampleswerecollected in clean plastic bags fromdecomposed banana agro-waste (dumpsite and plantations) at the Tzaneen Allesbeestefarm Limpopo SouthAfrica and fromdecomposed Strelitziaalbaplant at theUniversity of Limpopo campus SouthAfricaFungal isolation was done by serial dilution method Three-gram samples of soil from each site were suspended in 50mlsterile distilled water A 100 120583l aliquot of soil suspensionthat had been diluted ten times was spread plated onto aselective Avicel agar medium (2 Avicel 15 agar 5mlchloramphenicol (50mgml) and 067 Yeast Nitrogen Base(YNB) without amino acids The Avicel agar plates wereincubated at 30∘C until fungal growth was evident The fungiwere purified by hyphal tipmethod [11 12]The fungal isolateswere maintained on malt extract agar (30 g malt extract 20 gdextrose 3 g peptone and 15 g agar) at 4∘C

The enzymatic screening for cellulases was based ona carboxymethyl cellulose (CMC) plate assay whereby theagar medium was mixed with Congo Red (CR) dye [13]The composition of the medium was 1 CMC 15 agar067 YNB without amino acids and 001 Congo Red Tenfungal isolates obtained were cultured on CMC-CR agar andincubated at 30∘C for 72-96 h As growth develops on CMC-CR agar plate the hydrolysis of CMC releases the bound CRdye This is revealed by the appearance of pale yellow halozone surrounding the fungal colony

The second screening was based on cellulolytic hydrolysisof filter paper (total cellulase activity) Six fungal isolates werecultured in a synthetic medium described by Peixoto [14]and Avicel as the sole source of carbon The synthetic mediaconsisted of 2 gl K

2HPO4 05 gl KCl 001 gl FeSO

47H2O

20 gl Avicel 015 gl MgSO47H2O 7 gl KH

2PO4 1 gl

(NH4)SO4 and 1 gl yeast extractThe pH of the mediumwas

adjusted to 55 prior to sterilization Inoculation was doneby cutting approximately 05 times 05 cm of fungal growth onagar medium and adding it into 100ml medium in 250mlErlenmeyer flasks All the flasks (Triplicates) were incubatedat 30∘C for 7 days while shaking at 150 rpm A 5ml samplewas removed after every 24 h of incubation and used fordetermining total cellulase activity as expressed in filter paperunits (FPU)

22 Molecular Identification of Fungal Isolates Identificationof fungi was based on the PCR amplification of the conservednucleotide sequence of ribosomal internal transcribed spacer(ITS) of the gene coding for the 18S 58S and 28S rRNA[15] Isolation and purification of fungal genomic DNA wasperformed by following the procedure outlined in the ZRFungalBacterial DNA Kit (Zymo Research Catalogue NoD6005)

The ITS target region (ie ITS-58S-ITS fragment) wasamplified using EconoTaq PLUS GREEN 2X Master Mix(Lucigen) using the following primers ITS1 51015840-TCCGTA-GGTGAACCTGAGG-31015840 and ITS4 51015840-TCCTCCGCTTAT-TGATATGC-31015840 [15 16] The following PCR conditions wereused 35 cycles including an initial denaturation step at 95∘Cfor 2 minutes Subsequent denaturation was at 95∘C 30seconds annealing at 50∘C for 30 seconds and extension at72∘C for 1minute A final extension at 72∘C for 10minutes wasfollowed by holding at 4∘CThe PCR products were analysedon a 1 agarose gel

DNA sequencing was done using ABI V31 Big dyeaccording to manufacturerrsquos instructions on the ABI 3500XL Genetic Analyzer (Applied Biosystems ThermoFisherScientific Specieswere identified by searching databases usingBLAST (httpwwwncbinlmnihgovBLAST)

23 Effect of Initial Medium pH and Incubation Temperatureon Cellulase Production The effect of initial pH of the mediaon the production of cellulases was studied by adjustingthe pH of the media using either 1M NaOH or 1M HClsolutions to pH values in the range of 45 to 70 The effect oftemperature on the production of cellulasewas investigated at30 35 and 40∘Cwith pH being adjusted to 65 or 70 based oncellulase activity determined during pH studies Inoculationand incubation conditions were maintained as describedabove in secondary screening section Samples of 5m l wereharvested every 24 h of incubation and used for enzymeassays Crude enzyme was prepared by centrifugation ofharvested sample using a Beckman Coulter microfuge 16centrifuge at 13000 rpm for 10min at room temperature

24 Cellulase Activity Assay The total cellulase activity wasdetermined by filter paper assay (FPase) usingWhatman No1 filter paper strip with a dimension of 1 times 60 cm equivalentto 50mg of substrate according to Ghose (1987) At leasttwo dilutions were made one dilution that release slightlyless than 20mg and the other dilution releasing more than20mg The reaction mixture contained 10ml of 005MNa-citrate pH 50 filter paper strip and 05ml of crudeenzyme diluted accordingly The mixture was incubated at50∘C for 1 h The released reducing sugar was estimatedby addition of 35-dinitrosalicylic acid (DNS) with glucoseas standard The absorbance was read at 540 nm by usingBeckman Coulter DU 720 UVVis spectrophotometer Theassay was performed in triplicate including controls Filterpaper activity (FPU) is defined as 037 divided by the amountof enzyme required to liberate 20mg of glucose from filterpaper strip (asymp 50mg) in 1 h

Enzyme Research 3

Table 1 Fungal isolation and screening on Avicel and CMC-Congo Red agar

Sampling site Isolate code no Isolate taxaBanana plantation site Allesbeeste farmTzaneen

aLMLBP07 13-5 Trichoderma harzianumaLMLBP06 13-3 Aspergillus fumigatus

Decomposing banana pseudostemAllesbeeste farm Tzaneen

bLMLPS 13-1 Aspergillus fumigatusbLMLPS 13-4 Aspergillus fumigatus

Banana dumpsite Allesbeeste farmTzaneen

cLMLBS02 13-2 Aspergillus fumigatus

Decomposing Strelizia alba University ofLimpopo

dLMLSAUL 14-1 Trichoderma longibrachiatum

aBP refers to banana plantation site bPS refers to Pseudostem cBS refers to banana dumpsite outside plantation dSAUL refers to Strelitzia alba at Universityof Limpopo

25 Endoglucanase Assay Endoglucanase activity in theculture supernatant was determined according to themethoddescribed by Ghose (1987) The reaction mixture contained05ml of 1 CMC in 005M Na-acetate buffer pH 50 and05ml of appropriately diluted crude enzyme The mixturewas incubated at 50∘C for 30min and the released reducingsugar was estimated as indicated in the assay for total activityabove One unit of endoglucanase activity was defined as theamount of enzyme liberating one 120583mole of reducing sugarfrom CMC under the assay conditions

26 120573-Glucosidase Assay 120573-Glucosidase activity was deter-mined according to the method described by Herr [17] Thereaction mixture contained 02ml of 001M 120588-nitrophenyl120573-d-glucopyranoside (pNPG) in 005M citrate buffer pH48 and 02ml of appropriately diluted enzyme solutionThe substrate control contained 04ml of 001M pNPGprepared in 005M citrate buffer at pH 48 The mixtureswere incubated at 50∘C for 30min The reaction was stoppedby adding 4ml of a 50mM NaOH-Glycine buffer pH 106The activity of the enzyme indicated by the release of120588-nitrophenol was determined at 420 nm using BeckmanCoulter DU 720 UVVis spectrophotometer [18 19] Oneunit of 120573-glucosidase activity was defined as the amount ofenzyme liberating one 120583mole of 120588-nitrophenol under theassay conditions

27 Calculations of Enzyme Activity Cellulase activity wasdetermined by filter paper assay using Whatman No 1according to Ghose (1987)

271 Cellulases (FPase)

FPA(FPUmL) = 037[Enz] (1)

where [Enz] is the concentration of enzyme that releases20mg of glucose from filter paper in 60 minutes

272 Endoglucanase (CMCase) and 120573-Glucosidase To esti-mate the activities of either endoglucanase andor 120573-glucosidase based on the released reducing sugars or 120588-nitrophenol the following equation was used [20]

120573 minus glucosidase or CMCase ( UmL) = E times Vf times Df120576 times t times Venz (2)

wherebyΔE is absorbance value at 540 nm Vf is final volume120576 is extinction coefficient of glucose (slope) Δt is incubationtime Venz is volume of crude enzyme and DF is dilutionfactor (if applicable)

3 Results

Ten fungal isolates were obtained based on their ability togrow on Avicel as sole source of carbon on solid mediaThese fungal isolateswere further screened for endoglucanaseactivity by culturing on CMC-CR agar plates at 30∘C for 72 -96 h The results revealed that all the isolated fungi were ableto grow and secrete endoglucanase which hydrolysed CMCbound to CR dye This was revealed by appearance of pale-yellow ldquohalo zonerdquo around the fungal growth or colony and itwas indication of endoglucanase activity or CMC hydrolysisHowever only six fungal isolates indicated larger halo zonesaround growth These six fungal isolates were subsequentlyidentified using ITS sequencing The identification of theselected six isolates revealed two different genera namelyTrichoderma and Aspergillus (Table 1) The Trichodermaspecies were T longibrachiatum and T harzianum and theAspergillus species were A fumigatus The six fungal isolateswere further evaluated for cellulase production in submergedfermentation

A quantitative evaluation of cellulase production bythe selected fungi (Table 1) was carried out in submergedfermentation using Avicel as a substrate at an initial pH of55 at 30∘CThe production levels of cellulases weremeasuredby the activity of the enzymes under specified conditionsMaximum cellulase activity was observed after 96 h for all thefungal strains (Figure 1) T longibrachiatum LMLSAUL 14-1 produced 41 FPUml followed by T harzianum LMLBP0713-5 with activity of 31 FPUml A fumigatus LMLPS 13-4with activity of 21 FPUml A fumigatus LMLPS 13-1 withactivity of 19 FPUml A fumigatus LMLBS02 13-2 activity of17 FPUml and A fumigatus LMLBP06 13-3 activity of 16FPUml

The influence of pH on the production of cellulase wasassessed for the six fungal strains at 30∘C (Figure 2) Max-imum cellulase activity was detected for all fungal isolates

4 Enzyme Research

005

115

225

335

445

5

0 20 40 60 80 100 120 140 160 180

)lmUPF(

sealulleclatoT

Time (h)

T longibrachiatum LMLSAUL 14 - 1 A fumigatus LMLPS 13 - 4T harzianum LMLBP07 13 - 5 A fumigatus LMLBS02 13 - 2A fumigatus LMLBP06 13 - 3 A fumigatus LMLPS 13 - 1

Figure 1 Time course for cellulase production by isolated fungal strains at 30∘C and initial pH 55

A fumigatus LMLPS 13 - 4 A fumigatus LMLBP06 13 - 3A fumigatus LMLBS02 13 - 2 T harzianum LMLBP07 13 - 5T longibrachiatum LMLSAUL 14 - 1 A fumigatus LMLPS 13 - 1

5 55 6 65 7 7545pH

0

1

2

3

4

5

6

7

8

9

Tota

l cel

lula

se (F

PUm

l)

Figure 2 The effect of initial medium pH on the production of cellulases by isolated fungi at 30∘C

when the initial pHof themediumwas 65 (Figure 2)TheTri-choderma longibrachiatum LMLSAUL 14-1 strain producedthe highest total cellulase activity of 81 FPUml This wasfollowed by T harzianum LMLBP07 13-5 with activity of58 FPUml A fumigatus LMLBP06 13-3 and A fumigatusLMLPS 13-1 produced maximum cellulase activity of 31FPUml with the lowest cellulase activity of 20 FPUml beingproduced by A fumigatus LMLPS 13-4

The production of endoglucanase activity was highest atpH 65 for all the fungal strains (Figure 3) T longibrachiatumLMLSAUL 14 - 1 and T harzianum LMLBP07 13-5 producedactivities of 230 Uml and 160 Uml respectively whereasA fumigatus LMLPS 13-1 LMLBS02 13-2 and LMLBP06 13-3 produced amounts of 140 Uml The lowest activity of 121Uml was noted for A fumigatus LMLPS 13-4

The levels of 120573-glucosidase produced with respect toinitial medium pH differed between the fungal strains Afumigatus LMLPS 13-4 and A fumigatus LMLBS02 13-2 pro-duced higher activities of 380 Uml and 341 Uml at pH 70respectively compared to the other isolates tested (Figure 4)Some fungi were able to secrete higher 120573-glucosidase atvarious pH values T harzianum LMLBP07 13-5 produced120573-glucosidase activity of 257 Uml over a pH range of 6- 70 and A fumigatus LMLPS 13-1 produced 120573-glucosidaseactivity of 235 Uml over a pH range of 65 - 70 (Figure 4)The lowest 120573-glucosidase activity produced was 206 Uml byT longibrachiatum LMLSAUL 14 - 1

The production of cellulases with respect to changesin incubation temperature was also investigated at initialmedium pH of 65 and 70 (Figures 5 and 6) These pH values

Enzyme Research 5

45 5 55 6 65 7 75pH

A fumigatus LMLPS 13 - 4 A fumigatus LMLBP06 13 - 3

A fumigatus LMLBS02 13 - 2 T harzianum LMLBP07 13 - 5

T longibrachiatum LMLSAUL 14 -1 A fumigatus LMLPS 13 - 1

000

500

1000

1500

2000

2500

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 3 The effect of initial medium pH on the production of endoglucanases by the isolated fungal strains at 30∘C

000

5001000

15002000

2500

30003500

4000

45 5 55 6 65 7 75

-g

luco

sidas

e (U

ml)

pHA fumigatus LMLPS13 - 4 A fumigatus LMLBP06 13 - 3A fumigatus LMLBS02 13 - 2 T harzianum LMLBP07 13 - 5T longibrachiatum LMLSAUL 14 - 1 A fumigatus LMLPS 13 -1

Figure 4 The effect of initial medium pH on the production of 120573-glucosidase by the isolated fungi at 30∘C

have shown to favour production of 120573-glucosidase by speciesof Aspergillus and Trichoderma At initial medium pH of 65an increase in growth temperature led to a decrease in thelevel of total cellulase activity for T longibrachiatum LMLUL14-1 at 35∘C (Figure 5) The A fumigatus strains were able toproduce cellulase at both 30 and 35∘C with the exception ofT harzianum LMLUL 13-5 Cellulase activity in A fumigatusLMLUL 13-1 increased by 18-fold at 35∘C (Figure 5) Increas-ing the incubation temperature to 40∘C drastically reducedthe cellulase production for T longibrachiatum LMLUL 14-1 A fumigatus LMLUL 13-1 and A fumigatus LMLUL 13-3(Figure 5) Generally most fungal species showed cellulaseactivity levels decreasing as the temperature increased to40∘C

At initial medium pH of 70 and a temperature of 30∘Cfavoured high production of cellulase by T harzianumLMLBP07 14-5 T longibrachiatum LMLUL 14-1 and Afumigatus LMLBS02 13-2 (Figure 6)

The cellulase production by A fumigatus LMLPS 13-1A fumigatus LMLBP06 13-3 and A fumigatus LMLPS 13-4

improved at 35∘C irrespective of initial pH tested (Figures 5and 6) At production temperature 40∘C the production ofcellulases was reduced for all strains tested All the Tricho-derma andAspergillus strains producedmore endoglucanasesat 30∘C with both initial medium pH of 65 and 70 (Figures 7and 8) but initial medium pH of 65 favoured more enzymeproduction An increase in temperature to 35∘C and 40∘Cresulted in a 2-fold decrease of the endoglucanase activity inall fungal species (Figure 7)

In an experiment with initial medium pH 70 a greaterimprovement in the production of endoglucanases was seenat 35∘C (Figure 8) T harzianum LMLBP07 13-5 produced2-fold higher endoglucanase activity at initial medium pHof 70 while other fungal species attained slight increases(Figure 8)

At both initial medium pH of 65 and 70 higher tempera-tures caused a reduction in endoglucanase activity and severereduction occurred at 40∘C as illustrated in Figures 7 and 8

The production of 120573-glucosidase at an initial pH of 65and 70 was dependent on the fungal species and production

6 Enzyme Research

0123456789

10

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Fpase 30 degCFpase 35 degCFpase 40 degC

Tota

l cel

lula

se ac

tivity

(FPU

ml)

Figure 5 Effect of incubation temperature on the production of cellulase (total cellulase activity) by fungal strains at initial medium pH 65

0

1

2

3

4

5

6

7

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Fpase 30 degCFpase 35 degCFpase 40 degC

Tota

l cel

lula

se ac

tivity

(FPU

ml)

Figure 6 Effect of incubation temperature on the production of cellulases (total cellulase activity) by fungal strains at initial medium pH 70

temperature In a medium with an initial pH of 65 onlyA fumigatus LMLPS 13-1 and T harzianum LMLBP07 13-5produced high levels of 120573-glucosidase activity 240 Uml and250 Uml respectively at 30∘C An increase in temperaturefrom 30 to 40∘C led to improved production of 120573-glucosidaseby some fungal species For instance A fumigatus LMLBP0613-3 showed 5-fold increase ie from 54 to 254 Uml of120573-glucosidase activity at 40∘C and 52-fold increase of 120573-glucosidase at 35∘C (Figure 9)

The production of 120573-glucosidase by T harzianumLMLBP07 13-5 was optimal between 35 and 40∘C whilstT longibrachiatum LMLSAUL 14-1 produced 120573-glucosidaseoptimally at 40∘C A fumigatus LMLPS 13-4 attained maxi-mum 120573-glucosidase activity at 30∘C (Figure 9) Generally at40∘C the production of 120573-glucosidase by all fungal specieswas much higher than at 30∘C with the exception of Afumigatus LMLPS 13-4 In a medium with initial pH 70 anincrease in temperature led to improvement in the produc-tion of 120573-glucosidase (Figure 10) A maximum level of 120573-glucosidase level was attained at 35∘CwithT longibrachiatumLMLSAUL 14-1 and T harzianum LMLBP07 13-5 showing

155-fold increase and 113-fold increase respectively Afumigatus LMLPS 13-4 and A fumigatus LMLPS 13-1 alsoshowed an increase of 120573-glucosidase activity at 35∘C (176-fold and 16-fold respectively) A fumigatus LMLBP06 13-3showed a proportional increase of 127-fold with maximum120573-glucosidase produced at 40∘C Conversely 40∘C led to a126-fold decrease of 120573-glucosidase produced byA fumigatusLMLBS02 13 - 2 (Figure 10)

Generally 120573-glucosidase activity was the highest at 35∘Cand pH 70 for all strains tested

4 Discussion

41 Fungal Screening Fungi and bacteria are associated withsoil and decaying plant materials In nature fungi colonisethe plant debris and in symbiotic relationship with othermicroorganisms they secrete an assortment of proteinsand a complex of hydrolytic enzymes to hydrolyse plantpolysaccharides for their survival However not all themicroorganisms are able to secrete significant amount ofhydrolytic enzymes for biotechnological applications Hence

Enzyme Research 7

0

5

10

15

20

25

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Endoglucanase 30 degCEndoglucanase 35 degCEndoglucanase 40 degC

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 7 Effect of incubation temperature on the production of endoglucanase by fungal strains at initial medium pH 65

0

5

10

15

20

25

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Endoglucanase 30 degCEndoglucanase 35 degCEndoglucanase 40 degC

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 8 Effect of incubation temperature on the production of endoglucanase by fungal strains at initial medium pH 70

0

5

10

15

20

25

30

35

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 9 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial medium pH 65

there is a need to screen and select hypersecreting hydrolyticenzymes In this study three different methods for screeningfor cellulolytic activity (1)microbial growth on cellulose agar(2) clearing of cellulose in agar and (3) reducing sugar pro-duction (or glucose) were performed Growth on cellulose

containing agar was useful for isolation of cellulolytic fungalstrains Ten fungal strains were selected based on fast andabundant growth on Avicel agar plates Several authors havealso reported the use of microcrystalline cellulose to screenfor cellulase producing microorganisms [21ndash23] CMC-CR

8 Enzyme Research

05

10152025303540

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 10 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial pH 70

(001) agar plate has been used to detect endoglucanaseactivity as indicated by the presence of a pale yellow zonearound the colony According to Yoon et al [13] the platescreening methods with dye coupled substrates provides arelatively straight forward and easy tool for specific detectionof fungi that produce endoglucanase Larger halos are theresults of higher enzyme activity [2] However at high CR dyeconcentrations fungal growth is suppressed

42 Fungal Identification The application of rDNA genesfor identification of fungal species is based on the detectionof conserved sequences in 58S rDNA and 28S rDNA thatenables the amplification of the ITS2 region between them[24] The identification of the fungal species based on theamplification of the ribosomal ITS region of the ribosomalDNA revealed that the organisms are A fumigatus strainsT harzianum and T longibrachiatum The ITS regions areregarded as primary DNA barcode for the fungal kingdomand exhibit high reliability [25]

43 Cellulases Production and Optimisation Subsequent toscreening is process optimisation with various factors such asnutrient requirements temperature pH and agitation speedbeen investigated (Gautam et al 2010 [26]) In this studythe effect of initial pH and temperatures on the productionlevels of cellulase were investigated Fungal strains belongingto Trichoderma and Aspergillus exhibited the potential toproduce cellulase over a pH 45-70 and temperature (30-40) range although the cellulase production levels variedfrom one fungal strain to another under the conditionsused It has been reported that expression of fungal genesis regulated by extracellular pH and many fungi exhibitgrowth and enzyme secretion over a wide pH range [27]Temperature also influences microbial growth and enzymeproduction [20 28] and an optimal environment conduciveto the production of cellulase will also be influenced bymedianutritional composition [26]

The initial pH of 45-55 drastically reduced the levelsof cellulases and endoglucanases produced by all strainsThis implied that acidity of the medium possibly affected

fungal growth and metabolism Acidic medium pH effect onthe production of cellulases by Trichoderma and Aspergilluswas also reported by Gautam et al [29] and Delabona etal [23] Contrary to these results other authors reportedsuch acidic condition to be favourable for production ofcellulases [22 30ndash34] Our results showed that a less acidicmedium with a pH 55 and above favoured the productionof cellulases by all Trichoderma and Aspergillus strains withoptimum production occurring at a pH of 65 Beyond pH65 a drastic decrease in cellulases levels was observed Gilnaamp Khaleel [35] and Gautam et al ([29] 2010) also reportedmaximum production of cellulases at pH 65 Aboul-Fotouhet al [36] reported maximum production of cellulases byAspergillus niger at initial medium pH of 6 when cultured on10 rice straw Other findings by Ncube et al [37] showed nosignificant differences in the production of endoglucanasesby A niger FGSCA 733 over a pH range of 3-7These findingsillustrate that an optimum pH for maximum production ofcellulase is dependent on fungal species and to some extenton the particular strain evaluated

There was effect of temperature on the productionof cellulases The production of cellulases decreased withincreasing temperature At 40∘C a significant reduction inendoglucanases activity was observed in all strains tested(Figures 8 and 9) On the contrary Ncube et al [37]reported maximum endoglucanase activity at 40∘C by Aniger FGSCA 733 Other studies reported optimal cellulaseand endoglucanase production at 45∘C [29] and 60∘C [2238] A temperature of 40∘C did not significantly affect 120573-glucosidase production (Figures 9 and 10) Leghlili et al(2013) reported the production of cellulases endoglucanasesand 120573-glucosidase T longibrachiatum (GHL) at both 30∘Cand 35∘C with higher production levels obtained at 35∘COther authors reported maximum production levels of 120573-glucosidase by T longibrachiatum at 35∘C [39] and A nigerMS82 at 25∘C [30] Aspergillus niger MS82 also producedsufficient endoglucanases at 30∘C and 35∘C in acidic initialmedium pH 40 [30]

These discrepancies with regard to the optimum initialpH and production temperature on the levels of cellulases as

Enzyme Research 9

measured by filter paper activity (FPUml) can be attributedto genetic make-up of the fungal strains as a result ofadaptations to different habitats Furthermore our reportedhigh levels of cellulases endoglucanases and low level of 120573-glucosidase by the Trichoderma isolates and vice versa highlevels of 120573-glucosidase by A fumigatus are in agreement withthe reported levels of these fungal species by Stewart andParry [38]

5 Conclusion

In conclusion all the Trichoderma and Aspergillus strainsproduced substantial levels of all the three enzymes (ieexoglucanase endoglucanase and 120573-glucosidase) requiredfor complete hydrolysis of cellulosic material Trichodermastrains produced higher cellulases and endoglucanases levelswhile A fumigatus strains produced higher 120573-glucosidaselevels The production of all the cellulases componentsseemed to be strongly influenced by the interactive effectof initial pH and incubation temperature on the microor-ganisms Hence the observed maximum production ofcellulases by the fungi depended on the chosen initial pHof the medium and incubation temperature The cellulasesproduced by these fungal strainswill be assessed for efficiencyin hydrolysing agricultural lignocellulose wastes includingbanana pseudostem for the production of bioethanol

Data Availability

The experimental data used to support the findings of thisstudy are available from the corresponding author uponrequest

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors are grateful to the National Research Foundation(NRF) and the Flemish Inter-University Council (VLIR-UOS) for the financial support towards the research

References

[1] A C OrsquoSullivan ldquoCellulose the structure slowly unravelsrdquoCellulose vol 4 no 3 pp 173ndash207 1997

[2] Y-H P Zhang M E Himmel and J R Mielenz ldquoOutlookfor cellulase improvement screening and selection strategiesrdquoBiotechnology Advances vol 24 no 5 pp 452ndash481 2006

[3] P Beguin and J P Aubert ldquoThe biological degradation ofcelluloserdquo FEMS Microbiology Review vol 13 pp 25ndash28 1994

[4] M K Bhat and S Bhat ldquoCellulose degrading enzymes and theirpotential industrial applicationsrdquo Biotechnology Advances vol15 no 3-4 pp 583ndash620 1997

[5] J Zhuang M A Marchant S E Nokes and H J StrobelldquoEconomic analysis of cellulase production methods for bio-ethanolrdquo Applied Engineering in Agriculture vol 23 no 5 pp679ndash687 2007

[6] S T Merino and J Cherry ldquoProgress and challenges in enzymedevelopment for biomass utilizationrdquo Advances in BiochemicalEngineeringBiotechnology vol 108 pp 95ndash120 2007

[7] M Dashtban H Schraft and W Qin ldquoFungal bioconversionof lignocellulosic residues Opportunities amp perspectivesrdquo Inter-national Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009

[8] B S Padam H S Tin F Y Chye and M I Abdullah ldquoBananaby-products an under-utilized renewable food biomass withgreat potentialrdquo Journal of Food Science and Technology vol 51no 12 pp 3527ndash3545 2014

[9] H P S Abdul Khalil M S Alwani and A K M OmarldquoChemical composition anatomy lignin distribution and cellwall structure ofMalaysian plantwaste fibresrdquoBioResources vol1 no 2 pp 220ndash232 2006

[10] K Li S Fu H Zhan Y Zhan and L A Lucia ldquoAnalysis of thechemical composition and morphological structure of bananapseudostemrdquo Bioresources vol 5 no 2 pp 576ndash585 2010

[11] K Ibatsam S H Muhammad M Sobia and M Irum ldquoIso-lation and screening of highly cellulolytic filamentous fungirdquoJournal of Applied Science and Environmental Management vol16 no 3 pp 223ndash226 2012

[12] G Nunez-Trujillo R Cabrera A Cosoveanu T T Martin CGimenez and G Nunez-Trujillo ldquoSurvey of banana endophyticfungi isolated in artificial culture media from an appliedviewpointrdquo Journal of Horticulture Forestry and Biotechnologyvol 17 no 2 pp 22ndash25 2013

[13] J H Yoon J E Park D Y Suh S B Hong S J Ko and S HKim ldquoComparison of dyes for easy detection of extracellularcellulases in fungirdquoMycobiology vol 35 no 1 pp 21ndash24 2007

[14] A B Peixoto Study of the production of enzymes and gumsby wild yeasts collected in various regions of Brazil [MSThesis] Faculty of Food Engineering University of CampinasCampinas Brazil 2006

[15] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods AndApplications M A Innis D H Gelfand J J Sninsky et al Edspp 315ndash322 Academic Press Inc New York NY USA 1990

[16] B Benoliel F A Goncalves Torres and L M P de MoraesldquoA novel promising Trichoderma harzianum strain for theproduction of a cellulolytic complex using sugarcane bagasse innaturardquo SpringerPlus vol 2 p 256 2013

[17] D Herr ldquoSecretion of cellulase and 120573-glucosidase byTricho-derma viride ITCC-1433 in submerged culture on differentsubstratesrdquo Biotechnology and Bioengineering vol 21 no 8 pp1361ndash1371 1979

[18] A S Lakshmi and G Narasimha ldquoProduction of cellulases byfungal cultures isolated from forest litter soilrdquo Annals of ForestResearch vol 55 no 1 pp 85ndash92 2012

[19] X Han W Song G Liu Z Li P Yang and Y Qu ldquoImprov-ing cellulase productivity of Penicillium oxalicum RE-10 byrepeated fed-batch fermentation strategyrdquo Bioresource Technol-ogy vol 227 pp 155ndash163 2017

[20] M Rubeena K Neethu S Sajith et al ldquoLignocellulolyticactivities of a novel strain of TrichodermaharzianumrdquoAdvancesin Bioscience and Biotechnology vol 4 pp 212ndash221 2013

[21] A L Grigorevski-Lima F N M Da Vinha D T Souzaet al ldquoAspergillus fumigatus thermophilic and acidophilicendoglucanasesrdquo Applied Biochemistry and Biotechnology vol155 no 1-3 pp 321ndash329 2009

10 Enzyme Research

[22] J P Andrade A S D R Bispo P A S Marbach and RP Do Nascimento ldquoProduction and partial characterizationof cellulases from Trichoderma sp IS-05 isolated from SandyCoastal plains of Northeast Brazilrdquo Enzyme Research vol 2011Article ID 167248 7 pages 2011

[23] P D S Delabona C S Farinas M R da Silva S F Azzoniand J G D C Pradella ldquoUse of a new Trichoderma harzianumstrain isolated from the Amazon rainforest with pretreatedsugar cane bagasse for on-site cellulase productionrdquo BioresourceTechnology vol 107 pp 517ndash521 2012

[24] C Y Turenne S E Sanche D J Hoban J A Karlowskyand A M Kabani ldquoRapid identification of fungi by using theITS2 genetic region and an automated fluorescent capillaryelectrophoresis systemrdquo Journal of Clinical Microbiology vol 37no 6 pp 1846ndash1851 1999

[25] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcode marker for fungirdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 109 no 27 pp6241ndash6246 2012

[26] M Shahriarinour M N A Wahab R Mohamad S Mustafaand A B Ariff ldquoEffect of medium composition and culturalcondition on cellulase production by Aspergillus terreusrdquoAfrican Journal of Biotechnology vol 10 no 38 pp 7459ndash74672011

[27] D B Archer and J F Peberdy ldquoThe molecular biology ofsecreted enzyme production by fungirdquo Critical Reviews inBiotechnology vol 17 no 4 pp 273ndash306 1997

[28] R Soni A Nazir and B S Chadha ldquoOptimization of cellulaseproduction by a versatile Aspergillus fumigatus fresenius strain(AMA) capable of efficient deinking and enzymatic hydrolysisof Solka floc and bagasserdquo Industrial Crops and Products vol 31no 2 pp 277ndash283 2010

[29] S P Gautam P S Bundela A K Pandey J Khan M KAwasthi and S Sarsaiya ldquoOptimization for the production ofcellulase enzyme from Municipal solid waste residue by twonovel cellulolytic fungirdquo Biotechnology Research Internationalpp 1ndash8 2011

[30] M Sohail R Siddiqi A Ahmad and S A Khan ldquoCellulaseproduction from Aspergillus niger MS82 effect of temperatureand pHrdquo New Biotechnology vol 25 no 6 pp 437ndash441 2009

[31] S Ahmed A Bashir H Saleem M Saadia and A JamilldquoProduction and purification of cellulose-degrading enzymesfrom a filamentous fungus Trichoderma harzianumrdquo PakistanJournal of Botany vol 41 no 3 pp 1411ndash1419 2009

[32] A Das T Paul S K Halder et al ldquoStudy on regulation ofgrowth and biosynthesis of cellulolytic enzymes from newlyisolated Aspergillus fumigatus ABK9rdquo Polish Journal of Micro-biology vol 62 no 1 pp 31ndash43 2013

[33] S Shafique R Bajwa and S Shafique ldquoCellulase biosynthesis byselected Trichoderma speciesrdquo Pakistan Journal of Botany vol41 no 2 pp 907ndash916 2009

[34] N Sarkar and K Aikat ldquoAspergillus fumigatus NITDGPKA3provides for increased cellulase productionrdquo International Jour-nal of Chemical Engineering vol 2014 9 pages 2014

[35] V V Gilna and K M Khaleel ldquoCellulase enzyme activity ofAspergillus fumigatus from mangrove soil on lignocellulosicsubstraterdquo Recent Research in Science and Technology vol 3 no1 pp 132ndash134 2011

[36] G E Aboul-Fotouh G M El-Garhy H H Azzaz A MAbd El-Mola and G A Mousa ldquoFungal cellulase production

optimisation and its utilisation in Goatrsquos relations degradationrdquoAsian Journal of Animal and Veterinary Advances vol 11 no 12pp 824ndash831 2016

[37] TNcube R LHoward EKAbotsi E L J vanRensburg and INcube ldquoJatropha curcas seed cake as substrate for production ofxylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentationrdquo Industrial Crops and Products vol 37 no 1pp 118ndash123 2012

[38] J C Stewart and J B Parry ldquoFactors influencing the productionof cellulase by Aspergillus fumigatus (Fresenius)rdquo Journal ofGeneral Microbiology vol 125 no 1 pp 33ndash39 1981

[39] H Leghlimi Z Meraihi H Boukhalfa-Lezzar E Copinet andF Duchiron ldquoProduction and characterization of cellulolyticactivities produced by Trichoderma longibrachiatum (GHL)rdquoAfrican Journal of Biotechnology vol 12 no 5 pp 465ndash475 2013

Hindawiwwwhindawicom

International Journal of

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Hindawiwwwhindawicom Volume 2018

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GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

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Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

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Hindawiwwwhindawicom Volume 2018

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Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

4 Enzyme Research

005

115

225

335

445

5

0 20 40 60 80 100 120 140 160 180

)lmUPF(

sealulleclatoT

Time (h)

T longibrachiatum LMLSAUL 14 - 1 A fumigatus LMLPS 13 - 4T harzianum LMLBP07 13 - 5 A fumigatus LMLBS02 13 - 2A fumigatus LMLBP06 13 - 3 A fumigatus LMLPS 13 - 1

Figure 1 Time course for cellulase production by isolated fungal strains at 30∘C and initial pH 55

A fumigatus LMLPS 13 - 4 A fumigatus LMLBP06 13 - 3A fumigatus LMLBS02 13 - 2 T harzianum LMLBP07 13 - 5T longibrachiatum LMLSAUL 14 - 1 A fumigatus LMLPS 13 - 1

5 55 6 65 7 7545pH

0

1

2

3

4

5

6

7

8

9

Tota

l cel

lula

se (F

PUm

l)

Figure 2 The effect of initial medium pH on the production of cellulases by isolated fungi at 30∘C

when the initial pHof themediumwas 65 (Figure 2)TheTri-choderma longibrachiatum LMLSAUL 14-1 strain producedthe highest total cellulase activity of 81 FPUml This wasfollowed by T harzianum LMLBP07 13-5 with activity of58 FPUml A fumigatus LMLBP06 13-3 and A fumigatusLMLPS 13-1 produced maximum cellulase activity of 31FPUml with the lowest cellulase activity of 20 FPUml beingproduced by A fumigatus LMLPS 13-4

The production of endoglucanase activity was highest atpH 65 for all the fungal strains (Figure 3) T longibrachiatumLMLSAUL 14 - 1 and T harzianum LMLBP07 13-5 producedactivities of 230 Uml and 160 Uml respectively whereasA fumigatus LMLPS 13-1 LMLBS02 13-2 and LMLBP06 13-3 produced amounts of 140 Uml The lowest activity of 121Uml was noted for A fumigatus LMLPS 13-4

The levels of 120573-glucosidase produced with respect toinitial medium pH differed between the fungal strains Afumigatus LMLPS 13-4 and A fumigatus LMLBS02 13-2 pro-duced higher activities of 380 Uml and 341 Uml at pH 70respectively compared to the other isolates tested (Figure 4)Some fungi were able to secrete higher 120573-glucosidase atvarious pH values T harzianum LMLBP07 13-5 produced120573-glucosidase activity of 257 Uml over a pH range of 6- 70 and A fumigatus LMLPS 13-1 produced 120573-glucosidaseactivity of 235 Uml over a pH range of 65 - 70 (Figure 4)The lowest 120573-glucosidase activity produced was 206 Uml byT longibrachiatum LMLSAUL 14 - 1

The production of cellulases with respect to changesin incubation temperature was also investigated at initialmedium pH of 65 and 70 (Figures 5 and 6) These pH values

Enzyme Research 5

45 5 55 6 65 7 75pH

A fumigatus LMLPS 13 - 4 A fumigatus LMLBP06 13 - 3

A fumigatus LMLBS02 13 - 2 T harzianum LMLBP07 13 - 5

T longibrachiatum LMLSAUL 14 -1 A fumigatus LMLPS 13 - 1

000

500

1000

1500

2000

2500

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 3 The effect of initial medium pH on the production of endoglucanases by the isolated fungal strains at 30∘C

000

5001000

15002000

2500

30003500

4000

45 5 55 6 65 7 75

-g

luco

sidas

e (U

ml)

pHA fumigatus LMLPS13 - 4 A fumigatus LMLBP06 13 - 3A fumigatus LMLBS02 13 - 2 T harzianum LMLBP07 13 - 5T longibrachiatum LMLSAUL 14 - 1 A fumigatus LMLPS 13 -1

Figure 4 The effect of initial medium pH on the production of 120573-glucosidase by the isolated fungi at 30∘C

have shown to favour production of 120573-glucosidase by speciesof Aspergillus and Trichoderma At initial medium pH of 65an increase in growth temperature led to a decrease in thelevel of total cellulase activity for T longibrachiatum LMLUL14-1 at 35∘C (Figure 5) The A fumigatus strains were able toproduce cellulase at both 30 and 35∘C with the exception ofT harzianum LMLUL 13-5 Cellulase activity in A fumigatusLMLUL 13-1 increased by 18-fold at 35∘C (Figure 5) Increas-ing the incubation temperature to 40∘C drastically reducedthe cellulase production for T longibrachiatum LMLUL 14-1 A fumigatus LMLUL 13-1 and A fumigatus LMLUL 13-3(Figure 5) Generally most fungal species showed cellulaseactivity levels decreasing as the temperature increased to40∘C

At initial medium pH of 70 and a temperature of 30∘Cfavoured high production of cellulase by T harzianumLMLBP07 14-5 T longibrachiatum LMLUL 14-1 and Afumigatus LMLBS02 13-2 (Figure 6)

The cellulase production by A fumigatus LMLPS 13-1A fumigatus LMLBP06 13-3 and A fumigatus LMLPS 13-4

improved at 35∘C irrespective of initial pH tested (Figures 5and 6) At production temperature 40∘C the production ofcellulases was reduced for all strains tested All the Tricho-derma andAspergillus strains producedmore endoglucanasesat 30∘C with both initial medium pH of 65 and 70 (Figures 7and 8) but initial medium pH of 65 favoured more enzymeproduction An increase in temperature to 35∘C and 40∘Cresulted in a 2-fold decrease of the endoglucanase activity inall fungal species (Figure 7)

In an experiment with initial medium pH 70 a greaterimprovement in the production of endoglucanases was seenat 35∘C (Figure 8) T harzianum LMLBP07 13-5 produced2-fold higher endoglucanase activity at initial medium pHof 70 while other fungal species attained slight increases(Figure 8)

At both initial medium pH of 65 and 70 higher tempera-tures caused a reduction in endoglucanase activity and severereduction occurred at 40∘C as illustrated in Figures 7 and 8

The production of 120573-glucosidase at an initial pH of 65and 70 was dependent on the fungal species and production

6 Enzyme Research

0123456789

10

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Fpase 30 degCFpase 35 degCFpase 40 degC

Tota

l cel

lula

se ac

tivity

(FPU

ml)

Figure 5 Effect of incubation temperature on the production of cellulase (total cellulase activity) by fungal strains at initial medium pH 65

0

1

2

3

4

5

6

7

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Fpase 30 degCFpase 35 degCFpase 40 degC

Tota

l cel

lula

se ac

tivity

(FPU

ml)

Figure 6 Effect of incubation temperature on the production of cellulases (total cellulase activity) by fungal strains at initial medium pH 70

temperature In a medium with an initial pH of 65 onlyA fumigatus LMLPS 13-1 and T harzianum LMLBP07 13-5produced high levels of 120573-glucosidase activity 240 Uml and250 Uml respectively at 30∘C An increase in temperaturefrom 30 to 40∘C led to improved production of 120573-glucosidaseby some fungal species For instance A fumigatus LMLBP0613-3 showed 5-fold increase ie from 54 to 254 Uml of120573-glucosidase activity at 40∘C and 52-fold increase of 120573-glucosidase at 35∘C (Figure 9)

The production of 120573-glucosidase by T harzianumLMLBP07 13-5 was optimal between 35 and 40∘C whilstT longibrachiatum LMLSAUL 14-1 produced 120573-glucosidaseoptimally at 40∘C A fumigatus LMLPS 13-4 attained maxi-mum 120573-glucosidase activity at 30∘C (Figure 9) Generally at40∘C the production of 120573-glucosidase by all fungal specieswas much higher than at 30∘C with the exception of Afumigatus LMLPS 13-4 In a medium with initial pH 70 anincrease in temperature led to improvement in the produc-tion of 120573-glucosidase (Figure 10) A maximum level of 120573-glucosidase level was attained at 35∘CwithT longibrachiatumLMLSAUL 14-1 and T harzianum LMLBP07 13-5 showing

155-fold increase and 113-fold increase respectively Afumigatus LMLPS 13-4 and A fumigatus LMLPS 13-1 alsoshowed an increase of 120573-glucosidase activity at 35∘C (176-fold and 16-fold respectively) A fumigatus LMLBP06 13-3showed a proportional increase of 127-fold with maximum120573-glucosidase produced at 40∘C Conversely 40∘C led to a126-fold decrease of 120573-glucosidase produced byA fumigatusLMLBS02 13 - 2 (Figure 10)

Generally 120573-glucosidase activity was the highest at 35∘Cand pH 70 for all strains tested

4 Discussion

41 Fungal Screening Fungi and bacteria are associated withsoil and decaying plant materials In nature fungi colonisethe plant debris and in symbiotic relationship with othermicroorganisms they secrete an assortment of proteinsand a complex of hydrolytic enzymes to hydrolyse plantpolysaccharides for their survival However not all themicroorganisms are able to secrete significant amount ofhydrolytic enzymes for biotechnological applications Hence

Enzyme Research 7

0

5

10

15

20

25

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Endoglucanase 30 degCEndoglucanase 35 degCEndoglucanase 40 degC

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 7 Effect of incubation temperature on the production of endoglucanase by fungal strains at initial medium pH 65

0

5

10

15

20

25

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Endoglucanase 30 degCEndoglucanase 35 degCEndoglucanase 40 degC

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 8 Effect of incubation temperature on the production of endoglucanase by fungal strains at initial medium pH 70

0

5

10

15

20

25

30

35

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 9 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial medium pH 65

there is a need to screen and select hypersecreting hydrolyticenzymes In this study three different methods for screeningfor cellulolytic activity (1)microbial growth on cellulose agar(2) clearing of cellulose in agar and (3) reducing sugar pro-duction (or glucose) were performed Growth on cellulose

containing agar was useful for isolation of cellulolytic fungalstrains Ten fungal strains were selected based on fast andabundant growth on Avicel agar plates Several authors havealso reported the use of microcrystalline cellulose to screenfor cellulase producing microorganisms [21ndash23] CMC-CR

8 Enzyme Research

05

10152025303540

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 10 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial pH 70

(001) agar plate has been used to detect endoglucanaseactivity as indicated by the presence of a pale yellow zonearound the colony According to Yoon et al [13] the platescreening methods with dye coupled substrates provides arelatively straight forward and easy tool for specific detectionof fungi that produce endoglucanase Larger halos are theresults of higher enzyme activity [2] However at high CR dyeconcentrations fungal growth is suppressed

42 Fungal Identification The application of rDNA genesfor identification of fungal species is based on the detectionof conserved sequences in 58S rDNA and 28S rDNA thatenables the amplification of the ITS2 region between them[24] The identification of the fungal species based on theamplification of the ribosomal ITS region of the ribosomalDNA revealed that the organisms are A fumigatus strainsT harzianum and T longibrachiatum The ITS regions areregarded as primary DNA barcode for the fungal kingdomand exhibit high reliability [25]

43 Cellulases Production and Optimisation Subsequent toscreening is process optimisation with various factors such asnutrient requirements temperature pH and agitation speedbeen investigated (Gautam et al 2010 [26]) In this studythe effect of initial pH and temperatures on the productionlevels of cellulase were investigated Fungal strains belongingto Trichoderma and Aspergillus exhibited the potential toproduce cellulase over a pH 45-70 and temperature (30-40) range although the cellulase production levels variedfrom one fungal strain to another under the conditionsused It has been reported that expression of fungal genesis regulated by extracellular pH and many fungi exhibitgrowth and enzyme secretion over a wide pH range [27]Temperature also influences microbial growth and enzymeproduction [20 28] and an optimal environment conduciveto the production of cellulase will also be influenced bymedianutritional composition [26]

The initial pH of 45-55 drastically reduced the levelsof cellulases and endoglucanases produced by all strainsThis implied that acidity of the medium possibly affected

fungal growth and metabolism Acidic medium pH effect onthe production of cellulases by Trichoderma and Aspergilluswas also reported by Gautam et al [29] and Delabona etal [23] Contrary to these results other authors reportedsuch acidic condition to be favourable for production ofcellulases [22 30ndash34] Our results showed that a less acidicmedium with a pH 55 and above favoured the productionof cellulases by all Trichoderma and Aspergillus strains withoptimum production occurring at a pH of 65 Beyond pH65 a drastic decrease in cellulases levels was observed Gilnaamp Khaleel [35] and Gautam et al ([29] 2010) also reportedmaximum production of cellulases at pH 65 Aboul-Fotouhet al [36] reported maximum production of cellulases byAspergillus niger at initial medium pH of 6 when cultured on10 rice straw Other findings by Ncube et al [37] showed nosignificant differences in the production of endoglucanasesby A niger FGSCA 733 over a pH range of 3-7These findingsillustrate that an optimum pH for maximum production ofcellulase is dependent on fungal species and to some extenton the particular strain evaluated

There was effect of temperature on the productionof cellulases The production of cellulases decreased withincreasing temperature At 40∘C a significant reduction inendoglucanases activity was observed in all strains tested(Figures 8 and 9) On the contrary Ncube et al [37]reported maximum endoglucanase activity at 40∘C by Aniger FGSCA 733 Other studies reported optimal cellulaseand endoglucanase production at 45∘C [29] and 60∘C [2238] A temperature of 40∘C did not significantly affect 120573-glucosidase production (Figures 9 and 10) Leghlili et al(2013) reported the production of cellulases endoglucanasesand 120573-glucosidase T longibrachiatum (GHL) at both 30∘Cand 35∘C with higher production levels obtained at 35∘COther authors reported maximum production levels of 120573-glucosidase by T longibrachiatum at 35∘C [39] and A nigerMS82 at 25∘C [30] Aspergillus niger MS82 also producedsufficient endoglucanases at 30∘C and 35∘C in acidic initialmedium pH 40 [30]

These discrepancies with regard to the optimum initialpH and production temperature on the levels of cellulases as

Enzyme Research 9

measured by filter paper activity (FPUml) can be attributedto genetic make-up of the fungal strains as a result ofadaptations to different habitats Furthermore our reportedhigh levels of cellulases endoglucanases and low level of 120573-glucosidase by the Trichoderma isolates and vice versa highlevels of 120573-glucosidase by A fumigatus are in agreement withthe reported levels of these fungal species by Stewart andParry [38]

5 Conclusion

In conclusion all the Trichoderma and Aspergillus strainsproduced substantial levels of all the three enzymes (ieexoglucanase endoglucanase and 120573-glucosidase) requiredfor complete hydrolysis of cellulosic material Trichodermastrains produced higher cellulases and endoglucanases levelswhile A fumigatus strains produced higher 120573-glucosidaselevels The production of all the cellulases componentsseemed to be strongly influenced by the interactive effectof initial pH and incubation temperature on the microor-ganisms Hence the observed maximum production ofcellulases by the fungi depended on the chosen initial pHof the medium and incubation temperature The cellulasesproduced by these fungal strainswill be assessed for efficiencyin hydrolysing agricultural lignocellulose wastes includingbanana pseudostem for the production of bioethanol

Data Availability

The experimental data used to support the findings of thisstudy are available from the corresponding author uponrequest

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors are grateful to the National Research Foundation(NRF) and the Flemish Inter-University Council (VLIR-UOS) for the financial support towards the research

References

[1] A C OrsquoSullivan ldquoCellulose the structure slowly unravelsrdquoCellulose vol 4 no 3 pp 173ndash207 1997

[2] Y-H P Zhang M E Himmel and J R Mielenz ldquoOutlookfor cellulase improvement screening and selection strategiesrdquoBiotechnology Advances vol 24 no 5 pp 452ndash481 2006

[3] P Beguin and J P Aubert ldquoThe biological degradation ofcelluloserdquo FEMS Microbiology Review vol 13 pp 25ndash28 1994

[4] M K Bhat and S Bhat ldquoCellulose degrading enzymes and theirpotential industrial applicationsrdquo Biotechnology Advances vol15 no 3-4 pp 583ndash620 1997

[5] J Zhuang M A Marchant S E Nokes and H J StrobelldquoEconomic analysis of cellulase production methods for bio-ethanolrdquo Applied Engineering in Agriculture vol 23 no 5 pp679ndash687 2007

[6] S T Merino and J Cherry ldquoProgress and challenges in enzymedevelopment for biomass utilizationrdquo Advances in BiochemicalEngineeringBiotechnology vol 108 pp 95ndash120 2007

[7] M Dashtban H Schraft and W Qin ldquoFungal bioconversionof lignocellulosic residues Opportunities amp perspectivesrdquo Inter-national Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009

[8] B S Padam H S Tin F Y Chye and M I Abdullah ldquoBananaby-products an under-utilized renewable food biomass withgreat potentialrdquo Journal of Food Science and Technology vol 51no 12 pp 3527ndash3545 2014

[9] H P S Abdul Khalil M S Alwani and A K M OmarldquoChemical composition anatomy lignin distribution and cellwall structure ofMalaysian plantwaste fibresrdquoBioResources vol1 no 2 pp 220ndash232 2006

[10] K Li S Fu H Zhan Y Zhan and L A Lucia ldquoAnalysis of thechemical composition and morphological structure of bananapseudostemrdquo Bioresources vol 5 no 2 pp 576ndash585 2010

[11] K Ibatsam S H Muhammad M Sobia and M Irum ldquoIso-lation and screening of highly cellulolytic filamentous fungirdquoJournal of Applied Science and Environmental Management vol16 no 3 pp 223ndash226 2012

[12] G Nunez-Trujillo R Cabrera A Cosoveanu T T Martin CGimenez and G Nunez-Trujillo ldquoSurvey of banana endophyticfungi isolated in artificial culture media from an appliedviewpointrdquo Journal of Horticulture Forestry and Biotechnologyvol 17 no 2 pp 22ndash25 2013

[13] J H Yoon J E Park D Y Suh S B Hong S J Ko and S HKim ldquoComparison of dyes for easy detection of extracellularcellulases in fungirdquoMycobiology vol 35 no 1 pp 21ndash24 2007

[14] A B Peixoto Study of the production of enzymes and gumsby wild yeasts collected in various regions of Brazil [MSThesis] Faculty of Food Engineering University of CampinasCampinas Brazil 2006

[15] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods AndApplications M A Innis D H Gelfand J J Sninsky et al Edspp 315ndash322 Academic Press Inc New York NY USA 1990

[16] B Benoliel F A Goncalves Torres and L M P de MoraesldquoA novel promising Trichoderma harzianum strain for theproduction of a cellulolytic complex using sugarcane bagasse innaturardquo SpringerPlus vol 2 p 256 2013

[17] D Herr ldquoSecretion of cellulase and 120573-glucosidase byTricho-derma viride ITCC-1433 in submerged culture on differentsubstratesrdquo Biotechnology and Bioengineering vol 21 no 8 pp1361ndash1371 1979

[18] A S Lakshmi and G Narasimha ldquoProduction of cellulases byfungal cultures isolated from forest litter soilrdquo Annals of ForestResearch vol 55 no 1 pp 85ndash92 2012

[19] X Han W Song G Liu Z Li P Yang and Y Qu ldquoImprov-ing cellulase productivity of Penicillium oxalicum RE-10 byrepeated fed-batch fermentation strategyrdquo Bioresource Technol-ogy vol 227 pp 155ndash163 2017

[20] M Rubeena K Neethu S Sajith et al ldquoLignocellulolyticactivities of a novel strain of TrichodermaharzianumrdquoAdvancesin Bioscience and Biotechnology vol 4 pp 212ndash221 2013

[21] A L Grigorevski-Lima F N M Da Vinha D T Souzaet al ldquoAspergillus fumigatus thermophilic and acidophilicendoglucanasesrdquo Applied Biochemistry and Biotechnology vol155 no 1-3 pp 321ndash329 2009

10 Enzyme Research

[22] J P Andrade A S D R Bispo P A S Marbach and RP Do Nascimento ldquoProduction and partial characterizationof cellulases from Trichoderma sp IS-05 isolated from SandyCoastal plains of Northeast Brazilrdquo Enzyme Research vol 2011Article ID 167248 7 pages 2011

[23] P D S Delabona C S Farinas M R da Silva S F Azzoniand J G D C Pradella ldquoUse of a new Trichoderma harzianumstrain isolated from the Amazon rainforest with pretreatedsugar cane bagasse for on-site cellulase productionrdquo BioresourceTechnology vol 107 pp 517ndash521 2012

[24] C Y Turenne S E Sanche D J Hoban J A Karlowskyand A M Kabani ldquoRapid identification of fungi by using theITS2 genetic region and an automated fluorescent capillaryelectrophoresis systemrdquo Journal of Clinical Microbiology vol 37no 6 pp 1846ndash1851 1999

[25] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcode marker for fungirdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 109 no 27 pp6241ndash6246 2012

[26] M Shahriarinour M N A Wahab R Mohamad S Mustafaand A B Ariff ldquoEffect of medium composition and culturalcondition on cellulase production by Aspergillus terreusrdquoAfrican Journal of Biotechnology vol 10 no 38 pp 7459ndash74672011

[27] D B Archer and J F Peberdy ldquoThe molecular biology ofsecreted enzyme production by fungirdquo Critical Reviews inBiotechnology vol 17 no 4 pp 273ndash306 1997

[28] R Soni A Nazir and B S Chadha ldquoOptimization of cellulaseproduction by a versatile Aspergillus fumigatus fresenius strain(AMA) capable of efficient deinking and enzymatic hydrolysisof Solka floc and bagasserdquo Industrial Crops and Products vol 31no 2 pp 277ndash283 2010

[29] S P Gautam P S Bundela A K Pandey J Khan M KAwasthi and S Sarsaiya ldquoOptimization for the production ofcellulase enzyme from Municipal solid waste residue by twonovel cellulolytic fungirdquo Biotechnology Research Internationalpp 1ndash8 2011

[30] M Sohail R Siddiqi A Ahmad and S A Khan ldquoCellulaseproduction from Aspergillus niger MS82 effect of temperatureand pHrdquo New Biotechnology vol 25 no 6 pp 437ndash441 2009

[31] S Ahmed A Bashir H Saleem M Saadia and A JamilldquoProduction and purification of cellulose-degrading enzymesfrom a filamentous fungus Trichoderma harzianumrdquo PakistanJournal of Botany vol 41 no 3 pp 1411ndash1419 2009

[32] A Das T Paul S K Halder et al ldquoStudy on regulation ofgrowth and biosynthesis of cellulolytic enzymes from newlyisolated Aspergillus fumigatus ABK9rdquo Polish Journal of Micro-biology vol 62 no 1 pp 31ndash43 2013

[33] S Shafique R Bajwa and S Shafique ldquoCellulase biosynthesis byselected Trichoderma speciesrdquo Pakistan Journal of Botany vol41 no 2 pp 907ndash916 2009

[34] N Sarkar and K Aikat ldquoAspergillus fumigatus NITDGPKA3provides for increased cellulase productionrdquo International Jour-nal of Chemical Engineering vol 2014 9 pages 2014

[35] V V Gilna and K M Khaleel ldquoCellulase enzyme activity ofAspergillus fumigatus from mangrove soil on lignocellulosicsubstraterdquo Recent Research in Science and Technology vol 3 no1 pp 132ndash134 2011

[36] G E Aboul-Fotouh G M El-Garhy H H Azzaz A MAbd El-Mola and G A Mousa ldquoFungal cellulase production

optimisation and its utilisation in Goatrsquos relations degradationrdquoAsian Journal of Animal and Veterinary Advances vol 11 no 12pp 824ndash831 2016

[37] TNcube R LHoward EKAbotsi E L J vanRensburg and INcube ldquoJatropha curcas seed cake as substrate for production ofxylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentationrdquo Industrial Crops and Products vol 37 no 1pp 118ndash123 2012

[38] J C Stewart and J B Parry ldquoFactors influencing the productionof cellulase by Aspergillus fumigatus (Fresenius)rdquo Journal ofGeneral Microbiology vol 125 no 1 pp 33ndash39 1981

[39] H Leghlimi Z Meraihi H Boukhalfa-Lezzar E Copinet andF Duchiron ldquoProduction and characterization of cellulolyticactivities produced by Trichoderma longibrachiatum (GHL)rdquoAfrican Journal of Biotechnology vol 12 no 5 pp 465ndash475 2013

Hindawiwwwhindawicom

International Journal of

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GenomicsInternational Journal of

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

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Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

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Cell BiologyInternational Journal of

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Enzyme Research

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International Journal of

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Nucleic AcidsJournal of

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Submit your manuscripts atwwwhindawicom

Enzyme Research 5

45 5 55 6 65 7 75pH

A fumigatus LMLPS 13 - 4 A fumigatus LMLBP06 13 - 3

A fumigatus LMLBS02 13 - 2 T harzianum LMLBP07 13 - 5

T longibrachiatum LMLSAUL 14 -1 A fumigatus LMLPS 13 - 1

000

500

1000

1500

2000

2500

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 3 The effect of initial medium pH on the production of endoglucanases by the isolated fungal strains at 30∘C

000

5001000

15002000

2500

30003500

4000

45 5 55 6 65 7 75

-g

luco

sidas

e (U

ml)

pHA fumigatus LMLPS13 - 4 A fumigatus LMLBP06 13 - 3A fumigatus LMLBS02 13 - 2 T harzianum LMLBP07 13 - 5T longibrachiatum LMLSAUL 14 - 1 A fumigatus LMLPS 13 -1

Figure 4 The effect of initial medium pH on the production of 120573-glucosidase by the isolated fungi at 30∘C

have shown to favour production of 120573-glucosidase by speciesof Aspergillus and Trichoderma At initial medium pH of 65an increase in growth temperature led to a decrease in thelevel of total cellulase activity for T longibrachiatum LMLUL14-1 at 35∘C (Figure 5) The A fumigatus strains were able toproduce cellulase at both 30 and 35∘C with the exception ofT harzianum LMLUL 13-5 Cellulase activity in A fumigatusLMLUL 13-1 increased by 18-fold at 35∘C (Figure 5) Increas-ing the incubation temperature to 40∘C drastically reducedthe cellulase production for T longibrachiatum LMLUL 14-1 A fumigatus LMLUL 13-1 and A fumigatus LMLUL 13-3(Figure 5) Generally most fungal species showed cellulaseactivity levels decreasing as the temperature increased to40∘C

At initial medium pH of 70 and a temperature of 30∘Cfavoured high production of cellulase by T harzianumLMLBP07 14-5 T longibrachiatum LMLUL 14-1 and Afumigatus LMLBS02 13-2 (Figure 6)

The cellulase production by A fumigatus LMLPS 13-1A fumigatus LMLBP06 13-3 and A fumigatus LMLPS 13-4

improved at 35∘C irrespective of initial pH tested (Figures 5and 6) At production temperature 40∘C the production ofcellulases was reduced for all strains tested All the Tricho-derma andAspergillus strains producedmore endoglucanasesat 30∘C with both initial medium pH of 65 and 70 (Figures 7and 8) but initial medium pH of 65 favoured more enzymeproduction An increase in temperature to 35∘C and 40∘Cresulted in a 2-fold decrease of the endoglucanase activity inall fungal species (Figure 7)

In an experiment with initial medium pH 70 a greaterimprovement in the production of endoglucanases was seenat 35∘C (Figure 8) T harzianum LMLBP07 13-5 produced2-fold higher endoglucanase activity at initial medium pHof 70 while other fungal species attained slight increases(Figure 8)

At both initial medium pH of 65 and 70 higher tempera-tures caused a reduction in endoglucanase activity and severereduction occurred at 40∘C as illustrated in Figures 7 and 8

The production of 120573-glucosidase at an initial pH of 65and 70 was dependent on the fungal species and production

6 Enzyme Research

0123456789

10

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Fpase 30 degCFpase 35 degCFpase 40 degC

Tota

l cel

lula

se ac

tivity

(FPU

ml)

Figure 5 Effect of incubation temperature on the production of cellulase (total cellulase activity) by fungal strains at initial medium pH 65

0

1

2

3

4

5

6

7

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Fpase 30 degCFpase 35 degCFpase 40 degC

Tota

l cel

lula

se ac

tivity

(FPU

ml)

Figure 6 Effect of incubation temperature on the production of cellulases (total cellulase activity) by fungal strains at initial medium pH 70

temperature In a medium with an initial pH of 65 onlyA fumigatus LMLPS 13-1 and T harzianum LMLBP07 13-5produced high levels of 120573-glucosidase activity 240 Uml and250 Uml respectively at 30∘C An increase in temperaturefrom 30 to 40∘C led to improved production of 120573-glucosidaseby some fungal species For instance A fumigatus LMLBP0613-3 showed 5-fold increase ie from 54 to 254 Uml of120573-glucosidase activity at 40∘C and 52-fold increase of 120573-glucosidase at 35∘C (Figure 9)

The production of 120573-glucosidase by T harzianumLMLBP07 13-5 was optimal between 35 and 40∘C whilstT longibrachiatum LMLSAUL 14-1 produced 120573-glucosidaseoptimally at 40∘C A fumigatus LMLPS 13-4 attained maxi-mum 120573-glucosidase activity at 30∘C (Figure 9) Generally at40∘C the production of 120573-glucosidase by all fungal specieswas much higher than at 30∘C with the exception of Afumigatus LMLPS 13-4 In a medium with initial pH 70 anincrease in temperature led to improvement in the produc-tion of 120573-glucosidase (Figure 10) A maximum level of 120573-glucosidase level was attained at 35∘CwithT longibrachiatumLMLSAUL 14-1 and T harzianum LMLBP07 13-5 showing

155-fold increase and 113-fold increase respectively Afumigatus LMLPS 13-4 and A fumigatus LMLPS 13-1 alsoshowed an increase of 120573-glucosidase activity at 35∘C (176-fold and 16-fold respectively) A fumigatus LMLBP06 13-3showed a proportional increase of 127-fold with maximum120573-glucosidase produced at 40∘C Conversely 40∘C led to a126-fold decrease of 120573-glucosidase produced byA fumigatusLMLBS02 13 - 2 (Figure 10)

Generally 120573-glucosidase activity was the highest at 35∘Cand pH 70 for all strains tested

4 Discussion

41 Fungal Screening Fungi and bacteria are associated withsoil and decaying plant materials In nature fungi colonisethe plant debris and in symbiotic relationship with othermicroorganisms they secrete an assortment of proteinsand a complex of hydrolytic enzymes to hydrolyse plantpolysaccharides for their survival However not all themicroorganisms are able to secrete significant amount ofhydrolytic enzymes for biotechnological applications Hence

Enzyme Research 7

0

5

10

15

20

25

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Endoglucanase 30 degCEndoglucanase 35 degCEndoglucanase 40 degC

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 7 Effect of incubation temperature on the production of endoglucanase by fungal strains at initial medium pH 65

0

5

10

15

20

25

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Endoglucanase 30 degCEndoglucanase 35 degCEndoglucanase 40 degC

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 8 Effect of incubation temperature on the production of endoglucanase by fungal strains at initial medium pH 70

0

5

10

15

20

25

30

35

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 9 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial medium pH 65

there is a need to screen and select hypersecreting hydrolyticenzymes In this study three different methods for screeningfor cellulolytic activity (1)microbial growth on cellulose agar(2) clearing of cellulose in agar and (3) reducing sugar pro-duction (or glucose) were performed Growth on cellulose

containing agar was useful for isolation of cellulolytic fungalstrains Ten fungal strains were selected based on fast andabundant growth on Avicel agar plates Several authors havealso reported the use of microcrystalline cellulose to screenfor cellulase producing microorganisms [21ndash23] CMC-CR

8 Enzyme Research

05

10152025303540

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 10 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial pH 70

(001) agar plate has been used to detect endoglucanaseactivity as indicated by the presence of a pale yellow zonearound the colony According to Yoon et al [13] the platescreening methods with dye coupled substrates provides arelatively straight forward and easy tool for specific detectionof fungi that produce endoglucanase Larger halos are theresults of higher enzyme activity [2] However at high CR dyeconcentrations fungal growth is suppressed

42 Fungal Identification The application of rDNA genesfor identification of fungal species is based on the detectionof conserved sequences in 58S rDNA and 28S rDNA thatenables the amplification of the ITS2 region between them[24] The identification of the fungal species based on theamplification of the ribosomal ITS region of the ribosomalDNA revealed that the organisms are A fumigatus strainsT harzianum and T longibrachiatum The ITS regions areregarded as primary DNA barcode for the fungal kingdomand exhibit high reliability [25]

43 Cellulases Production and Optimisation Subsequent toscreening is process optimisation with various factors such asnutrient requirements temperature pH and agitation speedbeen investigated (Gautam et al 2010 [26]) In this studythe effect of initial pH and temperatures on the productionlevels of cellulase were investigated Fungal strains belongingto Trichoderma and Aspergillus exhibited the potential toproduce cellulase over a pH 45-70 and temperature (30-40) range although the cellulase production levels variedfrom one fungal strain to another under the conditionsused It has been reported that expression of fungal genesis regulated by extracellular pH and many fungi exhibitgrowth and enzyme secretion over a wide pH range [27]Temperature also influences microbial growth and enzymeproduction [20 28] and an optimal environment conduciveto the production of cellulase will also be influenced bymedianutritional composition [26]

The initial pH of 45-55 drastically reduced the levelsof cellulases and endoglucanases produced by all strainsThis implied that acidity of the medium possibly affected

fungal growth and metabolism Acidic medium pH effect onthe production of cellulases by Trichoderma and Aspergilluswas also reported by Gautam et al [29] and Delabona etal [23] Contrary to these results other authors reportedsuch acidic condition to be favourable for production ofcellulases [22 30ndash34] Our results showed that a less acidicmedium with a pH 55 and above favoured the productionof cellulases by all Trichoderma and Aspergillus strains withoptimum production occurring at a pH of 65 Beyond pH65 a drastic decrease in cellulases levels was observed Gilnaamp Khaleel [35] and Gautam et al ([29] 2010) also reportedmaximum production of cellulases at pH 65 Aboul-Fotouhet al [36] reported maximum production of cellulases byAspergillus niger at initial medium pH of 6 when cultured on10 rice straw Other findings by Ncube et al [37] showed nosignificant differences in the production of endoglucanasesby A niger FGSCA 733 over a pH range of 3-7These findingsillustrate that an optimum pH for maximum production ofcellulase is dependent on fungal species and to some extenton the particular strain evaluated

There was effect of temperature on the productionof cellulases The production of cellulases decreased withincreasing temperature At 40∘C a significant reduction inendoglucanases activity was observed in all strains tested(Figures 8 and 9) On the contrary Ncube et al [37]reported maximum endoglucanase activity at 40∘C by Aniger FGSCA 733 Other studies reported optimal cellulaseand endoglucanase production at 45∘C [29] and 60∘C [2238] A temperature of 40∘C did not significantly affect 120573-glucosidase production (Figures 9 and 10) Leghlili et al(2013) reported the production of cellulases endoglucanasesand 120573-glucosidase T longibrachiatum (GHL) at both 30∘Cand 35∘C with higher production levels obtained at 35∘COther authors reported maximum production levels of 120573-glucosidase by T longibrachiatum at 35∘C [39] and A nigerMS82 at 25∘C [30] Aspergillus niger MS82 also producedsufficient endoglucanases at 30∘C and 35∘C in acidic initialmedium pH 40 [30]

These discrepancies with regard to the optimum initialpH and production temperature on the levels of cellulases as

Enzyme Research 9

measured by filter paper activity (FPUml) can be attributedto genetic make-up of the fungal strains as a result ofadaptations to different habitats Furthermore our reportedhigh levels of cellulases endoglucanases and low level of 120573-glucosidase by the Trichoderma isolates and vice versa highlevels of 120573-glucosidase by A fumigatus are in agreement withthe reported levels of these fungal species by Stewart andParry [38]

5 Conclusion

In conclusion all the Trichoderma and Aspergillus strainsproduced substantial levels of all the three enzymes (ieexoglucanase endoglucanase and 120573-glucosidase) requiredfor complete hydrolysis of cellulosic material Trichodermastrains produced higher cellulases and endoglucanases levelswhile A fumigatus strains produced higher 120573-glucosidaselevels The production of all the cellulases componentsseemed to be strongly influenced by the interactive effectof initial pH and incubation temperature on the microor-ganisms Hence the observed maximum production ofcellulases by the fungi depended on the chosen initial pHof the medium and incubation temperature The cellulasesproduced by these fungal strainswill be assessed for efficiencyin hydrolysing agricultural lignocellulose wastes includingbanana pseudostem for the production of bioethanol

Data Availability

The experimental data used to support the findings of thisstudy are available from the corresponding author uponrequest

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors are grateful to the National Research Foundation(NRF) and the Flemish Inter-University Council (VLIR-UOS) for the financial support towards the research

References

[1] A C OrsquoSullivan ldquoCellulose the structure slowly unravelsrdquoCellulose vol 4 no 3 pp 173ndash207 1997

[2] Y-H P Zhang M E Himmel and J R Mielenz ldquoOutlookfor cellulase improvement screening and selection strategiesrdquoBiotechnology Advances vol 24 no 5 pp 452ndash481 2006

[3] P Beguin and J P Aubert ldquoThe biological degradation ofcelluloserdquo FEMS Microbiology Review vol 13 pp 25ndash28 1994

[4] M K Bhat and S Bhat ldquoCellulose degrading enzymes and theirpotential industrial applicationsrdquo Biotechnology Advances vol15 no 3-4 pp 583ndash620 1997

[5] J Zhuang M A Marchant S E Nokes and H J StrobelldquoEconomic analysis of cellulase production methods for bio-ethanolrdquo Applied Engineering in Agriculture vol 23 no 5 pp679ndash687 2007

[6] S T Merino and J Cherry ldquoProgress and challenges in enzymedevelopment for biomass utilizationrdquo Advances in BiochemicalEngineeringBiotechnology vol 108 pp 95ndash120 2007

[7] M Dashtban H Schraft and W Qin ldquoFungal bioconversionof lignocellulosic residues Opportunities amp perspectivesrdquo Inter-national Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009

[8] B S Padam H S Tin F Y Chye and M I Abdullah ldquoBananaby-products an under-utilized renewable food biomass withgreat potentialrdquo Journal of Food Science and Technology vol 51no 12 pp 3527ndash3545 2014

[9] H P S Abdul Khalil M S Alwani and A K M OmarldquoChemical composition anatomy lignin distribution and cellwall structure ofMalaysian plantwaste fibresrdquoBioResources vol1 no 2 pp 220ndash232 2006

[10] K Li S Fu H Zhan Y Zhan and L A Lucia ldquoAnalysis of thechemical composition and morphological structure of bananapseudostemrdquo Bioresources vol 5 no 2 pp 576ndash585 2010

[11] K Ibatsam S H Muhammad M Sobia and M Irum ldquoIso-lation and screening of highly cellulolytic filamentous fungirdquoJournal of Applied Science and Environmental Management vol16 no 3 pp 223ndash226 2012

[12] G Nunez-Trujillo R Cabrera A Cosoveanu T T Martin CGimenez and G Nunez-Trujillo ldquoSurvey of banana endophyticfungi isolated in artificial culture media from an appliedviewpointrdquo Journal of Horticulture Forestry and Biotechnologyvol 17 no 2 pp 22ndash25 2013

[13] J H Yoon J E Park D Y Suh S B Hong S J Ko and S HKim ldquoComparison of dyes for easy detection of extracellularcellulases in fungirdquoMycobiology vol 35 no 1 pp 21ndash24 2007

[14] A B Peixoto Study of the production of enzymes and gumsby wild yeasts collected in various regions of Brazil [MSThesis] Faculty of Food Engineering University of CampinasCampinas Brazil 2006

[15] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods AndApplications M A Innis D H Gelfand J J Sninsky et al Edspp 315ndash322 Academic Press Inc New York NY USA 1990

[16] B Benoliel F A Goncalves Torres and L M P de MoraesldquoA novel promising Trichoderma harzianum strain for theproduction of a cellulolytic complex using sugarcane bagasse innaturardquo SpringerPlus vol 2 p 256 2013

[17] D Herr ldquoSecretion of cellulase and 120573-glucosidase byTricho-derma viride ITCC-1433 in submerged culture on differentsubstratesrdquo Biotechnology and Bioengineering vol 21 no 8 pp1361ndash1371 1979

[18] A S Lakshmi and G Narasimha ldquoProduction of cellulases byfungal cultures isolated from forest litter soilrdquo Annals of ForestResearch vol 55 no 1 pp 85ndash92 2012

[19] X Han W Song G Liu Z Li P Yang and Y Qu ldquoImprov-ing cellulase productivity of Penicillium oxalicum RE-10 byrepeated fed-batch fermentation strategyrdquo Bioresource Technol-ogy vol 227 pp 155ndash163 2017

[20] M Rubeena K Neethu S Sajith et al ldquoLignocellulolyticactivities of a novel strain of TrichodermaharzianumrdquoAdvancesin Bioscience and Biotechnology vol 4 pp 212ndash221 2013

[21] A L Grigorevski-Lima F N M Da Vinha D T Souzaet al ldquoAspergillus fumigatus thermophilic and acidophilicendoglucanasesrdquo Applied Biochemistry and Biotechnology vol155 no 1-3 pp 321ndash329 2009

10 Enzyme Research

[22] J P Andrade A S D R Bispo P A S Marbach and RP Do Nascimento ldquoProduction and partial characterizationof cellulases from Trichoderma sp IS-05 isolated from SandyCoastal plains of Northeast Brazilrdquo Enzyme Research vol 2011Article ID 167248 7 pages 2011

[23] P D S Delabona C S Farinas M R da Silva S F Azzoniand J G D C Pradella ldquoUse of a new Trichoderma harzianumstrain isolated from the Amazon rainforest with pretreatedsugar cane bagasse for on-site cellulase productionrdquo BioresourceTechnology vol 107 pp 517ndash521 2012

[24] C Y Turenne S E Sanche D J Hoban J A Karlowskyand A M Kabani ldquoRapid identification of fungi by using theITS2 genetic region and an automated fluorescent capillaryelectrophoresis systemrdquo Journal of Clinical Microbiology vol 37no 6 pp 1846ndash1851 1999

[25] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcode marker for fungirdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 109 no 27 pp6241ndash6246 2012

[26] M Shahriarinour M N A Wahab R Mohamad S Mustafaand A B Ariff ldquoEffect of medium composition and culturalcondition on cellulase production by Aspergillus terreusrdquoAfrican Journal of Biotechnology vol 10 no 38 pp 7459ndash74672011

[27] D B Archer and J F Peberdy ldquoThe molecular biology ofsecreted enzyme production by fungirdquo Critical Reviews inBiotechnology vol 17 no 4 pp 273ndash306 1997

[28] R Soni A Nazir and B S Chadha ldquoOptimization of cellulaseproduction by a versatile Aspergillus fumigatus fresenius strain(AMA) capable of efficient deinking and enzymatic hydrolysisof Solka floc and bagasserdquo Industrial Crops and Products vol 31no 2 pp 277ndash283 2010

[29] S P Gautam P S Bundela A K Pandey J Khan M KAwasthi and S Sarsaiya ldquoOptimization for the production ofcellulase enzyme from Municipal solid waste residue by twonovel cellulolytic fungirdquo Biotechnology Research Internationalpp 1ndash8 2011

[30] M Sohail R Siddiqi A Ahmad and S A Khan ldquoCellulaseproduction from Aspergillus niger MS82 effect of temperatureand pHrdquo New Biotechnology vol 25 no 6 pp 437ndash441 2009

[31] S Ahmed A Bashir H Saleem M Saadia and A JamilldquoProduction and purification of cellulose-degrading enzymesfrom a filamentous fungus Trichoderma harzianumrdquo PakistanJournal of Botany vol 41 no 3 pp 1411ndash1419 2009

[32] A Das T Paul S K Halder et al ldquoStudy on regulation ofgrowth and biosynthesis of cellulolytic enzymes from newlyisolated Aspergillus fumigatus ABK9rdquo Polish Journal of Micro-biology vol 62 no 1 pp 31ndash43 2013

[33] S Shafique R Bajwa and S Shafique ldquoCellulase biosynthesis byselected Trichoderma speciesrdquo Pakistan Journal of Botany vol41 no 2 pp 907ndash916 2009

[34] N Sarkar and K Aikat ldquoAspergillus fumigatus NITDGPKA3provides for increased cellulase productionrdquo International Jour-nal of Chemical Engineering vol 2014 9 pages 2014

[35] V V Gilna and K M Khaleel ldquoCellulase enzyme activity ofAspergillus fumigatus from mangrove soil on lignocellulosicsubstraterdquo Recent Research in Science and Technology vol 3 no1 pp 132ndash134 2011

[36] G E Aboul-Fotouh G M El-Garhy H H Azzaz A MAbd El-Mola and G A Mousa ldquoFungal cellulase production

optimisation and its utilisation in Goatrsquos relations degradationrdquoAsian Journal of Animal and Veterinary Advances vol 11 no 12pp 824ndash831 2016

[37] TNcube R LHoward EKAbotsi E L J vanRensburg and INcube ldquoJatropha curcas seed cake as substrate for production ofxylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentationrdquo Industrial Crops and Products vol 37 no 1pp 118ndash123 2012

[38] J C Stewart and J B Parry ldquoFactors influencing the productionof cellulase by Aspergillus fumigatus (Fresenius)rdquo Journal ofGeneral Microbiology vol 125 no 1 pp 33ndash39 1981

[39] H Leghlimi Z Meraihi H Boukhalfa-Lezzar E Copinet andF Duchiron ldquoProduction and characterization of cellulolyticactivities produced by Trichoderma longibrachiatum (GHL)rdquoAfrican Journal of Biotechnology vol 12 no 5 pp 465ndash475 2013

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

6 Enzyme Research

0123456789

10

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Fpase 30 degCFpase 35 degCFpase 40 degC

Tota

l cel

lula

se ac

tivity

(FPU

ml)

Figure 5 Effect of incubation temperature on the production of cellulase (total cellulase activity) by fungal strains at initial medium pH 65

0

1

2

3

4

5

6

7

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Fpase 30 degCFpase 35 degCFpase 40 degC

Tota

l cel

lula

se ac

tivity

(FPU

ml)

Figure 6 Effect of incubation temperature on the production of cellulases (total cellulase activity) by fungal strains at initial medium pH 70

temperature In a medium with an initial pH of 65 onlyA fumigatus LMLPS 13-1 and T harzianum LMLBP07 13-5produced high levels of 120573-glucosidase activity 240 Uml and250 Uml respectively at 30∘C An increase in temperaturefrom 30 to 40∘C led to improved production of 120573-glucosidaseby some fungal species For instance A fumigatus LMLBP0613-3 showed 5-fold increase ie from 54 to 254 Uml of120573-glucosidase activity at 40∘C and 52-fold increase of 120573-glucosidase at 35∘C (Figure 9)

The production of 120573-glucosidase by T harzianumLMLBP07 13-5 was optimal between 35 and 40∘C whilstT longibrachiatum LMLSAUL 14-1 produced 120573-glucosidaseoptimally at 40∘C A fumigatus LMLPS 13-4 attained maxi-mum 120573-glucosidase activity at 30∘C (Figure 9) Generally at40∘C the production of 120573-glucosidase by all fungal specieswas much higher than at 30∘C with the exception of Afumigatus LMLPS 13-4 In a medium with initial pH 70 anincrease in temperature led to improvement in the produc-tion of 120573-glucosidase (Figure 10) A maximum level of 120573-glucosidase level was attained at 35∘CwithT longibrachiatumLMLSAUL 14-1 and T harzianum LMLBP07 13-5 showing

155-fold increase and 113-fold increase respectively Afumigatus LMLPS 13-4 and A fumigatus LMLPS 13-1 alsoshowed an increase of 120573-glucosidase activity at 35∘C (176-fold and 16-fold respectively) A fumigatus LMLBP06 13-3showed a proportional increase of 127-fold with maximum120573-glucosidase produced at 40∘C Conversely 40∘C led to a126-fold decrease of 120573-glucosidase produced byA fumigatusLMLBS02 13 - 2 (Figure 10)

Generally 120573-glucosidase activity was the highest at 35∘Cand pH 70 for all strains tested

4 Discussion

41 Fungal Screening Fungi and bacteria are associated withsoil and decaying plant materials In nature fungi colonisethe plant debris and in symbiotic relationship with othermicroorganisms they secrete an assortment of proteinsand a complex of hydrolytic enzymes to hydrolyse plantpolysaccharides for their survival However not all themicroorganisms are able to secrete significant amount ofhydrolytic enzymes for biotechnological applications Hence

Enzyme Research 7

0

5

10

15

20

25

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Endoglucanase 30 degCEndoglucanase 35 degCEndoglucanase 40 degC

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 7 Effect of incubation temperature on the production of endoglucanase by fungal strains at initial medium pH 65

0

5

10

15

20

25

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Endoglucanase 30 degCEndoglucanase 35 degCEndoglucanase 40 degC

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 8 Effect of incubation temperature on the production of endoglucanase by fungal strains at initial medium pH 70

0

5

10

15

20

25

30

35

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 9 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial medium pH 65

there is a need to screen and select hypersecreting hydrolyticenzymes In this study three different methods for screeningfor cellulolytic activity (1)microbial growth on cellulose agar(2) clearing of cellulose in agar and (3) reducing sugar pro-duction (or glucose) were performed Growth on cellulose

containing agar was useful for isolation of cellulolytic fungalstrains Ten fungal strains were selected based on fast andabundant growth on Avicel agar plates Several authors havealso reported the use of microcrystalline cellulose to screenfor cellulase producing microorganisms [21ndash23] CMC-CR

8 Enzyme Research

05

10152025303540

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 10 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial pH 70

(001) agar plate has been used to detect endoglucanaseactivity as indicated by the presence of a pale yellow zonearound the colony According to Yoon et al [13] the platescreening methods with dye coupled substrates provides arelatively straight forward and easy tool for specific detectionof fungi that produce endoglucanase Larger halos are theresults of higher enzyme activity [2] However at high CR dyeconcentrations fungal growth is suppressed

42 Fungal Identification The application of rDNA genesfor identification of fungal species is based on the detectionof conserved sequences in 58S rDNA and 28S rDNA thatenables the amplification of the ITS2 region between them[24] The identification of the fungal species based on theamplification of the ribosomal ITS region of the ribosomalDNA revealed that the organisms are A fumigatus strainsT harzianum and T longibrachiatum The ITS regions areregarded as primary DNA barcode for the fungal kingdomand exhibit high reliability [25]

43 Cellulases Production and Optimisation Subsequent toscreening is process optimisation with various factors such asnutrient requirements temperature pH and agitation speedbeen investigated (Gautam et al 2010 [26]) In this studythe effect of initial pH and temperatures on the productionlevels of cellulase were investigated Fungal strains belongingto Trichoderma and Aspergillus exhibited the potential toproduce cellulase over a pH 45-70 and temperature (30-40) range although the cellulase production levels variedfrom one fungal strain to another under the conditionsused It has been reported that expression of fungal genesis regulated by extracellular pH and many fungi exhibitgrowth and enzyme secretion over a wide pH range [27]Temperature also influences microbial growth and enzymeproduction [20 28] and an optimal environment conduciveto the production of cellulase will also be influenced bymedianutritional composition [26]

The initial pH of 45-55 drastically reduced the levelsof cellulases and endoglucanases produced by all strainsThis implied that acidity of the medium possibly affected

fungal growth and metabolism Acidic medium pH effect onthe production of cellulases by Trichoderma and Aspergilluswas also reported by Gautam et al [29] and Delabona etal [23] Contrary to these results other authors reportedsuch acidic condition to be favourable for production ofcellulases [22 30ndash34] Our results showed that a less acidicmedium with a pH 55 and above favoured the productionof cellulases by all Trichoderma and Aspergillus strains withoptimum production occurring at a pH of 65 Beyond pH65 a drastic decrease in cellulases levels was observed Gilnaamp Khaleel [35] and Gautam et al ([29] 2010) also reportedmaximum production of cellulases at pH 65 Aboul-Fotouhet al [36] reported maximum production of cellulases byAspergillus niger at initial medium pH of 6 when cultured on10 rice straw Other findings by Ncube et al [37] showed nosignificant differences in the production of endoglucanasesby A niger FGSCA 733 over a pH range of 3-7These findingsillustrate that an optimum pH for maximum production ofcellulase is dependent on fungal species and to some extenton the particular strain evaluated

There was effect of temperature on the productionof cellulases The production of cellulases decreased withincreasing temperature At 40∘C a significant reduction inendoglucanases activity was observed in all strains tested(Figures 8 and 9) On the contrary Ncube et al [37]reported maximum endoglucanase activity at 40∘C by Aniger FGSCA 733 Other studies reported optimal cellulaseand endoglucanase production at 45∘C [29] and 60∘C [2238] A temperature of 40∘C did not significantly affect 120573-glucosidase production (Figures 9 and 10) Leghlili et al(2013) reported the production of cellulases endoglucanasesand 120573-glucosidase T longibrachiatum (GHL) at both 30∘Cand 35∘C with higher production levels obtained at 35∘COther authors reported maximum production levels of 120573-glucosidase by T longibrachiatum at 35∘C [39] and A nigerMS82 at 25∘C [30] Aspergillus niger MS82 also producedsufficient endoglucanases at 30∘C and 35∘C in acidic initialmedium pH 40 [30]

These discrepancies with regard to the optimum initialpH and production temperature on the levels of cellulases as

Enzyme Research 9

measured by filter paper activity (FPUml) can be attributedto genetic make-up of the fungal strains as a result ofadaptations to different habitats Furthermore our reportedhigh levels of cellulases endoglucanases and low level of 120573-glucosidase by the Trichoderma isolates and vice versa highlevels of 120573-glucosidase by A fumigatus are in agreement withthe reported levels of these fungal species by Stewart andParry [38]

5 Conclusion

In conclusion all the Trichoderma and Aspergillus strainsproduced substantial levels of all the three enzymes (ieexoglucanase endoglucanase and 120573-glucosidase) requiredfor complete hydrolysis of cellulosic material Trichodermastrains produced higher cellulases and endoglucanases levelswhile A fumigatus strains produced higher 120573-glucosidaselevels The production of all the cellulases componentsseemed to be strongly influenced by the interactive effectof initial pH and incubation temperature on the microor-ganisms Hence the observed maximum production ofcellulases by the fungi depended on the chosen initial pHof the medium and incubation temperature The cellulasesproduced by these fungal strainswill be assessed for efficiencyin hydrolysing agricultural lignocellulose wastes includingbanana pseudostem for the production of bioethanol

Data Availability

The experimental data used to support the findings of thisstudy are available from the corresponding author uponrequest

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors are grateful to the National Research Foundation(NRF) and the Flemish Inter-University Council (VLIR-UOS) for the financial support towards the research

References

[1] A C OrsquoSullivan ldquoCellulose the structure slowly unravelsrdquoCellulose vol 4 no 3 pp 173ndash207 1997

[2] Y-H P Zhang M E Himmel and J R Mielenz ldquoOutlookfor cellulase improvement screening and selection strategiesrdquoBiotechnology Advances vol 24 no 5 pp 452ndash481 2006

[3] P Beguin and J P Aubert ldquoThe biological degradation ofcelluloserdquo FEMS Microbiology Review vol 13 pp 25ndash28 1994

[4] M K Bhat and S Bhat ldquoCellulose degrading enzymes and theirpotential industrial applicationsrdquo Biotechnology Advances vol15 no 3-4 pp 583ndash620 1997

[5] J Zhuang M A Marchant S E Nokes and H J StrobelldquoEconomic analysis of cellulase production methods for bio-ethanolrdquo Applied Engineering in Agriculture vol 23 no 5 pp679ndash687 2007

[6] S T Merino and J Cherry ldquoProgress and challenges in enzymedevelopment for biomass utilizationrdquo Advances in BiochemicalEngineeringBiotechnology vol 108 pp 95ndash120 2007

[7] M Dashtban H Schraft and W Qin ldquoFungal bioconversionof lignocellulosic residues Opportunities amp perspectivesrdquo Inter-national Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009

[8] B S Padam H S Tin F Y Chye and M I Abdullah ldquoBananaby-products an under-utilized renewable food biomass withgreat potentialrdquo Journal of Food Science and Technology vol 51no 12 pp 3527ndash3545 2014

[9] H P S Abdul Khalil M S Alwani and A K M OmarldquoChemical composition anatomy lignin distribution and cellwall structure ofMalaysian plantwaste fibresrdquoBioResources vol1 no 2 pp 220ndash232 2006

[10] K Li S Fu H Zhan Y Zhan and L A Lucia ldquoAnalysis of thechemical composition and morphological structure of bananapseudostemrdquo Bioresources vol 5 no 2 pp 576ndash585 2010

[11] K Ibatsam S H Muhammad M Sobia and M Irum ldquoIso-lation and screening of highly cellulolytic filamentous fungirdquoJournal of Applied Science and Environmental Management vol16 no 3 pp 223ndash226 2012

[12] G Nunez-Trujillo R Cabrera A Cosoveanu T T Martin CGimenez and G Nunez-Trujillo ldquoSurvey of banana endophyticfungi isolated in artificial culture media from an appliedviewpointrdquo Journal of Horticulture Forestry and Biotechnologyvol 17 no 2 pp 22ndash25 2013

[13] J H Yoon J E Park D Y Suh S B Hong S J Ko and S HKim ldquoComparison of dyes for easy detection of extracellularcellulases in fungirdquoMycobiology vol 35 no 1 pp 21ndash24 2007

[14] A B Peixoto Study of the production of enzymes and gumsby wild yeasts collected in various regions of Brazil [MSThesis] Faculty of Food Engineering University of CampinasCampinas Brazil 2006

[15] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods AndApplications M A Innis D H Gelfand J J Sninsky et al Edspp 315ndash322 Academic Press Inc New York NY USA 1990

[16] B Benoliel F A Goncalves Torres and L M P de MoraesldquoA novel promising Trichoderma harzianum strain for theproduction of a cellulolytic complex using sugarcane bagasse innaturardquo SpringerPlus vol 2 p 256 2013

[17] D Herr ldquoSecretion of cellulase and 120573-glucosidase byTricho-derma viride ITCC-1433 in submerged culture on differentsubstratesrdquo Biotechnology and Bioengineering vol 21 no 8 pp1361ndash1371 1979

[18] A S Lakshmi and G Narasimha ldquoProduction of cellulases byfungal cultures isolated from forest litter soilrdquo Annals of ForestResearch vol 55 no 1 pp 85ndash92 2012

[19] X Han W Song G Liu Z Li P Yang and Y Qu ldquoImprov-ing cellulase productivity of Penicillium oxalicum RE-10 byrepeated fed-batch fermentation strategyrdquo Bioresource Technol-ogy vol 227 pp 155ndash163 2017

[20] M Rubeena K Neethu S Sajith et al ldquoLignocellulolyticactivities of a novel strain of TrichodermaharzianumrdquoAdvancesin Bioscience and Biotechnology vol 4 pp 212ndash221 2013

[21] A L Grigorevski-Lima F N M Da Vinha D T Souzaet al ldquoAspergillus fumigatus thermophilic and acidophilicendoglucanasesrdquo Applied Biochemistry and Biotechnology vol155 no 1-3 pp 321ndash329 2009

10 Enzyme Research

[22] J P Andrade A S D R Bispo P A S Marbach and RP Do Nascimento ldquoProduction and partial characterizationof cellulases from Trichoderma sp IS-05 isolated from SandyCoastal plains of Northeast Brazilrdquo Enzyme Research vol 2011Article ID 167248 7 pages 2011

[23] P D S Delabona C S Farinas M R da Silva S F Azzoniand J G D C Pradella ldquoUse of a new Trichoderma harzianumstrain isolated from the Amazon rainforest with pretreatedsugar cane bagasse for on-site cellulase productionrdquo BioresourceTechnology vol 107 pp 517ndash521 2012

[24] C Y Turenne S E Sanche D J Hoban J A Karlowskyand A M Kabani ldquoRapid identification of fungi by using theITS2 genetic region and an automated fluorescent capillaryelectrophoresis systemrdquo Journal of Clinical Microbiology vol 37no 6 pp 1846ndash1851 1999

[25] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcode marker for fungirdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 109 no 27 pp6241ndash6246 2012

[26] M Shahriarinour M N A Wahab R Mohamad S Mustafaand A B Ariff ldquoEffect of medium composition and culturalcondition on cellulase production by Aspergillus terreusrdquoAfrican Journal of Biotechnology vol 10 no 38 pp 7459ndash74672011

[27] D B Archer and J F Peberdy ldquoThe molecular biology ofsecreted enzyme production by fungirdquo Critical Reviews inBiotechnology vol 17 no 4 pp 273ndash306 1997

[28] R Soni A Nazir and B S Chadha ldquoOptimization of cellulaseproduction by a versatile Aspergillus fumigatus fresenius strain(AMA) capable of efficient deinking and enzymatic hydrolysisof Solka floc and bagasserdquo Industrial Crops and Products vol 31no 2 pp 277ndash283 2010

[29] S P Gautam P S Bundela A K Pandey J Khan M KAwasthi and S Sarsaiya ldquoOptimization for the production ofcellulase enzyme from Municipal solid waste residue by twonovel cellulolytic fungirdquo Biotechnology Research Internationalpp 1ndash8 2011

[30] M Sohail R Siddiqi A Ahmad and S A Khan ldquoCellulaseproduction from Aspergillus niger MS82 effect of temperatureand pHrdquo New Biotechnology vol 25 no 6 pp 437ndash441 2009

[31] S Ahmed A Bashir H Saleem M Saadia and A JamilldquoProduction and purification of cellulose-degrading enzymesfrom a filamentous fungus Trichoderma harzianumrdquo PakistanJournal of Botany vol 41 no 3 pp 1411ndash1419 2009

[32] A Das T Paul S K Halder et al ldquoStudy on regulation ofgrowth and biosynthesis of cellulolytic enzymes from newlyisolated Aspergillus fumigatus ABK9rdquo Polish Journal of Micro-biology vol 62 no 1 pp 31ndash43 2013

[33] S Shafique R Bajwa and S Shafique ldquoCellulase biosynthesis byselected Trichoderma speciesrdquo Pakistan Journal of Botany vol41 no 2 pp 907ndash916 2009

[34] N Sarkar and K Aikat ldquoAspergillus fumigatus NITDGPKA3provides for increased cellulase productionrdquo International Jour-nal of Chemical Engineering vol 2014 9 pages 2014

[35] V V Gilna and K M Khaleel ldquoCellulase enzyme activity ofAspergillus fumigatus from mangrove soil on lignocellulosicsubstraterdquo Recent Research in Science and Technology vol 3 no1 pp 132ndash134 2011

[36] G E Aboul-Fotouh G M El-Garhy H H Azzaz A MAbd El-Mola and G A Mousa ldquoFungal cellulase production

optimisation and its utilisation in Goatrsquos relations degradationrdquoAsian Journal of Animal and Veterinary Advances vol 11 no 12pp 824ndash831 2016

[37] TNcube R LHoward EKAbotsi E L J vanRensburg and INcube ldquoJatropha curcas seed cake as substrate for production ofxylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentationrdquo Industrial Crops and Products vol 37 no 1pp 118ndash123 2012

[38] J C Stewart and J B Parry ldquoFactors influencing the productionof cellulase by Aspergillus fumigatus (Fresenius)rdquo Journal ofGeneral Microbiology vol 125 no 1 pp 33ndash39 1981

[39] H Leghlimi Z Meraihi H Boukhalfa-Lezzar E Copinet andF Duchiron ldquoProduction and characterization of cellulolyticactivities produced by Trichoderma longibrachiatum (GHL)rdquoAfrican Journal of Biotechnology vol 12 no 5 pp 465ndash475 2013

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Enzyme Research 7

0

5

10

15

20

25

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Endoglucanase 30 degCEndoglucanase 35 degCEndoglucanase 40 degC

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 7 Effect of incubation temperature on the production of endoglucanase by fungal strains at initial medium pH 65

0

5

10

15

20

25

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Endoglucanase 30 degCEndoglucanase 35 degCEndoglucanase 40 degC

Endo

gluc

anas

e act

ivity

(Um

l)

Figure 8 Effect of incubation temperature on the production of endoglucanase by fungal strains at initial medium pH 70

0

5

10

15

20

25

30

35

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 9 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial medium pH 65

there is a need to screen and select hypersecreting hydrolyticenzymes In this study three different methods for screeningfor cellulolytic activity (1)microbial growth on cellulose agar(2) clearing of cellulose in agar and (3) reducing sugar pro-duction (or glucose) were performed Growth on cellulose

containing agar was useful for isolation of cellulolytic fungalstrains Ten fungal strains were selected based on fast andabundant growth on Avicel agar plates Several authors havealso reported the use of microcrystalline cellulose to screenfor cellulase producing microorganisms [21ndash23] CMC-CR

8 Enzyme Research

05

10152025303540

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 10 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial pH 70

(001) agar plate has been used to detect endoglucanaseactivity as indicated by the presence of a pale yellow zonearound the colony According to Yoon et al [13] the platescreening methods with dye coupled substrates provides arelatively straight forward and easy tool for specific detectionof fungi that produce endoglucanase Larger halos are theresults of higher enzyme activity [2] However at high CR dyeconcentrations fungal growth is suppressed

42 Fungal Identification The application of rDNA genesfor identification of fungal species is based on the detectionof conserved sequences in 58S rDNA and 28S rDNA thatenables the amplification of the ITS2 region between them[24] The identification of the fungal species based on theamplification of the ribosomal ITS region of the ribosomalDNA revealed that the organisms are A fumigatus strainsT harzianum and T longibrachiatum The ITS regions areregarded as primary DNA barcode for the fungal kingdomand exhibit high reliability [25]

43 Cellulases Production and Optimisation Subsequent toscreening is process optimisation with various factors such asnutrient requirements temperature pH and agitation speedbeen investigated (Gautam et al 2010 [26]) In this studythe effect of initial pH and temperatures on the productionlevels of cellulase were investigated Fungal strains belongingto Trichoderma and Aspergillus exhibited the potential toproduce cellulase over a pH 45-70 and temperature (30-40) range although the cellulase production levels variedfrom one fungal strain to another under the conditionsused It has been reported that expression of fungal genesis regulated by extracellular pH and many fungi exhibitgrowth and enzyme secretion over a wide pH range [27]Temperature also influences microbial growth and enzymeproduction [20 28] and an optimal environment conduciveto the production of cellulase will also be influenced bymedianutritional composition [26]

The initial pH of 45-55 drastically reduced the levelsof cellulases and endoglucanases produced by all strainsThis implied that acidity of the medium possibly affected

fungal growth and metabolism Acidic medium pH effect onthe production of cellulases by Trichoderma and Aspergilluswas also reported by Gautam et al [29] and Delabona etal [23] Contrary to these results other authors reportedsuch acidic condition to be favourable for production ofcellulases [22 30ndash34] Our results showed that a less acidicmedium with a pH 55 and above favoured the productionof cellulases by all Trichoderma and Aspergillus strains withoptimum production occurring at a pH of 65 Beyond pH65 a drastic decrease in cellulases levels was observed Gilnaamp Khaleel [35] and Gautam et al ([29] 2010) also reportedmaximum production of cellulases at pH 65 Aboul-Fotouhet al [36] reported maximum production of cellulases byAspergillus niger at initial medium pH of 6 when cultured on10 rice straw Other findings by Ncube et al [37] showed nosignificant differences in the production of endoglucanasesby A niger FGSCA 733 over a pH range of 3-7These findingsillustrate that an optimum pH for maximum production ofcellulase is dependent on fungal species and to some extenton the particular strain evaluated

There was effect of temperature on the productionof cellulases The production of cellulases decreased withincreasing temperature At 40∘C a significant reduction inendoglucanases activity was observed in all strains tested(Figures 8 and 9) On the contrary Ncube et al [37]reported maximum endoglucanase activity at 40∘C by Aniger FGSCA 733 Other studies reported optimal cellulaseand endoglucanase production at 45∘C [29] and 60∘C [2238] A temperature of 40∘C did not significantly affect 120573-glucosidase production (Figures 9 and 10) Leghlili et al(2013) reported the production of cellulases endoglucanasesand 120573-glucosidase T longibrachiatum (GHL) at both 30∘Cand 35∘C with higher production levels obtained at 35∘COther authors reported maximum production levels of 120573-glucosidase by T longibrachiatum at 35∘C [39] and A nigerMS82 at 25∘C [30] Aspergillus niger MS82 also producedsufficient endoglucanases at 30∘C and 35∘C in acidic initialmedium pH 40 [30]

These discrepancies with regard to the optimum initialpH and production temperature on the levels of cellulases as

Enzyme Research 9

measured by filter paper activity (FPUml) can be attributedto genetic make-up of the fungal strains as a result ofadaptations to different habitats Furthermore our reportedhigh levels of cellulases endoglucanases and low level of 120573-glucosidase by the Trichoderma isolates and vice versa highlevels of 120573-glucosidase by A fumigatus are in agreement withthe reported levels of these fungal species by Stewart andParry [38]

5 Conclusion

In conclusion all the Trichoderma and Aspergillus strainsproduced substantial levels of all the three enzymes (ieexoglucanase endoglucanase and 120573-glucosidase) requiredfor complete hydrolysis of cellulosic material Trichodermastrains produced higher cellulases and endoglucanases levelswhile A fumigatus strains produced higher 120573-glucosidaselevels The production of all the cellulases componentsseemed to be strongly influenced by the interactive effectof initial pH and incubation temperature on the microor-ganisms Hence the observed maximum production ofcellulases by the fungi depended on the chosen initial pHof the medium and incubation temperature The cellulasesproduced by these fungal strainswill be assessed for efficiencyin hydrolysing agricultural lignocellulose wastes includingbanana pseudostem for the production of bioethanol

Data Availability

The experimental data used to support the findings of thisstudy are available from the corresponding author uponrequest

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors are grateful to the National Research Foundation(NRF) and the Flemish Inter-University Council (VLIR-UOS) for the financial support towards the research

References

[1] A C OrsquoSullivan ldquoCellulose the structure slowly unravelsrdquoCellulose vol 4 no 3 pp 173ndash207 1997

[2] Y-H P Zhang M E Himmel and J R Mielenz ldquoOutlookfor cellulase improvement screening and selection strategiesrdquoBiotechnology Advances vol 24 no 5 pp 452ndash481 2006

[3] P Beguin and J P Aubert ldquoThe biological degradation ofcelluloserdquo FEMS Microbiology Review vol 13 pp 25ndash28 1994

[4] M K Bhat and S Bhat ldquoCellulose degrading enzymes and theirpotential industrial applicationsrdquo Biotechnology Advances vol15 no 3-4 pp 583ndash620 1997

[5] J Zhuang M A Marchant S E Nokes and H J StrobelldquoEconomic analysis of cellulase production methods for bio-ethanolrdquo Applied Engineering in Agriculture vol 23 no 5 pp679ndash687 2007

[6] S T Merino and J Cherry ldquoProgress and challenges in enzymedevelopment for biomass utilizationrdquo Advances in BiochemicalEngineeringBiotechnology vol 108 pp 95ndash120 2007

[7] M Dashtban H Schraft and W Qin ldquoFungal bioconversionof lignocellulosic residues Opportunities amp perspectivesrdquo Inter-national Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009

[8] B S Padam H S Tin F Y Chye and M I Abdullah ldquoBananaby-products an under-utilized renewable food biomass withgreat potentialrdquo Journal of Food Science and Technology vol 51no 12 pp 3527ndash3545 2014

[9] H P S Abdul Khalil M S Alwani and A K M OmarldquoChemical composition anatomy lignin distribution and cellwall structure ofMalaysian plantwaste fibresrdquoBioResources vol1 no 2 pp 220ndash232 2006

[10] K Li S Fu H Zhan Y Zhan and L A Lucia ldquoAnalysis of thechemical composition and morphological structure of bananapseudostemrdquo Bioresources vol 5 no 2 pp 576ndash585 2010

[11] K Ibatsam S H Muhammad M Sobia and M Irum ldquoIso-lation and screening of highly cellulolytic filamentous fungirdquoJournal of Applied Science and Environmental Management vol16 no 3 pp 223ndash226 2012

[12] G Nunez-Trujillo R Cabrera A Cosoveanu T T Martin CGimenez and G Nunez-Trujillo ldquoSurvey of banana endophyticfungi isolated in artificial culture media from an appliedviewpointrdquo Journal of Horticulture Forestry and Biotechnologyvol 17 no 2 pp 22ndash25 2013

[13] J H Yoon J E Park D Y Suh S B Hong S J Ko and S HKim ldquoComparison of dyes for easy detection of extracellularcellulases in fungirdquoMycobiology vol 35 no 1 pp 21ndash24 2007

[14] A B Peixoto Study of the production of enzymes and gumsby wild yeasts collected in various regions of Brazil [MSThesis] Faculty of Food Engineering University of CampinasCampinas Brazil 2006

[15] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods AndApplications M A Innis D H Gelfand J J Sninsky et al Edspp 315ndash322 Academic Press Inc New York NY USA 1990

[16] B Benoliel F A Goncalves Torres and L M P de MoraesldquoA novel promising Trichoderma harzianum strain for theproduction of a cellulolytic complex using sugarcane bagasse innaturardquo SpringerPlus vol 2 p 256 2013

[17] D Herr ldquoSecretion of cellulase and 120573-glucosidase byTricho-derma viride ITCC-1433 in submerged culture on differentsubstratesrdquo Biotechnology and Bioengineering vol 21 no 8 pp1361ndash1371 1979

[18] A S Lakshmi and G Narasimha ldquoProduction of cellulases byfungal cultures isolated from forest litter soilrdquo Annals of ForestResearch vol 55 no 1 pp 85ndash92 2012

[19] X Han W Song G Liu Z Li P Yang and Y Qu ldquoImprov-ing cellulase productivity of Penicillium oxalicum RE-10 byrepeated fed-batch fermentation strategyrdquo Bioresource Technol-ogy vol 227 pp 155ndash163 2017

[20] M Rubeena K Neethu S Sajith et al ldquoLignocellulolyticactivities of a novel strain of TrichodermaharzianumrdquoAdvancesin Bioscience and Biotechnology vol 4 pp 212ndash221 2013

[21] A L Grigorevski-Lima F N M Da Vinha D T Souzaet al ldquoAspergillus fumigatus thermophilic and acidophilicendoglucanasesrdquo Applied Biochemistry and Biotechnology vol155 no 1-3 pp 321ndash329 2009

10 Enzyme Research

[22] J P Andrade A S D R Bispo P A S Marbach and RP Do Nascimento ldquoProduction and partial characterizationof cellulases from Trichoderma sp IS-05 isolated from SandyCoastal plains of Northeast Brazilrdquo Enzyme Research vol 2011Article ID 167248 7 pages 2011

[23] P D S Delabona C S Farinas M R da Silva S F Azzoniand J G D C Pradella ldquoUse of a new Trichoderma harzianumstrain isolated from the Amazon rainforest with pretreatedsugar cane bagasse for on-site cellulase productionrdquo BioresourceTechnology vol 107 pp 517ndash521 2012

[24] C Y Turenne S E Sanche D J Hoban J A Karlowskyand A M Kabani ldquoRapid identification of fungi by using theITS2 genetic region and an automated fluorescent capillaryelectrophoresis systemrdquo Journal of Clinical Microbiology vol 37no 6 pp 1846ndash1851 1999

[25] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcode marker for fungirdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 109 no 27 pp6241ndash6246 2012

[26] M Shahriarinour M N A Wahab R Mohamad S Mustafaand A B Ariff ldquoEffect of medium composition and culturalcondition on cellulase production by Aspergillus terreusrdquoAfrican Journal of Biotechnology vol 10 no 38 pp 7459ndash74672011

[27] D B Archer and J F Peberdy ldquoThe molecular biology ofsecreted enzyme production by fungirdquo Critical Reviews inBiotechnology vol 17 no 4 pp 273ndash306 1997

[28] R Soni A Nazir and B S Chadha ldquoOptimization of cellulaseproduction by a versatile Aspergillus fumigatus fresenius strain(AMA) capable of efficient deinking and enzymatic hydrolysisof Solka floc and bagasserdquo Industrial Crops and Products vol 31no 2 pp 277ndash283 2010

[29] S P Gautam P S Bundela A K Pandey J Khan M KAwasthi and S Sarsaiya ldquoOptimization for the production ofcellulase enzyme from Municipal solid waste residue by twonovel cellulolytic fungirdquo Biotechnology Research Internationalpp 1ndash8 2011

[30] M Sohail R Siddiqi A Ahmad and S A Khan ldquoCellulaseproduction from Aspergillus niger MS82 effect of temperatureand pHrdquo New Biotechnology vol 25 no 6 pp 437ndash441 2009

[31] S Ahmed A Bashir H Saleem M Saadia and A JamilldquoProduction and purification of cellulose-degrading enzymesfrom a filamentous fungus Trichoderma harzianumrdquo PakistanJournal of Botany vol 41 no 3 pp 1411ndash1419 2009

[32] A Das T Paul S K Halder et al ldquoStudy on regulation ofgrowth and biosynthesis of cellulolytic enzymes from newlyisolated Aspergillus fumigatus ABK9rdquo Polish Journal of Micro-biology vol 62 no 1 pp 31ndash43 2013

[33] S Shafique R Bajwa and S Shafique ldquoCellulase biosynthesis byselected Trichoderma speciesrdquo Pakistan Journal of Botany vol41 no 2 pp 907ndash916 2009

[34] N Sarkar and K Aikat ldquoAspergillus fumigatus NITDGPKA3provides for increased cellulase productionrdquo International Jour-nal of Chemical Engineering vol 2014 9 pages 2014

[35] V V Gilna and K M Khaleel ldquoCellulase enzyme activity ofAspergillus fumigatus from mangrove soil on lignocellulosicsubstraterdquo Recent Research in Science and Technology vol 3 no1 pp 132ndash134 2011

[36] G E Aboul-Fotouh G M El-Garhy H H Azzaz A MAbd El-Mola and G A Mousa ldquoFungal cellulase production

optimisation and its utilisation in Goatrsquos relations degradationrdquoAsian Journal of Animal and Veterinary Advances vol 11 no 12pp 824ndash831 2016

[37] TNcube R LHoward EKAbotsi E L J vanRensburg and INcube ldquoJatropha curcas seed cake as substrate for production ofxylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentationrdquo Industrial Crops and Products vol 37 no 1pp 118ndash123 2012

[38] J C Stewart and J B Parry ldquoFactors influencing the productionof cellulase by Aspergillus fumigatus (Fresenius)rdquo Journal ofGeneral Microbiology vol 125 no 1 pp 33ndash39 1981

[39] H Leghlimi Z Meraihi H Boukhalfa-Lezzar E Copinet andF Duchiron ldquoProduction and characterization of cellulolyticactivities produced by Trichoderma longibrachiatum (GHL)rdquoAfrican Journal of Biotechnology vol 12 no 5 pp 465ndash475 2013

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

8 Enzyme Research

05

10152025303540

T harzianumLMLBP07 13 - 5

T longibrachiatumLMLSAUL 14 -1

A fumigatusLMLBP06 13 - 3

A fumigatusLMLBS02 13 - 2

A fumigatusLMLPS 13 - 4

A fumigatusLMLPS 13 - 1

Fungal isolates

Β-glucosidase 30 degCΒ-glucosidase 35 degCΒ-glucosidase 40 degC

-g

luco

sidas

e act

ivity

(Um

l)

Figure 10 Effect of incubation temperature on the production of 120573-glucosidase by fungal strains at initial pH 70

(001) agar plate has been used to detect endoglucanaseactivity as indicated by the presence of a pale yellow zonearound the colony According to Yoon et al [13] the platescreening methods with dye coupled substrates provides arelatively straight forward and easy tool for specific detectionof fungi that produce endoglucanase Larger halos are theresults of higher enzyme activity [2] However at high CR dyeconcentrations fungal growth is suppressed

42 Fungal Identification The application of rDNA genesfor identification of fungal species is based on the detectionof conserved sequences in 58S rDNA and 28S rDNA thatenables the amplification of the ITS2 region between them[24] The identification of the fungal species based on theamplification of the ribosomal ITS region of the ribosomalDNA revealed that the organisms are A fumigatus strainsT harzianum and T longibrachiatum The ITS regions areregarded as primary DNA barcode for the fungal kingdomand exhibit high reliability [25]

43 Cellulases Production and Optimisation Subsequent toscreening is process optimisation with various factors such asnutrient requirements temperature pH and agitation speedbeen investigated (Gautam et al 2010 [26]) In this studythe effect of initial pH and temperatures on the productionlevels of cellulase were investigated Fungal strains belongingto Trichoderma and Aspergillus exhibited the potential toproduce cellulase over a pH 45-70 and temperature (30-40) range although the cellulase production levels variedfrom one fungal strain to another under the conditionsused It has been reported that expression of fungal genesis regulated by extracellular pH and many fungi exhibitgrowth and enzyme secretion over a wide pH range [27]Temperature also influences microbial growth and enzymeproduction [20 28] and an optimal environment conduciveto the production of cellulase will also be influenced bymedianutritional composition [26]

The initial pH of 45-55 drastically reduced the levelsof cellulases and endoglucanases produced by all strainsThis implied that acidity of the medium possibly affected

fungal growth and metabolism Acidic medium pH effect onthe production of cellulases by Trichoderma and Aspergilluswas also reported by Gautam et al [29] and Delabona etal [23] Contrary to these results other authors reportedsuch acidic condition to be favourable for production ofcellulases [22 30ndash34] Our results showed that a less acidicmedium with a pH 55 and above favoured the productionof cellulases by all Trichoderma and Aspergillus strains withoptimum production occurring at a pH of 65 Beyond pH65 a drastic decrease in cellulases levels was observed Gilnaamp Khaleel [35] and Gautam et al ([29] 2010) also reportedmaximum production of cellulases at pH 65 Aboul-Fotouhet al [36] reported maximum production of cellulases byAspergillus niger at initial medium pH of 6 when cultured on10 rice straw Other findings by Ncube et al [37] showed nosignificant differences in the production of endoglucanasesby A niger FGSCA 733 over a pH range of 3-7These findingsillustrate that an optimum pH for maximum production ofcellulase is dependent on fungal species and to some extenton the particular strain evaluated

There was effect of temperature on the productionof cellulases The production of cellulases decreased withincreasing temperature At 40∘C a significant reduction inendoglucanases activity was observed in all strains tested(Figures 8 and 9) On the contrary Ncube et al [37]reported maximum endoglucanase activity at 40∘C by Aniger FGSCA 733 Other studies reported optimal cellulaseand endoglucanase production at 45∘C [29] and 60∘C [2238] A temperature of 40∘C did not significantly affect 120573-glucosidase production (Figures 9 and 10) Leghlili et al(2013) reported the production of cellulases endoglucanasesand 120573-glucosidase T longibrachiatum (GHL) at both 30∘Cand 35∘C with higher production levels obtained at 35∘COther authors reported maximum production levels of 120573-glucosidase by T longibrachiatum at 35∘C [39] and A nigerMS82 at 25∘C [30] Aspergillus niger MS82 also producedsufficient endoglucanases at 30∘C and 35∘C in acidic initialmedium pH 40 [30]

These discrepancies with regard to the optimum initialpH and production temperature on the levels of cellulases as

Enzyme Research 9

measured by filter paper activity (FPUml) can be attributedto genetic make-up of the fungal strains as a result ofadaptations to different habitats Furthermore our reportedhigh levels of cellulases endoglucanases and low level of 120573-glucosidase by the Trichoderma isolates and vice versa highlevels of 120573-glucosidase by A fumigatus are in agreement withthe reported levels of these fungal species by Stewart andParry [38]

5 Conclusion

In conclusion all the Trichoderma and Aspergillus strainsproduced substantial levels of all the three enzymes (ieexoglucanase endoglucanase and 120573-glucosidase) requiredfor complete hydrolysis of cellulosic material Trichodermastrains produced higher cellulases and endoglucanases levelswhile A fumigatus strains produced higher 120573-glucosidaselevels The production of all the cellulases componentsseemed to be strongly influenced by the interactive effectof initial pH and incubation temperature on the microor-ganisms Hence the observed maximum production ofcellulases by the fungi depended on the chosen initial pHof the medium and incubation temperature The cellulasesproduced by these fungal strainswill be assessed for efficiencyin hydrolysing agricultural lignocellulose wastes includingbanana pseudostem for the production of bioethanol

Data Availability

The experimental data used to support the findings of thisstudy are available from the corresponding author uponrequest

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors are grateful to the National Research Foundation(NRF) and the Flemish Inter-University Council (VLIR-UOS) for the financial support towards the research

References

[1] A C OrsquoSullivan ldquoCellulose the structure slowly unravelsrdquoCellulose vol 4 no 3 pp 173ndash207 1997

[2] Y-H P Zhang M E Himmel and J R Mielenz ldquoOutlookfor cellulase improvement screening and selection strategiesrdquoBiotechnology Advances vol 24 no 5 pp 452ndash481 2006

[3] P Beguin and J P Aubert ldquoThe biological degradation ofcelluloserdquo FEMS Microbiology Review vol 13 pp 25ndash28 1994

[4] M K Bhat and S Bhat ldquoCellulose degrading enzymes and theirpotential industrial applicationsrdquo Biotechnology Advances vol15 no 3-4 pp 583ndash620 1997

[5] J Zhuang M A Marchant S E Nokes and H J StrobelldquoEconomic analysis of cellulase production methods for bio-ethanolrdquo Applied Engineering in Agriculture vol 23 no 5 pp679ndash687 2007

[6] S T Merino and J Cherry ldquoProgress and challenges in enzymedevelopment for biomass utilizationrdquo Advances in BiochemicalEngineeringBiotechnology vol 108 pp 95ndash120 2007

[7] M Dashtban H Schraft and W Qin ldquoFungal bioconversionof lignocellulosic residues Opportunities amp perspectivesrdquo Inter-national Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009

[8] B S Padam H S Tin F Y Chye and M I Abdullah ldquoBananaby-products an under-utilized renewable food biomass withgreat potentialrdquo Journal of Food Science and Technology vol 51no 12 pp 3527ndash3545 2014

[9] H P S Abdul Khalil M S Alwani and A K M OmarldquoChemical composition anatomy lignin distribution and cellwall structure ofMalaysian plantwaste fibresrdquoBioResources vol1 no 2 pp 220ndash232 2006

[10] K Li S Fu H Zhan Y Zhan and L A Lucia ldquoAnalysis of thechemical composition and morphological structure of bananapseudostemrdquo Bioresources vol 5 no 2 pp 576ndash585 2010

[11] K Ibatsam S H Muhammad M Sobia and M Irum ldquoIso-lation and screening of highly cellulolytic filamentous fungirdquoJournal of Applied Science and Environmental Management vol16 no 3 pp 223ndash226 2012

[12] G Nunez-Trujillo R Cabrera A Cosoveanu T T Martin CGimenez and G Nunez-Trujillo ldquoSurvey of banana endophyticfungi isolated in artificial culture media from an appliedviewpointrdquo Journal of Horticulture Forestry and Biotechnologyvol 17 no 2 pp 22ndash25 2013

[13] J H Yoon J E Park D Y Suh S B Hong S J Ko and S HKim ldquoComparison of dyes for easy detection of extracellularcellulases in fungirdquoMycobiology vol 35 no 1 pp 21ndash24 2007

[14] A B Peixoto Study of the production of enzymes and gumsby wild yeasts collected in various regions of Brazil [MSThesis] Faculty of Food Engineering University of CampinasCampinas Brazil 2006

[15] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods AndApplications M A Innis D H Gelfand J J Sninsky et al Edspp 315ndash322 Academic Press Inc New York NY USA 1990

[16] B Benoliel F A Goncalves Torres and L M P de MoraesldquoA novel promising Trichoderma harzianum strain for theproduction of a cellulolytic complex using sugarcane bagasse innaturardquo SpringerPlus vol 2 p 256 2013

[17] D Herr ldquoSecretion of cellulase and 120573-glucosidase byTricho-derma viride ITCC-1433 in submerged culture on differentsubstratesrdquo Biotechnology and Bioengineering vol 21 no 8 pp1361ndash1371 1979

[18] A S Lakshmi and G Narasimha ldquoProduction of cellulases byfungal cultures isolated from forest litter soilrdquo Annals of ForestResearch vol 55 no 1 pp 85ndash92 2012

[19] X Han W Song G Liu Z Li P Yang and Y Qu ldquoImprov-ing cellulase productivity of Penicillium oxalicum RE-10 byrepeated fed-batch fermentation strategyrdquo Bioresource Technol-ogy vol 227 pp 155ndash163 2017

[20] M Rubeena K Neethu S Sajith et al ldquoLignocellulolyticactivities of a novel strain of TrichodermaharzianumrdquoAdvancesin Bioscience and Biotechnology vol 4 pp 212ndash221 2013

[21] A L Grigorevski-Lima F N M Da Vinha D T Souzaet al ldquoAspergillus fumigatus thermophilic and acidophilicendoglucanasesrdquo Applied Biochemistry and Biotechnology vol155 no 1-3 pp 321ndash329 2009

10 Enzyme Research

[22] J P Andrade A S D R Bispo P A S Marbach and RP Do Nascimento ldquoProduction and partial characterizationof cellulases from Trichoderma sp IS-05 isolated from SandyCoastal plains of Northeast Brazilrdquo Enzyme Research vol 2011Article ID 167248 7 pages 2011

[23] P D S Delabona C S Farinas M R da Silva S F Azzoniand J G D C Pradella ldquoUse of a new Trichoderma harzianumstrain isolated from the Amazon rainforest with pretreatedsugar cane bagasse for on-site cellulase productionrdquo BioresourceTechnology vol 107 pp 517ndash521 2012

[24] C Y Turenne S E Sanche D J Hoban J A Karlowskyand A M Kabani ldquoRapid identification of fungi by using theITS2 genetic region and an automated fluorescent capillaryelectrophoresis systemrdquo Journal of Clinical Microbiology vol 37no 6 pp 1846ndash1851 1999

[25] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcode marker for fungirdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 109 no 27 pp6241ndash6246 2012

[26] M Shahriarinour M N A Wahab R Mohamad S Mustafaand A B Ariff ldquoEffect of medium composition and culturalcondition on cellulase production by Aspergillus terreusrdquoAfrican Journal of Biotechnology vol 10 no 38 pp 7459ndash74672011

[27] D B Archer and J F Peberdy ldquoThe molecular biology ofsecreted enzyme production by fungirdquo Critical Reviews inBiotechnology vol 17 no 4 pp 273ndash306 1997

[28] R Soni A Nazir and B S Chadha ldquoOptimization of cellulaseproduction by a versatile Aspergillus fumigatus fresenius strain(AMA) capable of efficient deinking and enzymatic hydrolysisof Solka floc and bagasserdquo Industrial Crops and Products vol 31no 2 pp 277ndash283 2010

[29] S P Gautam P S Bundela A K Pandey J Khan M KAwasthi and S Sarsaiya ldquoOptimization for the production ofcellulase enzyme from Municipal solid waste residue by twonovel cellulolytic fungirdquo Biotechnology Research Internationalpp 1ndash8 2011

[30] M Sohail R Siddiqi A Ahmad and S A Khan ldquoCellulaseproduction from Aspergillus niger MS82 effect of temperatureand pHrdquo New Biotechnology vol 25 no 6 pp 437ndash441 2009

[31] S Ahmed A Bashir H Saleem M Saadia and A JamilldquoProduction and purification of cellulose-degrading enzymesfrom a filamentous fungus Trichoderma harzianumrdquo PakistanJournal of Botany vol 41 no 3 pp 1411ndash1419 2009

[32] A Das T Paul S K Halder et al ldquoStudy on regulation ofgrowth and biosynthesis of cellulolytic enzymes from newlyisolated Aspergillus fumigatus ABK9rdquo Polish Journal of Micro-biology vol 62 no 1 pp 31ndash43 2013

[33] S Shafique R Bajwa and S Shafique ldquoCellulase biosynthesis byselected Trichoderma speciesrdquo Pakistan Journal of Botany vol41 no 2 pp 907ndash916 2009

[34] N Sarkar and K Aikat ldquoAspergillus fumigatus NITDGPKA3provides for increased cellulase productionrdquo International Jour-nal of Chemical Engineering vol 2014 9 pages 2014

[35] V V Gilna and K M Khaleel ldquoCellulase enzyme activity ofAspergillus fumigatus from mangrove soil on lignocellulosicsubstraterdquo Recent Research in Science and Technology vol 3 no1 pp 132ndash134 2011

[36] G E Aboul-Fotouh G M El-Garhy H H Azzaz A MAbd El-Mola and G A Mousa ldquoFungal cellulase production

optimisation and its utilisation in Goatrsquos relations degradationrdquoAsian Journal of Animal and Veterinary Advances vol 11 no 12pp 824ndash831 2016

[37] TNcube R LHoward EKAbotsi E L J vanRensburg and INcube ldquoJatropha curcas seed cake as substrate for production ofxylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentationrdquo Industrial Crops and Products vol 37 no 1pp 118ndash123 2012

[38] J C Stewart and J B Parry ldquoFactors influencing the productionof cellulase by Aspergillus fumigatus (Fresenius)rdquo Journal ofGeneral Microbiology vol 125 no 1 pp 33ndash39 1981

[39] H Leghlimi Z Meraihi H Boukhalfa-Lezzar E Copinet andF Duchiron ldquoProduction and characterization of cellulolyticactivities produced by Trichoderma longibrachiatum (GHL)rdquoAfrican Journal of Biotechnology vol 12 no 5 pp 465ndash475 2013

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Enzyme Research 9

measured by filter paper activity (FPUml) can be attributedto genetic make-up of the fungal strains as a result ofadaptations to different habitats Furthermore our reportedhigh levels of cellulases endoglucanases and low level of 120573-glucosidase by the Trichoderma isolates and vice versa highlevels of 120573-glucosidase by A fumigatus are in agreement withthe reported levels of these fungal species by Stewart andParry [38]

5 Conclusion

In conclusion all the Trichoderma and Aspergillus strainsproduced substantial levels of all the three enzymes (ieexoglucanase endoglucanase and 120573-glucosidase) requiredfor complete hydrolysis of cellulosic material Trichodermastrains produced higher cellulases and endoglucanases levelswhile A fumigatus strains produced higher 120573-glucosidaselevels The production of all the cellulases componentsseemed to be strongly influenced by the interactive effectof initial pH and incubation temperature on the microor-ganisms Hence the observed maximum production ofcellulases by the fungi depended on the chosen initial pHof the medium and incubation temperature The cellulasesproduced by these fungal strainswill be assessed for efficiencyin hydrolysing agricultural lignocellulose wastes includingbanana pseudostem for the production of bioethanol

Data Availability

The experimental data used to support the findings of thisstudy are available from the corresponding author uponrequest

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors are grateful to the National Research Foundation(NRF) and the Flemish Inter-University Council (VLIR-UOS) for the financial support towards the research

References

[1] A C OrsquoSullivan ldquoCellulose the structure slowly unravelsrdquoCellulose vol 4 no 3 pp 173ndash207 1997

[2] Y-H P Zhang M E Himmel and J R Mielenz ldquoOutlookfor cellulase improvement screening and selection strategiesrdquoBiotechnology Advances vol 24 no 5 pp 452ndash481 2006

[3] P Beguin and J P Aubert ldquoThe biological degradation ofcelluloserdquo FEMS Microbiology Review vol 13 pp 25ndash28 1994

[4] M K Bhat and S Bhat ldquoCellulose degrading enzymes and theirpotential industrial applicationsrdquo Biotechnology Advances vol15 no 3-4 pp 583ndash620 1997

[5] J Zhuang M A Marchant S E Nokes and H J StrobelldquoEconomic analysis of cellulase production methods for bio-ethanolrdquo Applied Engineering in Agriculture vol 23 no 5 pp679ndash687 2007

[6] S T Merino and J Cherry ldquoProgress and challenges in enzymedevelopment for biomass utilizationrdquo Advances in BiochemicalEngineeringBiotechnology vol 108 pp 95ndash120 2007

[7] M Dashtban H Schraft and W Qin ldquoFungal bioconversionof lignocellulosic residues Opportunities amp perspectivesrdquo Inter-national Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009

[8] B S Padam H S Tin F Y Chye and M I Abdullah ldquoBananaby-products an under-utilized renewable food biomass withgreat potentialrdquo Journal of Food Science and Technology vol 51no 12 pp 3527ndash3545 2014

[9] H P S Abdul Khalil M S Alwani and A K M OmarldquoChemical composition anatomy lignin distribution and cellwall structure ofMalaysian plantwaste fibresrdquoBioResources vol1 no 2 pp 220ndash232 2006

[10] K Li S Fu H Zhan Y Zhan and L A Lucia ldquoAnalysis of thechemical composition and morphological structure of bananapseudostemrdquo Bioresources vol 5 no 2 pp 576ndash585 2010

[11] K Ibatsam S H Muhammad M Sobia and M Irum ldquoIso-lation and screening of highly cellulolytic filamentous fungirdquoJournal of Applied Science and Environmental Management vol16 no 3 pp 223ndash226 2012

[12] G Nunez-Trujillo R Cabrera A Cosoveanu T T Martin CGimenez and G Nunez-Trujillo ldquoSurvey of banana endophyticfungi isolated in artificial culture media from an appliedviewpointrdquo Journal of Horticulture Forestry and Biotechnologyvol 17 no 2 pp 22ndash25 2013

[13] J H Yoon J E Park D Y Suh S B Hong S J Ko and S HKim ldquoComparison of dyes for easy detection of extracellularcellulases in fungirdquoMycobiology vol 35 no 1 pp 21ndash24 2007

[14] A B Peixoto Study of the production of enzymes and gumsby wild yeasts collected in various regions of Brazil [MSThesis] Faculty of Food Engineering University of CampinasCampinas Brazil 2006

[15] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods AndApplications M A Innis D H Gelfand J J Sninsky et al Edspp 315ndash322 Academic Press Inc New York NY USA 1990

[16] B Benoliel F A Goncalves Torres and L M P de MoraesldquoA novel promising Trichoderma harzianum strain for theproduction of a cellulolytic complex using sugarcane bagasse innaturardquo SpringerPlus vol 2 p 256 2013

[17] D Herr ldquoSecretion of cellulase and 120573-glucosidase byTricho-derma viride ITCC-1433 in submerged culture on differentsubstratesrdquo Biotechnology and Bioengineering vol 21 no 8 pp1361ndash1371 1979

[18] A S Lakshmi and G Narasimha ldquoProduction of cellulases byfungal cultures isolated from forest litter soilrdquo Annals of ForestResearch vol 55 no 1 pp 85ndash92 2012

[19] X Han W Song G Liu Z Li P Yang and Y Qu ldquoImprov-ing cellulase productivity of Penicillium oxalicum RE-10 byrepeated fed-batch fermentation strategyrdquo Bioresource Technol-ogy vol 227 pp 155ndash163 2017

[20] M Rubeena K Neethu S Sajith et al ldquoLignocellulolyticactivities of a novel strain of TrichodermaharzianumrdquoAdvancesin Bioscience and Biotechnology vol 4 pp 212ndash221 2013

[21] A L Grigorevski-Lima F N M Da Vinha D T Souzaet al ldquoAspergillus fumigatus thermophilic and acidophilicendoglucanasesrdquo Applied Biochemistry and Biotechnology vol155 no 1-3 pp 321ndash329 2009

10 Enzyme Research

[22] J P Andrade A S D R Bispo P A S Marbach and RP Do Nascimento ldquoProduction and partial characterizationof cellulases from Trichoderma sp IS-05 isolated from SandyCoastal plains of Northeast Brazilrdquo Enzyme Research vol 2011Article ID 167248 7 pages 2011

[23] P D S Delabona C S Farinas M R da Silva S F Azzoniand J G D C Pradella ldquoUse of a new Trichoderma harzianumstrain isolated from the Amazon rainforest with pretreatedsugar cane bagasse for on-site cellulase productionrdquo BioresourceTechnology vol 107 pp 517ndash521 2012

[24] C Y Turenne S E Sanche D J Hoban J A Karlowskyand A M Kabani ldquoRapid identification of fungi by using theITS2 genetic region and an automated fluorescent capillaryelectrophoresis systemrdquo Journal of Clinical Microbiology vol 37no 6 pp 1846ndash1851 1999

[25] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcode marker for fungirdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 109 no 27 pp6241ndash6246 2012

[26] M Shahriarinour M N A Wahab R Mohamad S Mustafaand A B Ariff ldquoEffect of medium composition and culturalcondition on cellulase production by Aspergillus terreusrdquoAfrican Journal of Biotechnology vol 10 no 38 pp 7459ndash74672011

[27] D B Archer and J F Peberdy ldquoThe molecular biology ofsecreted enzyme production by fungirdquo Critical Reviews inBiotechnology vol 17 no 4 pp 273ndash306 1997

[28] R Soni A Nazir and B S Chadha ldquoOptimization of cellulaseproduction by a versatile Aspergillus fumigatus fresenius strain(AMA) capable of efficient deinking and enzymatic hydrolysisof Solka floc and bagasserdquo Industrial Crops and Products vol 31no 2 pp 277ndash283 2010

[29] S P Gautam P S Bundela A K Pandey J Khan M KAwasthi and S Sarsaiya ldquoOptimization for the production ofcellulase enzyme from Municipal solid waste residue by twonovel cellulolytic fungirdquo Biotechnology Research Internationalpp 1ndash8 2011

[30] M Sohail R Siddiqi A Ahmad and S A Khan ldquoCellulaseproduction from Aspergillus niger MS82 effect of temperatureand pHrdquo New Biotechnology vol 25 no 6 pp 437ndash441 2009

[31] S Ahmed A Bashir H Saleem M Saadia and A JamilldquoProduction and purification of cellulose-degrading enzymesfrom a filamentous fungus Trichoderma harzianumrdquo PakistanJournal of Botany vol 41 no 3 pp 1411ndash1419 2009

[32] A Das T Paul S K Halder et al ldquoStudy on regulation ofgrowth and biosynthesis of cellulolytic enzymes from newlyisolated Aspergillus fumigatus ABK9rdquo Polish Journal of Micro-biology vol 62 no 1 pp 31ndash43 2013

[33] S Shafique R Bajwa and S Shafique ldquoCellulase biosynthesis byselected Trichoderma speciesrdquo Pakistan Journal of Botany vol41 no 2 pp 907ndash916 2009

[34] N Sarkar and K Aikat ldquoAspergillus fumigatus NITDGPKA3provides for increased cellulase productionrdquo International Jour-nal of Chemical Engineering vol 2014 9 pages 2014

[35] V V Gilna and K M Khaleel ldquoCellulase enzyme activity ofAspergillus fumigatus from mangrove soil on lignocellulosicsubstraterdquo Recent Research in Science and Technology vol 3 no1 pp 132ndash134 2011

[36] G E Aboul-Fotouh G M El-Garhy H H Azzaz A MAbd El-Mola and G A Mousa ldquoFungal cellulase production

optimisation and its utilisation in Goatrsquos relations degradationrdquoAsian Journal of Animal and Veterinary Advances vol 11 no 12pp 824ndash831 2016

[37] TNcube R LHoward EKAbotsi E L J vanRensburg and INcube ldquoJatropha curcas seed cake as substrate for production ofxylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentationrdquo Industrial Crops and Products vol 37 no 1pp 118ndash123 2012

[38] J C Stewart and J B Parry ldquoFactors influencing the productionof cellulase by Aspergillus fumigatus (Fresenius)rdquo Journal ofGeneral Microbiology vol 125 no 1 pp 33ndash39 1981

[39] H Leghlimi Z Meraihi H Boukhalfa-Lezzar E Copinet andF Duchiron ldquoProduction and characterization of cellulolyticactivities produced by Trichoderma longibrachiatum (GHL)rdquoAfrican Journal of Biotechnology vol 12 no 5 pp 465ndash475 2013

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

10 Enzyme Research

[22] J P Andrade A S D R Bispo P A S Marbach and RP Do Nascimento ldquoProduction and partial characterizationof cellulases from Trichoderma sp IS-05 isolated from SandyCoastal plains of Northeast Brazilrdquo Enzyme Research vol 2011Article ID 167248 7 pages 2011

[23] P D S Delabona C S Farinas M R da Silva S F Azzoniand J G D C Pradella ldquoUse of a new Trichoderma harzianumstrain isolated from the Amazon rainforest with pretreatedsugar cane bagasse for on-site cellulase productionrdquo BioresourceTechnology vol 107 pp 517ndash521 2012

[24] C Y Turenne S E Sanche D J Hoban J A Karlowskyand A M Kabani ldquoRapid identification of fungi by using theITS2 genetic region and an automated fluorescent capillaryelectrophoresis systemrdquo Journal of Clinical Microbiology vol 37no 6 pp 1846ndash1851 1999

[25] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcode marker for fungirdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 109 no 27 pp6241ndash6246 2012

[26] M Shahriarinour M N A Wahab R Mohamad S Mustafaand A B Ariff ldquoEffect of medium composition and culturalcondition on cellulase production by Aspergillus terreusrdquoAfrican Journal of Biotechnology vol 10 no 38 pp 7459ndash74672011

[27] D B Archer and J F Peberdy ldquoThe molecular biology ofsecreted enzyme production by fungirdquo Critical Reviews inBiotechnology vol 17 no 4 pp 273ndash306 1997

[28] R Soni A Nazir and B S Chadha ldquoOptimization of cellulaseproduction by a versatile Aspergillus fumigatus fresenius strain(AMA) capable of efficient deinking and enzymatic hydrolysisof Solka floc and bagasserdquo Industrial Crops and Products vol 31no 2 pp 277ndash283 2010

[29] S P Gautam P S Bundela A K Pandey J Khan M KAwasthi and S Sarsaiya ldquoOptimization for the production ofcellulase enzyme from Municipal solid waste residue by twonovel cellulolytic fungirdquo Biotechnology Research Internationalpp 1ndash8 2011

[30] M Sohail R Siddiqi A Ahmad and S A Khan ldquoCellulaseproduction from Aspergillus niger MS82 effect of temperatureand pHrdquo New Biotechnology vol 25 no 6 pp 437ndash441 2009

[31] S Ahmed A Bashir H Saleem M Saadia and A JamilldquoProduction and purification of cellulose-degrading enzymesfrom a filamentous fungus Trichoderma harzianumrdquo PakistanJournal of Botany vol 41 no 3 pp 1411ndash1419 2009

[32] A Das T Paul S K Halder et al ldquoStudy on regulation ofgrowth and biosynthesis of cellulolytic enzymes from newlyisolated Aspergillus fumigatus ABK9rdquo Polish Journal of Micro-biology vol 62 no 1 pp 31ndash43 2013

[33] S Shafique R Bajwa and S Shafique ldquoCellulase biosynthesis byselected Trichoderma speciesrdquo Pakistan Journal of Botany vol41 no 2 pp 907ndash916 2009

[34] N Sarkar and K Aikat ldquoAspergillus fumigatus NITDGPKA3provides for increased cellulase productionrdquo International Jour-nal of Chemical Engineering vol 2014 9 pages 2014

[35] V V Gilna and K M Khaleel ldquoCellulase enzyme activity ofAspergillus fumigatus from mangrove soil on lignocellulosicsubstraterdquo Recent Research in Science and Technology vol 3 no1 pp 132ndash134 2011

[36] G E Aboul-Fotouh G M El-Garhy H H Azzaz A MAbd El-Mola and G A Mousa ldquoFungal cellulase production

optimisation and its utilisation in Goatrsquos relations degradationrdquoAsian Journal of Animal and Veterinary Advances vol 11 no 12pp 824ndash831 2016

[37] TNcube R LHoward EKAbotsi E L J vanRensburg and INcube ldquoJatropha curcas seed cake as substrate for production ofxylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentationrdquo Industrial Crops and Products vol 37 no 1pp 118ndash123 2012

[38] J C Stewart and J B Parry ldquoFactors influencing the productionof cellulase by Aspergillus fumigatus (Fresenius)rdquo Journal ofGeneral Microbiology vol 125 no 1 pp 33ndash39 1981

[39] H Leghlimi Z Meraihi H Boukhalfa-Lezzar E Copinet andF Duchiron ldquoProduction and characterization of cellulolyticactivities produced by Trichoderma longibrachiatum (GHL)rdquoAfrican Journal of Biotechnology vol 12 no 5 pp 465ndash475 2013

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom