bioresources.

PEER-REVIEWED ARTICLE

com
Selection of Fungal Isolates for Biopulping of Rice Straw
SaidM.BadrElDin,a,*ZeinabH.Kheiralla,bSaadM.AbdelMalek,aandDouaaH.Abdel
Aziz
a

Sixty-two fungal isolates were screened for lignin peroxidase production. The most potent isolates for
lignin peroxidase production were identified using the DNA sequence of the internal transcribed spacer
(ITS) region as Phanerochaete chrysosporium and Pleurotus ostreatus. The pretreatment of rice straw
with P. chrysosporium, Pl. ostreatus, or lignin peroxidase for use in the biopulping process was studied.
Great variations in the loss of pulp yield and kappa number were recorded with different fungal and
enzyme treatments. Pretreatment of rice straw with P. chrysosporium for 25 days resulted in a substantial
decrease in pulp yield (by 9.1%) and kappa number (by 25.6%). Losses of pulp yield and kappa number
were considerably lower with lignin peroxidase treatment (3.7 and 14.1%, respectively). However, the
pretreatment of rice straw with the Pl. ostreatus isolate caused moderate pulp yield losses (5.8%) and
preferential lignin degradation (kappa number losses of 34.6%). This indicated that the Pl. ostreatus
isolate might be superior to both the isolate of P. chrysosporium and lignin peroxidase for use in the
biopulping process or other processes in which preferential lignin degradation is desired.
Keywords:Ligninperoxidase;Biopulping;Ricestraw;Phanerochaetechrysosporium;Pleurotusostreatus
Contactinformation:a:AgriculturalMicrobiologyDepartment,NationalResearchCenter,Cairo,Egypt;b:Botany
Department,FacultyofWomenforArts,ScienceandEducation,AinShamsUniversity,Egypt;*Corresponding
author:badreldinsaid@hotmail.com

INTRODUCTION
Lignocellulosicmaterialcanbeobtainedfromwood,grass,agriculturalresidues,forestry
wastes, and municipal solid wastes. Several biological methods for lignocellulose recycling,
based on the enzymology of cellulose, hemicellulose, and lignin degradation, have been
suggested.Amongthem,compostinganduseasarawmaterialfortheproductionofethanolas
analternativecombustibleformseemtobethemosteconomicallyfeasible.Moreover,the
generaluseofalternative,environmentallyfriendlytechnologiesthatintroducelignocellulose
enzymes at different stages of pulp and paper manufacture as a pretreatment to pulping
(biopulping)andbleaching(biobleaching)haveallowedconsiderableelectricalpowersavings,
improvementsinpaperstrength,andareductionofpollutantsinthewastewaterfromthese
industries (Akhtar et al. 1998; Shukla et al. 2004; Singh et al. 2010; Isroi et al. 2011;
Garmaroodyetal.2011).Inaddition,pretreatmentofagriculturalwasteswithligninolyticfungi
enablestheuseofthewastesasrawmaterialforpapermanufacturing.
 Increase of demand for paper production and limited wood resources have directed
researcherstolookforappropriateadditionalresourcesofnonwoodmaterialsforpulpandpaper
manufacturing.Severalkindsofnonwoodlignocellulosicbyproductsofagriculturalcultivation
havebeeninvestigated,ofwhichwheat,rice,andbarleystraw
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4969
 bioresources. PEER-REVIEWED ARTICLE

com
arethemostprominent(Yaghoubietal.2008;Xuetal.2013;Wulandarietal.2013),
particularlyinthecountrieswithdeficientwoodresource,suchasChina,India,andEgypt.In
Egypt,hugeamountsofricestrawareproducedannuallyasfibrousbyproduct.Thedisposalof
thesebyproductsisbecomingamajorproblem.Themicrobialpretreatmentisanatural
process;therefore,noadverseenvironmentalconsequencesareforeseen.Biologicalpretreatment
employsmicroorganismsandtheirenzymaticmachineriestobreakdownligninandalter
lignocellulosicstructures.Someofthemostpromisingmicroorganismsforbiological
pretreatmentarewhiterotfungithatcanmineralizelignintoCO
2

andwaterinpureculture(Erikssonetal.1990;Akhtaretal.1998).
Several whiterot fungi such as Phanerochaete chrysosporium, Ceriporiopsis subvermispora,
Phlebiasubserialis,andPleurotusostreatusarecapableofefficientlymetabolizingligninina
varietyoflignocellulosicmaterials(Erikssonetal.1990;Martinezetal.1994;Doradoetal.
1999;Yaghoubietal.2008).Thefungihavebeenstudiedinconnectionwithseveralligninolytic
enzymes,suchasligninperoxidases(LiP),manganeseperoxidases(MnP),laccase(Lac),and
versatileperoxidases(VP)(Rodriguesetal.2008;Singhetal.2010;Isroietal.2011).
White rot fungi and their enzymes (especially ligninases and xylanases) have been
considered for wood chip treatment prior to pulping. Although ligninases attack the lignin
contentofwood,xylanasesdegradehemicellulosesandmakethepulpmorepermeableforthe
removalofresiduallignin.Thus,thebiopulpingprocessremovesnotonlyligninbutalsosome
of the wood extractives, thus reducing the pitch content and effluent toxicity (Ali and
Sreekrishnan2001).
Lignin degradation by white rot fungi is a complex secondary metabolic process
mediatedbytheactionofseveralextracellularenzymes,ofwhichligninperoxidasesarethemost
important(BuswellandOdier1987;Akhtaretal.1998;Rodriguesetal.2008;Isroietal.2011).
Consequently,theyandtheirextracellularligninolyticenzymeshavebeenconsideredforvarious
applicationsinenvironmentalbiotechnology.Thepresentworkaimstoisolateandselectfungal
strainswithhighligninperoxidasecontentstouseasapretreatmentforpulping(biopulping)of
ricestraw.

MATERIALS AND METHODS

Rice Straw
RicestrawwasobtainedfromKomHamadaCity,ElBehiraProvince,Egypt.Itwas2.0
to2.5mminlength.Themaincompositionofthericestrawwasasfollows:cellulose,40%;
hemicellulose,19%;lignin,25%;andash,16%.Thecomponents(cellulose,hemicellulose,and
lignin) of rice straw were analyzed according to the procedures of Goering and Van Soest
(1971).Ashcontentwasdeterminedbyignitedastrawsampleof2graminmufflefurnaceat
550oCtoconstantweight.

Microorganisms
In the present study, 62 local fungal strains were isolated from different rice straw
compostsamplesinEgypt.Theserialdilutionplatemethodwasused.Thepotatodextroseagar
(PDA)mediumwas pouredintosterilepetriplates,theninoculatedwith1.0mLfromeach
dilution.Theplateswereincubatedat30oCfor4days,andindividualcoloniesweregrownout
onslantsofPDAmedium.Theisolatedfungiwerepurified
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4970
 bioresources. PEER-REVIEWED ARTICLE

com
threetimesbyrestreakingonthesamemedium.Pureculturesoffungiweremaintainedonagar
slantsofPDAat4oC.Theisolatedpureculturesweretestedforenzymeactivity.

Selection of the Potent Isolates for Lignin Peroxidase Production

Erlenmeyerflasks(250mLcapacity)containing50mLofsterilepotatodextrosebroth
mediumwereautoclaved.Eachflaskwasinoculatedwith2fungaldiscs(0.8cm)ofa4dayold
cultureandincubatedat30
o

Cinrotaryshakersat100rpmfor2,3,4,5,and6days.
ThecultureswerefilteredusingWhatmanno.1filterpaperfortheremovaloffungalgrowth.The
filtratewasusedasthesourceofcrudeenzyme.

Lignin peroxidase Assays

Ligninperoxidaseactivitywasmeasuredbytheveratrylalcoholmethod(TienandKirk
1988).Theassaymixturecontainedinafinalvolumeof2.5mLwasasfollows:1.8mLofthe
dilutedsupernatant,0.1mLof50mMveratrylalcohol,0.5mLof0.5Msodiumtartratebuffer
(pH3.0),and0.1mLof10mMH
2

O
2

.Oxidationofthesubstrateatroom
temperature was monitored by an absorbance increase at 310 nm due to the formation of
veratraldehyde(
310

=9300M
1

cm
1

).Oneunitofenzymeactivitywasdefined
astheamountofenzymeneededtoproduce1molofveratraldehydeperminute.

Dry Biomass Measurement

FungalbiomassdeterminationwasconductedbyfiltrationthroughpreweighedWhatman
no.1filterpaper,washingwithdistilledwater,andthendryinginanovenuntiltwosuccessive
equalweightsforthesamesamplewereobtained.Thebiomasswascalculatedintermsofgram
perliterofgrowthmedium.

Extraction of Genomic DNA

Fungalmyceliaweregrownonpotatodextroseagar(PDA),harvestedusingaspatula,
transferredinto1.5mLEppendorftubes,suspendedin1mLofT
10

E
1

buffer (10 mM
TrisHCl,1mMEDTA[pH8.0]),andcentrifugedat13,000rpmfor5min.Thepelletwas
resuspendedin200Lof50mMNaOH,vortexed,andincubatedat95 Cfor10min.The
mixturewasthenneutralizedwith200Lof0.1mMTrisHCl(pH7.0)andcentrifugedfor5
minat13,000rpm.Thepelletwasresuspendedin500LofsterileH
2

O and
centrifugedat13,000rpmfor5min.Thesupernatantwasremoved,followedbytheadditionof
200Lofcrackingbuffer(2%TritonX100,1%sodiumdodecylsulfate,100mMNaCl,20mM
Tris[pH8.0],10mMEDTA[pH8.0]).ExtractionofgenomicDNAwasachievedbyadditionof
glassbeadsto3/4oftheliquidvolume,followedbytheadditionof200 Lofcoldphenol
chloroformisoamylalcoholina25:24:1ratio.Themixturewassubjectedtoconstantvortexing
for30min.Thesamplewasthencentrifugedat13,000rpmfor5min.Theaqueous(top)layer
wastransferredtoanewtube,and1mLof100%ethanolwasaddedforDNAprecipitation.The
samplewascentrifugedat13,000rpmfor2min.Thesupernatantwasremoved,andthepellet
wasresuspendedin400LofT
10

E
1

bufferplus30mgofRNaseA(Sigma,St.Louis,MO)fora1hwaterbathincubation
at35C.Thereactionwasstopped,andtheDNAwasprecipitatedwith10Lof3Msodium
acetateand1,000Lofcold100%ethanol.Thesamplewascentrifugedat13,000rpmfor2min,
thesupernatantwasremoved,andthepelletwasairdried.TheDNApelletwasresuspendedin
50LofTEbufferandwasusedastheDNAtemplateforamplificationafter30minorstoredat
20oCforfutureuse(Turenneetal.1999).
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4971
 bioresources. PEER-REVIEWED ARTICLE

com
PCR Amplification
TheprimersusedforuniversalfungalamplificationwereITS4(reverseprimer[5'tcctcc
gcttattgatatgc3'])(Whiteetal.1990)andfluorescentlylabeled(5'HEX)ITS86(forward
primer[5'gtgaatcatcgaatctttgaac3'])(LifeTechnologies,Burlington,Ontario,Canada).The
50LPCRreactionmixturecontained5LofDNAtemplate;5Lof25mMMgCl
2

10 x PCR buffer; 1.25 mM deoxynucleoside

triphosphate;dATP,dGTP,dCTP,andan8:1ratioofdUTPtodTTP;0.5Lof100mMofeach
primer;0.5unitsofuracilDNAglycosylase(UDG);2.5unitsofTaqDNApolymerase;and30
LofsteriledistilledH
2

O.ThePCRwasperformedinaGeneAmpPCR
system9600(PerkinElmerAppliedBiosystems,FosterCity,Calif.)withcyclesof37Cfor10
min(UDGactivation)and94Cfor10min(UDGinactivation)and30cyclesof94,55,and72
Cfor1mineach;themixturewasthenincubatedat72Cfor10minforafinalextension
(Turenneetal.1999).

DNA Sequencing
ThePCRreactionproductsweresequenceddirectlyusingabigDyeterminatorreagent
kitincludingTaqpolymeraseandtheprotocolrecommendedbythemanufacture(PerkinElmer).
The reactions were run on an ABI 310 automated DNA sequencer (Applied Biosystems
Incorporation,California,USA).

Phylogenetic Analysis
The blast program (www.ncbi.nlm.gov/blast) was used to assess DNA similar ities;
multiplesequencealignmentandmolecularphylogenywereperformedusingBioEditsoftware
(Hall1999).ThephylogenetictreewasdisplayedusingtheTreeViewprogram(Page1996).

Inoculum Preparation
Inpreparingtheliquidinoculumoftheselectedfungalisolates,1Lflaskscontaining200
mLofthefermentationmediumwereinoculatedwith10plugscutwithanumber8sizecork
porefromapotatodextroseagarplatecontaining10dayoldfungalcultures.Theflaskswere
thenincubatedat30
o

Cfor10days.Flasks containingthefungal
biomassweredecantedandwashedwithsterilizeddistilledwatertoremoveexcessmediumfrom
thefungalbiomass.Thefungalbiomasswasthenplacedinsterilizeddistilledwaterandblended
inaWaringblendertwicefor15s,eachtimeathighspeed.Distilledwaterwasthenaddedtothe
suspensiontoatotalvolume100mL(stock).About1.0mLoffungalsuspensionstockproduced
0.005%(dryweightbasis)inoculumofeachstrain.

Partial Purification of Lignin Peroxidase

Theculturefluidwascentrifugedat10,000rpmfor5minat4oCtoremovefungal
mycelium.(NH
4
o

C. The
precipitatedcrudeligninperoxidasefractionwasrecoveredbycentrifugation(10,000rpm,10
min).Thepelletwasresuspendedindeionizedwateranddialyzedseveraltimesagainst20mM
succinate(pH3.5)for2daysatroomtemperature.Theproteincontentandligninperoxidase
activityweredeterminedintheenzymefraction.Proteincontentwasdeterminedasdescribedby
Lowryetal.(1951)usingbovineserumalbuminasastandard.
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4972

)
2

SO
4

wasaddedtothesupernatantto85%saturationat4
 bioresources. PEER-REVIEWED ARTICLE

com
Fungal Pretreatment of Rice Straw
Wideneckjars(2L)containing100gofricestraw(dryweight),with2.02.5inlength,
wereautoclavedat121
o

Cfor20min.Aftercoolingthebioreactors,440mLofsterile
distilledwatercontaining25mgofmycelium(dryweight)ofPhanerochaetechrysosporiumor
Pleurotusostreatusor10mLofpartiallypurifiedenzymesolutionwereintroducedintoeachof
thejarstobringthefinalmoistureofricestrawtoabout60%(wetweightbasis)andweighed.
Ricestrawandfungalbiomassweremixedandincubatedat30oCfor5,10,15,20,and25days.
Themoisturecontentwaskeptforeachjarat60%ofwaterholdingcapacity(WHC)duringthe
experimentalperiod.Controlswerepreparedunderthesameconditionsbutwithoutinoculation.

Soda Pulping
Alkaline pulping was carried out by soaking straw (25 g oven dry weight) in a 1%
solutionofsodiumhydroxidesolutionata10:1liquortostrawratioat45oCfor10min.The
saturatedmasswassqueezedandcookedwithanadditionof12%sodiumhydroxideatastrawto
liquorratioof1:6and0.1ganthraquinone%ovendrystrawwithmaximumtemperaturesat127
o

Candcookingtimeupto60min(ElAshmawyetal.1984).Theresulting
pulpwaswashedoverfilterpaperanddried,andtheyieldandkappanumberweredetermined.
KappanumberwasdeterminedusingthemethoddescribedbyTasmanandBerzins(1957).

RESULTS

Screening for Lignin Peroxidase Production from Isolated Fungi

TheresultsinTable1showthatthedifferentfungalisolatesexhibitedagreatvariationin
theligninperoxidaseactivity.Of62fungalisolates,11isolateshadligninperoxidaseactivity.
Thehighestenzymeactivitieswererecordedafter4daysofincubation.Theenzymeactivitiesof
suchisolatesrangedfrom0.465to7.440U/mLafter4daysofincubation.Themostpotent
isolatesforligninperoxidaseproductionwereisolates21and43.Theenzymeactivitiesofthese
isolatesrangedfrom5.673to7.440U/mLafter4daysofincubation.Thus,thoseisolateswere
selectedforfurtherstudy.

Table 1. Lignin Peroxidase Activities for Fungal Isolates (U/mL)

Isolates No.
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4973

Time of incubation in days 2 3 4 5 6 1 0.372 0.465 0.465 0.279 0.093 2 0.558 0.744 1.116 0.837 0.651 5
0.372 0.651 0.837 0.558 0.279 14 0.093 0.372 0.465 0.279 0.093 16 0.279 0.558 0.651 0.465 0.186 21
1.767 2.976 5.673 3.999 3.255 34 0.093 0.186 0.651 0.744 0.372 43 3.720 6.975 7.440 5.859 2.883 51
0.651 0.744 0.930 0.558 0.279 59 0.186 0.372 0.465 0.186 0.093 62 0.615 0.701 0.880 0.511 0.324
 bioresources. PEER-REVIEWED ARTICLE

com
PCR and DNA Sequencing
PCR products of the ITS (internal transcribed spacer) region (ITS1 + 5.8S + ITS2)
amplifiedwithprimersITS1andITS4werevisualizedasasinglebandinagarosegels.The
DNA sequences of the ITS region for isolates 21 and 43 are represented in Figs. 1 and 2,
respectively.
AAAATACGATACTGAACAGGTTGTAAGCTGGCCTCTCGGGGCATGTGCACGCCTGGCTCAT
CCACTCTTCAACCTCTGTGCACTTGTTGTAGGTCGGTAGAAGAGCGAGCATCCTCTGATGCT
TTGCTTGGAAGCCTTCCTATGTTTTACTACAAACGCCTTCAGTTTAAGAATGTCTACCTGCGT
ACAACGCATCTATATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACG
CAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGC
ACCTTGCGCTCCCTGGTATTCCGGGGGAGCATGCCTGTTTGAGTGTCATGGTATCCTCAAC
CTTCATAACTTTTTTGTTACCGAAGGCTTGGACTTGGAAGGTTGTGCTGGCTTCAAGTCAAGT
CGGCTCCCCTTAAATGTATTAGCGGGGGGGGTAACGGAATCCCTTCGGGGGGAAAATTATC
TGCCCCCGGGGTCCTGAAGAAACAAAAGCCTGGGGCTTTCTAACCGTCCTTCAGTTGGACA
ACTTACTTTGACATCTGGCCCCAAATCAGGGAGAACACCCCCTGAACTTAACCTT

Fig. 1. DNA sequences of ITS region of isolate 21

CTTTCAACCACGAGAGAACTTTTGATAGATCTGGTGAAGTCGTCTCTCCAGTCGTCAGACTT
GGTTGCTGGGATTTAAACGTCTCGGTGTGACTACGCAGTCTATTTACTTACACACCCCAAAT
GTATGTCTACGAATGTCATTTAATGGGCCTTGTGCCTTTAAACCATAATACAACTTTCAACAA
CGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTAATTGC
AGAATTCAGTGGAATCATCGAATCTTTGAACGCACCTTGCGCCCCCTTGGTATTCCCGAGGG
GCATGCCTGGTTTGAGTGTCATTAAAATTTCTCAAACGCCACTTT

Fig. 2. DNA sequences of ITS region of isolate 43

Phylogenetic Analysis
Thesequencedataofisolates21and43werethenanalyzedusingtheBlastprogram
(www.ncbi.nlm.gov/blast) to assess the DNA similarities. Multiple sequence alignment and
molecular phylogeny were performed using BioEdit software; the phylogenetic trees are
displayed,usingtheTreeViewprogram,inFigs.3and4.
GanodermatornatumstrainCBS109679
PhanerochaetesordidastrainKUC8710
PhanerochaeteaustralisstrainFP102818sp
Phanerochaetesp.ATT215
Phanerochaetelaevis
Phanerochaetecalotricha
PhanerochaeteericinastrainFP101978
Corticiaceaesp.386
IsolateNo21
PhanerochaetechrysosporiumstrainBKMF1767
PhanerochaetechrysosporiumstrainFPL5175
PhanerochaetechrysosporiumstrainATCC24725

0.005 Fig. 3. The phylogenetic tree showing the relationship between isolate 21 and other
known sequences using the neighbor-joining method

Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4974
 bioresources. PEER-REVIEWED ARTICLE

com
PleurotuspopulinusstrainATCC52951
PleurotussubareolatusstrainATCC42990
PleurotusnebrodensisstrainCCMSSC1577
PleurotussapidusstrainACCC50155
PleurotuscornucopiaestrainACCC50375
Pleurotussp.Florida
PleurotusostreatusstrainH8
IsolateNo43

0.002 Fig. 4. The phylogenetic tree showing the relationship between isolate 43 and other
known sequences using the neighbor-joining method

The obtained results revealed that the sequence of isolate 21 showed the highest
similarity(95%)withPhanerochaetechrysosporium(P.chrysosporium),andthesequenceof
isolate43showedthehighestsimilarity(96%)withPleurotusostreatus(Pl.ostreatus).

Fungal Pretreatment of Rice Straw

TheresultsinTable2showedconsiderablevariationinthepulpyieldlossesofricestraw
withdifferentfungalandenzymetreatments.Inalltreatments,thepulpyieldofricestrawwas
decreasedgraduallyastimeincreased,ascomparedtothecontroltreatment.Thepulpyield
lossesrangedfrom3.7to9.1%ascomparedtothecontrolafter25daysincubationtime.The
greatest loss in pulp yield of rice straw was recorded after 25 days with P. chrysosporium
treatment.Thelowestlossinpulpyieldofricestrawwasdetectedafter25dayswithlignin
peroxidasetreatment.TreatmentofricestrawwithPl.ostreatuscausedmoderatelossesofpulp
yield.
Theincreaseinthepercentagesofashcontent(Table2)inthepulpyieldparalleledthe
lossesintotaldryweightinallthetreatmentsthroughouttheexperimentalperiod.However,the
actual amount of ash in all treatments did not show any remarkable changes. The highest
increaseinashcontentinpulpyieldoccurredwiththeP.chrysosporiumtreatment.Thelowest
increaseofashcontentwasobservedwithligninperoxidasetreatment.
The results also showed great variations in the kappa number of pulp with different
fungal and enzyme treatments. During the experimental period, the kappa number reduced
graduallyfromthatofthecontrolwithalltreatments.ThefungalstrainsP.chrysosporiumand
Pl.ostreatusreducedthekappanumberofpulpfromthatofthecontrolby25.6and34.6%,
respectively.Ligninperoxidasetreatmentreducedthekappanumberfromthatofthecontrolby
14.1%.
 Therefore,thepretreatmentofricestrawwiththePl.ostreatusisolatecausedmoderate
pulp yield losses and preferential lignin degradation. This indicated that the isolate of Pl.
ostreatusmightbesuperiortoboththeisolateofP.chrysosporiumandligninperoxidaseenzyme
foruseinbiopulpingprocessesorotherprocessesinwhichpreferentiallignindegradationis
desired.
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4975
 bioresources. PEER-REVIEWED ARTICLE

com
Table 2. Effect of Fungal and Lignin Peroxidase Pretreatment of Rice Straw on Pulp
Yield, Ash Content, and Kappa Number
Treatments Time in days

5 10 15 20 25 Pulp yield % Control 48.7 48.1 47.4 46.7 46.2 P. chrysosporium 48.2 46.5 45.0 43.4 42.0
Pl. ostreatus 48.3 47.0 45.8 44.6 43.5 Lignin peroxidase 48.4 47.3 46.2 45.3 44.5

Ash % Control 15.2 15.6 15.8 16.1 16.3 P. chrysosporium 16.2 16.9
17.5 18.2 18.7 Pl. ostreatus 15.8 16.5 17.0 17.6 18.1 Lignin peroxidase 15.5 15.8 16.2 16.5 16.8

Kappa number Control 8.6 8.4 8.3 8.1 7.8 P. chrysosporium 8.1 7.5 7.0
6.4 5.8 Pl. ostreatus 7.8 7.0 6.4 5.7 5.1 Lignin peroxidase 8.3 7.8 7.4 7.0 6.7

DISCUSSION
Thedecompositionoflignininnaturehasbeenbelievedforalongtimetooccurbythe
actionofwoodrotfungi,mostlytheBasidiomyceteclass.Thesemicroorganismssimultaneously
decomposeligninandwoodpolysaccharides.Lignindegradationbywhiterotfungiisacomplex
secondarymetabolicprocessmediatedbytheactionofseveralextracellularenzymes,ofwhich
ligninperoxidasesarethemostimportant.Inthecourseofscreeningfortheligninperoxidase
productionfromfungi,11isolatesoutof62fungalisolateshadligninperoxidaseactivity.The
mostpotentisolatesforligninperoxidaseproductionwereisolates21and43.
TheDNAsequencesoftheinternaltranscribedspacer(ITS)regionforisolates21and43
wereanalyzedbytheBlastprogramforidentificationoffungalisolates.Theobtainedresults
revealedthatthesequenceofisolate21showedthehighestsimilarity(95%)withPhanerochaete
chrysosporium, and the sequence of isolate 43 showed the highest similarity (96%) with
Pleurotusostreatus.Severalscreeningstudiestofindsuitablefungiforbiopulpingofwoodor
agricultural wastes have revealed that, under certain conditions, fungi preferentially degrade
lignin over cellulose. Such lignin selective fungi include P. chrysosporium, Ceriporiopsis
subvermispora(KirkandFarrell1987;Erikssonetal.1990;Taniguchietal.2005),Pycnoporus
cinnabarinus(AnderandEriksson1977),Pl.ostreatus(Martnezetal.1994;Yaghoubietal.
2008),Pl.eryngii(Martnezetal.1994;Doradoetal.1999),Phlebiaradiata(AnderandEriksson
1977), Phlebia tremellosa (Eriksson et al. 1990), Phlebia subserialis (Akhtar et al. 1998),
Phellinuspini(Erikssonetal.1990),andDichomitussqualens(Erikssonetal.1990).
Inrecentyears,ithasbeendemonstratedthatthepretreatmentofwoodandagricultural
wasteswithlignindegradingfungicouldbebeneficialnotonlytotheprocessofmechanical
pulping(Akhtaretal.1998;Scottetal.2002;SinghandChen2008)butalsoinchemicalpulping
(MessnerandSrebotnik1994;Akhtaretal.1998;Isroietal.
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4976
 bioresources. PEER-REVIEWED ARTICLE

com
2011).Reportsonbiochemicalpulpingindicateareductioninkappanumberatagivenpulp
yield(Oriaranetal.1990;Blanchetteetal.1992;Mosaietal.1999;Yaghoubietal.2008)as
well as improvements in certain physicochemical properties of paper handsheets, such as
brightnessandstrengthproperties(Akhtaretal.1998;Shuklaetal.2004;Singhetal.2010;Isroi
etal.2011;Garmaroodyetal.2011)andareductionofpollutantsinthewastewaterfromthese
industries(Yadavetal.2010).
Inthepresentstudy,greatvariationsinthelossofpulpyieldandkappanumberwere
recorded with different fungal and enzyme treatments. Pretreatment of rice straw with P.
chrysosporiumfor25daysresultedinasubstantialdecreaseinpulpyield(by9.1%)andkappa
number(by25.6%)fromthatofthecontrol.Lossesofpulpyieldandkappanumber(3.7and
14.1%fromthatofthecontrol,respectively)wereconsiderablylowerwithligninperoxidase
treatment of rice straw as compared with other fungal pretreatments. Pulp yield and kappa
numberwerereducedby5.8and34.6%fromthatofthecontrol,respectively,withpretreatment
byPl.ostreatusofricestrawfor25days.Similarly,Scottetal.(1996)reportedthatbiosulfite
pulpingwithC.subvermisporaSS3for2weeksreducedthekappanumberofpineby21%,
whereaspretreatmentofsprucewithC.subvermisporaCZ3for2weeksresultedina22%kappa
numberdecreasefromthatofthecontrol(Fischeretal.1994).Mosaietal.(1999)foundthat
pretreatmentofeucalyptuswoodchipswithC.subvermisporaSS3for10daysresultedina
decreaseinkappanumberby29%andincreaseinbrightnessby12%.Yaghoubietal.(2008)
usedC.subvermisporaforbiochemicalpulpingofagriculturalresidues,andtheresultswere
comparedwithchemicalpulping.Biologicaltreatmentofrice,wheat,andbarleystrawsamples
resultedinadecreaseofthekappanumberby34,21,and19%,respectively,ascomparedwith
controlsamples.
In the present study, pretreatment of rice straw with the Pl. ostreatus isolate caused
moderatepulpyieldlossesandpreferentiallignindegradation.Similarly,Taniguchietal.(2005)
reportedthatPl.ostreatuspreferentiallydegradedligninoverpolysaccharidesinricestraw,while
P.chrysosporiumandC.subvermisporadegradedbothligninandpolysaccharidesinthestraw.
This indicated that the isolate of Pl. ostreatus might be superior to both the isolate of P.
chrysosporiumandligninperoxidaseenzymeforuseinbiopulpingprocessesorotherprocesses
inwhichpreferentiallignindegradationisdesired.

CONCLUSIONS
Of62fungalisolates,themostpotentisolates forligninperoxidaseproductionwere
identifiedasPhanerochaetechrysosporiumandPleurotusostreatus.Greatvariationsintheloss
of pulp yield and kappa number were recorded with the pretreatment of rice straw with P.
chrysosporium,Pl.ostreatus,orligninperoxidase.ThePl.ostreatusisolatemaybesuperiorto
boththeisolateofP.chrysosporiumandligninperoxidaseforuseinthebiopulpingprocessor
otherprocessesinwhichpreferentiallignindegradationisdesired.
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4977
 bioresources. PEER-REVIEWED ARTICLE

com
REFERENCES CITED
Akhtar,M.,Blanchette,R.A.,Myers,G.C.,andKirk,T.K.(1998).Overviewof
biochemicalpulpingresearch,in:EnvironmentallyFriendlyTechnologiesforthePulpand
PaperIndustry,R.YoungandM.Akhtar(eds.),JohnWiley,NewYork.Ali,M.,and
Sreekrishnan,T.R.(2001).Aquatictoxicityfrompulpandpapermill
effluents:Areview,Adv.Environ.Res.5(2),175196.Ander,P.,andEriksson,K.E.
(1977).Selectivedegradationofwoodcomponentsby
whiterotfungi,Physiol.Plant.41(4),239248.Blanchette,R.A.,Burnes,T.A.,Eerdmans,
M.M.,andAkhtar,M.(1992).Evaluating
isolatesofPhanerochaetechrysosporiumandCeriporiopsissubvermisporaforuseinbiological
pulpingprocesses,Holzforschung46(2),109115.Buswell,J.A.,andOdier,E.(1987).Lignin
biodegradation,CRCCriticalReviewsin
Biotechnology6(1),160.Dorado,J.,Almendros,G.,Camarero,S.,Martinez,A.T.,Vares,
T.,andHatakka,A.
(1999).Transformationofwheatstrawinthecourseofsolidstatefermentationbyfour
ligninolyticbasidiomycetes,EnzymeMicrobialTechnol.25(8),605612.ElAshmawy,A.E.,
ElSaied,H.,andIbrahim,A.A.(1984).Alkalinepulpingof
bagassewithanthraquinone,Holzforschung38(5),289292.Eriksson,K.E.L.,Blanchette,
R.A.,andAnder,P.(1990).MicrobialandEnzymatic
DegradationofWoodandWoodComponents,SpringerVerlag,Berlin.Fischer,K.,Akhtar,
M.,Blanchette,R.A.,Burnes,T.A.,Messner,K.,andKirk,T.K.
(1994).Reductionofresincontentinwoodchipsduringexperimentalbiologicalpulping
processes,Holzforschung48(4),285290.Garmaroody,E.R.,Resalati,H.,Fadim,P.,Hosseini,
S.Z.,Rahnama,K.,Saraeeyan,A.
R.,andMirshokraee,S.A.(2011).Theeffectsoffungipretreatmentofpoplarchipsonthe
kraftfiberproperties,BioresourceTechnology102,41654170.Goering,H.K.,andVanSoest,
P.J.(1971).ForageFiberAnalysis,USDAARS
AgricultureHandbook,GovernmentPrintingOffice,Washington,DC.Hall,T.A.(1999).
BioEdit:Auserfriendlybiologicalsequencealignmenteditorand
analysisprogramforWindows95/98/NT,NucleicAcidsSymposiumSeries41,9598.Isroi,
Millati,R.,Syamsiah,S.,Niklasson,C.,Cahyanto,M.N.,Lundquist,K.,and
Taherzadeh,M.J.(2011).Biologicalpretreatmentoflignocelluloseswithwhiterotfungiandits
applications:Areview.BioResources6(4),52245259.Kirk,T.K.,andFarrell,R.L.(1987).
Enzymaticcombustion:Themicrobialdegradationoflignin,AnnualReviewof
Microbiology41(1),465501.Lowry,O.H.,Rosebrough,N.J.,Farr,A.L.,andRandall,R.J.
(1951).Proteinmeasurementwiththefolinphenolreagent,J.Biol.Chem.193(1),265275.
Martinez,A.T.,Camarero,S.,Guillen,F.,Gutierrez,A.,Munoz,C.,Varela,E.,
Martinez,M.J.,Barrasa,J.M.,Ruel,K.,andPelayo,J.M.(1994).Progressinbiopulpingof
nonwoodymaterials:Chemical,enzymaticandultrastructuralaspectofwheatstraw
delignificationwithligninolyticfungifromthegenusPleurotus,FEMSMicrobiologyReviews
13(2/3),265274.Messner,K.,andSrebotnik,E.(1994).Biopulping:Anoverviewof
developmentsinan
environmentallysafepapermakingtechnology,FEMSMicrobiologyReviews13(2/3),351
364.
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4978
 bioresources. PEER-REVIEWED ARTICLE

com
Mosai,S.,Wolfaardt,J.F.,Prior,B.A.,andChristov,L.P.(1999).Evaluationof
selectedwhiterotfungiforbiosulfitepulping,BioresourceTechnology68(1),8993.
Oriaran,T.P.,Labosky,P.,andBlankenhorn,P.R.(1990).Kraftpulppapermaking
propertiesofPhanerochaetechrysosporiumdegradedaspen,TappiJ.73(7),147152.Page,R.
D.M.(1996).Treeview:Anapplicationtodisplayphylogenetictreeson
personalcomputers,CABIOS12(4),357358.Rodrigues,M.A.M.,Pinto,P.,Bezerra,R.
M.F.,Dias,A.A.,andGuedes,C.V.M.(2008).Effectofenzymeextractsisolatedfromwhite
rotfungionchemicalcompositionandinvitrodigestibilityofwheatstraw,Anim.FeedSci.
Technol.141,326338.Shukla,O.P.,Rai,U.N.,andSubramanian,S.V.(2004).Biopulping
andbiobleaching:
AnenergyandenvironmentsavingtechnologyforIndianpulpandpaperindustry,EnviroNews
NewsletterofISEBIndia10(2),78.Singh,D.,andChen,S.(2008).Thewhiterotfungus
Phanerochaetechrysosporium:Conditionsfortheproductionoflignindegradingenzymes,
Appl.Microbiol.Biotechnol.81C,399417.Singh,P.,Sulaiman,O.,Hashim,R,Rupani,P.F.,
andPeng,L.C.(2010).Biopulping
oflignocellulosicmaterialusingdifferentfungalspecies:Areview,Rev.Environ.Sci.
Biotechnol.9,141151.Scott,G.M.,Akhtar,M.,Lentz,M.J.,Sykes,M.,andAbubakr,S.
(1996).BiosulphitepulpingusingCeriporiopsissubvermispora,in:BiotechnologyinthePulp
andPaperIndustry,E.SrebotnikandK.Messner(eds.),FacultasUniversitatsVerlag,Vienna.
Scott,G.M.,Akhtar,M.,Swaney,R.E.,andHoutman,C.G.(2002).Recent
developmentinbiopulpingtechnologyatMadison.WI,In:ProgressinBiotechnology,L.
ViikariandR.Lantto(eds).Elsevier,6171.Taniguchi,M.,Suzuki,H.,Watanaba,D.,Sakai,K.,
Hoshino,K.,andTanaka,T.(2005).EvaluationofpretreatmentwithPleurotusostreatusfor
enzymatichydrolysisofricestraw,JournalofBioscienceandBioengineering100(6),637643.
Tasman,J.E.,andBerzins,V.(1957).Thepermanganateconsumptionofpulpmaterials
II:Thekappanumber,TappiJ.40(9),695699.Tien,M.,andKirk,T.K.(1988).Lignin
peroxidaseofPhanerochaetechrysosporium,
MethodsinEnzymology161,238249.Turenne,C.Y.,Sanche,S.E.,Hoban,D.J.,
Karlowsky,J.A.,andKabani,A.M.(1999).RapididentificationoffungibyusingtheITS2
geneticregionandanautomatedfluorescentcapillaryelectrophoresissystem,Journalof
ClinicalMicrobiology37(6),18461851.White,T.J.,T.D.Burns,S.B.Lee,andJ.W.Taylor.
1990.Amplificationanddirect
sequencingoffungalribosomalRNAgenesforphylogenetics,in:M.A.Innis,D.H.Gelfand,J.
J.Sninsky,andT.J.White(eds.),PCRProtocols,AcademicPress,SanDiego,Calif,pp.315
322.Wulandari,A.P.,Triyana,T.,andAndayaningsih,P.(2013).Delignificationofrice
strawwithligninasefromnovelPenicilliumsp.strainapwtt2forbiopulping,International
JournalofBioscience,BiochemistryandBioinformatics3(1),4346.Xu,G.,Wang,X.,andHu,
J.(2013).Biobleachingofwheatstrawpulpusinglaccase
andxylanase,BioResources8(3),31813188.
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4979
 bioresources. PEER-REVIEWED ARTICLE

com
Yadav,R.D.,Chaudhry,S.,andDhiman,S.S.(2010).Biopulpinganditspotentialto
reduceeffluentloadsfrombleachingofhardwoodkraftpulp,BioResources3(1),159171.
Yaghoubi,K.,Pazouki,M.,andShojaosadati,S.A.(2008).Variableoptimizationfor
biopulpingofagriculturalresiduesbyCeriporiopsissubvermispora,BioresourceTechnology
99(10),43214328.
Articlesubmitted:June6,2013;Peerreviewcompleted:July18,2013;Revisedversion
received:August5,2013;Accepted:August6,2013;Published:August9,2013.
Badr El-Din et al. (2013). Fungi for straw biopulping, BioResources 8(4), 4969-4980. 4980