research article screening of lignocellulose-degrading superior...

Research ArticleScreening of Lignocellulose-Degrading Superior MushroomStrains and Determination of Their CMCase and Laccase Activity

Li Fen1 Zhu Xuwei1 Li Nanyi2 Zhang Puyu1 Zhang Shuang1 Zhao Xue1

Li Pengju1 Zhu Qichao3 and Lin Haiping1

1 The Nurturing Station for the State Key Laboratory of Subtropical Silviculture Provincial Engineering Laboratory ofBiopesticide Preparation Zhejiang AampF University Linrsquoan 311300 China

2 School of Agricultural and Food Science Zhejiang AampF University Linrsquoan 311300 China3Qingyuan Forestry Bureau of Zhejiang Qingyuan 323800 China

Correspondence should be addressed to Lin Haiping zjlxylhp163com

Received 10 November 2013 Accepted 24 December 2013 Published 12 February 2014

Academic Editors C Cravo-Laureau and K Ohmiya

Copyright copy 2014 Li Fen et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In order to screen lignocellulose-degrading superior mushroom strains ten strains of mushrooms (Lentinus edodes939 Pholiotanameko Lentinus edodes868 Coprinus comatus Macrolepiota procera Auricularia auricula Hericium erinaceus Grifola frondosaPleurotus nebrodensis and Shiraia bambusicola) were inoculated onto carboxymethylcellulose agar-Congo red plates to evaluatetheir ability to produce carbomethyl cellulase (CMCase) The results showed that the ratio of transparent circle to mycelium circleof Hericium erinaceus was 816 (119875 lt 001) higher than other strains The filter paper culture screening test showed that Hericiumerinaceus andMacrolepiota procera grew well and showed extreme decomposition of the filter paper When cultivated in guaiacolculture medium to detect their abilities to secrete laccase Hericium erinaceus showed the highest ability with the largest reddishbrown circles of 4330 cm CMCase activity determination indicated that Coprinus comatus andHericium erinaceus had the abilityto produce CMCasewith 3392UL on the 9th day and 2258UL on the 10th day respectively whileCoprinus comatus and Pleurotusnebrodensis had the ability to produce laccase with 49667UL and 48917UL on the 16th day and 18th day Based on the resultsCoprinus comatusmight be the most promising lignocellulose-degrading strain to produce both CMCase and laccase at high levels

1 Introduction

Lignocellulose is an abundant and renewable source of carbo-hydrates that can be converted into value-added products inthe earth It is estimated that 60 times 109 tons of lignocelluloseare generated by photosynthesis each year all over the worldbut only about 20 are used for conversion into energyand food [1] Many lignocellulose litters such as straws andsawdusts are produced each year in industry and agricultureTherefore it is of great importance to make these wastesavailable to reduce environmental pollution and stabilize thedevelopment of bioenergy [2]

Agricultural residues are rich in lignocellulosic com-pounds whose disposal and handling are problematic dueto their complex structure and decomposition properties [34] Lignin wraps around cellulose and hemicelluloses fiberswhich inhibits the degradation of both so it is necessary

to remove the lignin to facilitate the use of cellulose andhemicelluloses as a source for bioenergy Recently chemicaland physical methods of pretreating lignocellulosic com-pounds have been used to expose the underlying celluloseand hemicelluloses such as radicalization steam explosionpuffing acid and alkali [5] Unfortunately these methodsconsume high amounts of energy and cause pollutants [6]So it is pressing to find a high-efficiency energy-savingand environment-friendly way to break down cellulose andhemicelluloses

Somemicroorganisms especially some edible andmedic-inal mushrooms produce a complete set of enzymes capableof efficient degradation of native cellulose and lignin soscreening of lignocellulose-degrading superior mushroomstrains has become a significant project in the process ofreusing lignocellulose wastes Enzymes which are responsible

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 763108 6 pageshttpdxdoiorg1011552014763108

2 The Scientific World Journal

Table 1 Ratio of transparent circle to mycelium circle of 10 strains under CMC culture

Strains Transparent circle diameter (1198891)cm Mycelium circle diameter (1198892)cm 11988911198892Lentinus edodes939 387 plusmn 022 349 plusmn 009 111 plusmn 006C

Pholiota nameko 518 plusmn 011 504 plusmn 027 103 plusmn 006C

Lentinus edodes868 478 plusmn 022 384 plusmn 022 124 plusmn 006C

Coprinus comatus 334 plusmn 008 242 plusmn 022 139 plusmn 017C

Macrolepiota procera 432 plusmn 021 232 plusmn 030 188 plusmn 027BC

Auricularia auricula 141 plusmn 010 062 plusmn 017 230 plusmn 030B

Hericium erinaceus 495 plusmn 049 061 plusmn 009 816 plusmn 056A

Grifola frondosa 384 plusmn 019 170 plusmn 011 226 plusmn 005B

Pleurotus nebrodensis 000 plusmn 000 276 plusmn 051 000 plusmn 000D

Shiraia bambusicola 000 plusmn 000 591 plusmn 072 000 plusmn 000D

Different capital letters followed by the data in the table mean the significant difference at 001 levelThe data represent the average of three independent experiments

for cellulose degradation are hydrolytic [7] The cellulose-hydrolysing enzymes (ie cellulases) are divided into threemajor groups endoglucanases cellobiohydrolases (exoglu-canases) and 120573-glucosidases [8 9] Lignin degradationmainly depends on oxidative enzymes [10] including ligninperoxidase manganese-dependent peroxidase and laccasewhich are well studied In wood-destroying white rot fungilaccases are the most important components of the lignolyticcomplex responsible for decomposition of lignin whichis one of the most-difficult-to-be-decomposed polymers innature [11 12]

Many researches focus on the fields of screening oflignocellulose-degrading superior strains while many othersfocus on high production mushrooms all over the world butvery few put the two areas together At present microbesused in cellulose degradation are mainly fungi of which Tri-chodermaAspergillus Rhizopus andMyrothecium have beenstudied the most [3] Since edible and medicinal fungi canconvert lignincellulose to form fruiting bodies it indicatedthey can decompose lignocellulose to micromolecular nutri-ent substance for fungi to be utilizedThus studies on screen-ing of lignocellulose-degrading superior edible fungi play animportant role [13] In this study 10 strains of mushroomswere investigated for their ability to degrade lignocelluloseThe purpose and significance of the research are obtainingsome strains of mushrooms that have high ability to degradelignocellulose So in the process of cultivating them onlignocellulose littersmushroomproduction and rawmaterialto produce bioenergy could be obtained at the same time

2 Materials and Methods

21 Fungal Strains and Culture Media Ten commercialstrains (Lentinus edodes939 ACCC51655 Pholiota namekoACCC51896 Lentinus edodes868 ACCC51717 Coprinus com-atus ACCC51773 Macrolepiota procera ACCC51531 Auric-ularia auriculaACCC51604Hericium erinaceusACCC51322Grifola frondosa ACCC51962 Pleurotus nebrodensisACCC51498 and Shiraia bambusicolaACCC50027 (Agricul-tural Culture Collection of China)) were subjected to screen-ing experiments

All strains were seeded on potato dextrose agar medium(PDA potato 200mgmL glucose 20mgmL and agar 20mgmL) slants and discs and incubated at 26∘C for 7ndash10 days untilcolonies appeared which were prepared for the followingexperiments

Carboxymethylcellulose (CMC)-agar culture mediumcontained 10mgmL sodium CMC 4mgmL (NH

4)2SO4

2mgmL KH2PO4and 20mgmL agar [14] in which sodium

carboxymethylcellulose (CMCNa) was the sole carbon sourcefor the fungus

Filter paper culture medium contained 1mgmL(NH4)2SO4

1mgmL KH2PO4 07mgmL MgSO

4sdot7H2O

05mgmLNaCl and 6 cm times 1 cm filter paper [14 15]CMCase production medium contained 10mgmL

CMCNa 4mgmL (NH4)2SO4 2mgmL KH

2PO4 05mg

mL MgSO4sdot7H2O 10mgmL peptone and 5mgmL beef

extract [3]Guaiacol color culture medium contained 10mgmL

guaiacol 01mgmL C4H12N2O6 26mgmL peptone

05mgmL MgSO4sdot7H2O 1mgmL KH

2PO4 02mgmL

Na2HPO4 and 20mgmL agar [16]

Laccase production medium was equal to PDA withoutagar

All media were autoclaved at 121∘C for 20 minutes

22 CMC Culture Screening Five mm disc diameter wastaken from the PDA plates as inoculated on CMC platesand then grew at 26∘C for about one week until myceliumwas proper to measure Afterwards the plates were dyed byadding 20mL Congo red (1mgmL) and three hours later1molLNaCl was added to decolorize them The diametersof transparent circle and mycelium circle on each plate weremeasured

The ratio of transparent circle to colony diameter wasthen calculated to estimate the ability of the fungi to degradecellulose [17] The experiment was repeated three times Thedata presented in Table 1 represent mean values plusmn standarddeviation

23 Filter PaperCulture Screening Each 15times 150mmtest tubewas filled with 5mL filter paper culture medium so that there

The Scientific World Journal 3

005

115

225

335

445

5

a b c d e f g h i j

Dia

met

er o

f red

The kind of strains

Series1

brow

n ci

rcle

(cm

)

Figure 1 Diameter of red brown circle of 10 strains One week afterinoculating on guaiacol color culture media a Hericium erinaceusbCoprinus comatus c Lentinus edodes868 d Lentinus edodes939 ePleurotus nebrodensis fAuricularia auricular gPholiota nameko hMacrolepiota procera i Shiraia bambusicola and j Grifola frondosaThe data represent the average of three independent experiments

for sure was 1 cm of 6 times 1 cm filter paper out of the liquidStrains which were inoculated into the filter paper culturemediumwere kept at the surface of the liquid and in the filterpaper and then cultivated statically at 26∘C for about half amonth The experiment was repeated three times

24 Guaiacol Color Culture Screening One mycelium agardisc (five mm diameter) cut from the growing edge of eachstrain PDA culture was used to inoculate each plate andincubated at 26∘C for about one week and then monitoredfor the appearance of a reddish brown circle The diameterof the reddish brown circle was measured to identify laccasesecreting ability of the stains The experiment was repeatedthree timesThe data presented in Figure 1 represent values plusmnstandard deviation

25 Fermentation Superior cellulose degradation strainswere selected based on plate culture results and inoculatedinto 250mL Erlenmeyer flasks containing 50mL of CMCaseproduction medium Five mycelium agar discs (five mmdiameter) cut from the growing edge of each strain 7-dayPDA culture were used to inoculate each flask The strainswere cultivated in a rotary shaker at 120 revsdotminminus1 at 26∘C andwere sampled each day for five days All experiments wereperformed with three replicates

26 Enzyme Assay Carboxymethylcellulase (CMCase) wasestimated by using 1 carboxymethyl cellulose as the solecarbon sourceThe reactionmixture was composed of 02mLenzyme sample and 18mL CMC (1) dissolved in 01molLsodium acetate buffer (pH 48) The reaction was kept at50∘C for 30 minutes The amount of reducing sugar wasdetermined by the DNS(35-dinitrosalicylic) method [18]using glucose as a standard at 550 nm As a control 18mLof sodium acetate buffer was used instead of CMC followingthe same treatment [19] Enzymatic activity expressed asUmL was defined as the amount of enzyme producing 1 120583gof product per minute per mL of substrate extracted Each

05

10152025303540

5 6 7 8 9 10

CMCa

se ac

tiviti

es (U

L)

Time (day)

Coprinus comatusMacrolepiota proceraPholiota namekoGrifola frondosa

Heircium erinaceusLentinus edodes868Auricularia auricula

Figure 2 Time courses of CMCase activity of 7 strains Fivemycelium agar discs (5mm diameter) were used as the inoculumCultivated in CMC liquidmedium 1mL Zymotic fluid of the strainswas taken out of the flask for experiment each dayThedata representthe average of three independent experiments

0

100

200

300

400

500

600

4 6 8 10 12 14 16 18 20

Lacc

ase a

ctiv

ity (U

L)

Time (day)

Heircium erinaceusCoprinus comatus

Pleurotus nebrodensisLentinus edodes868

Figure 3 Time courses of laccase activity of 4 strains Fivemyceliumagar discs (5mm diameter) were used as the inoculum Cultivatedin potato dextrose liquid medium 1mL Zymotic fluid of the strainswas taken out of the flask for experiment each dayThedata representthe average of three independent experiments

same experiment was repeated three timesThe data in Figure2 represent the average of three independent experiments

The strains which showed the capacity to secrete laccasein guaiacol color culture screening were used to deter-mine laccase activity and were conducted in 3mL reac-tion mixtures consisting of 27mL of 01molL sodiumacetate buffer (pH 45) 02mL of 1mmolL 2201015840-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) solution and01mL extracellular culture supernatant The reaction wasmonitored bymeasuring the change in 420 nm for 3minOneunit of enzyme activity is defined as the amount of oxidationof ABTS followed by 01 increase in absorbance at 420 nmThe molar absorption coefficient of oxidized ABTS is 36 times104 L(molsdotcm) [1] Each same experiment was repeated threetimes The data in Figure 3 represent the average of threeindependent experiments


3 Results

31 CMC Culture and Filter Paper Culture Screening Mush-rooms were cultivated in CMC-agar culture to determinetheir ability to use CMCNa as the sole carbon source Allstrains tested showed different mycelial growth in CMCculture Based on estimations of their mycelium diame-ters Shiraia bambusicola grew best with mycelium growthdiameter of 591 cm followed by Pholiota nameko Lentinusedodes868 and Lentinus edodes939 where mycelium growthdiameters were 504 cm 384 cm and 349 cm respectivelyHericium erinaceus had the smallest mycelium growth diam-eter of 061 cm followed byAuricularia auricularwith 062 cmmycelium diameter However a rather different case wasobserved in the transparent circles that represent cellulosedegradation Shiraia bambusicola and Pleurotus nebrodensisshowed no transparent circle and Auricularia auricula with141 cm transparent circle became the third inferior candidatewhile Pholiota nameko followed by Hericium erinaceus hadthe largest transparent circles (518 cm and 495 cm resp)To sum up Hericium erinaceus had the highest ratio oftransparent circle diameter to mycelium circle diameter(d1d2) with the datum of 816 Big differences were observedamong d1d2 of 10 different strains (Table 1)

The filter paper culture screening test showed that allstrains grew slowly with evidence of mycelium extension inapproximately half a month The growth results also variedfrom strain to strain Hericium erinaceus and Macrolepiotaprocera grew well in the filter paper culture medium andshowed extreme decomposition of the filter paper whileGrifola frondosa Shiraia bambusicola and Coprinus coma-tus showed scarce biomass growth and without observabledegradation Some strains such as Lentinus edodes868 andMacrolepiota procera showed less than optimal growth butwere still able to decompose the filter paper All strainswere divided into four groups according to the degreeof decomposing the filter paper Hericium erinaceus andMacrolepiota procera decomposed the filter paper in a goodmanner Most of the strains decomposed the filter paperordinarily including Lentinus edodes868 Pleurotus nebroden-sis Lentinus edodes939 Auricularia auricula and Coprinuscomatus While Grifola frondosa and Pholiota nameko faintlydecomposed the filter paper Shiraia bambusicola showed nodegradation ofthe filter paper (Table 2) As shown in Tables1 and 2 the results of CMC culture and filter paper culturescreening experiment are approximately consistent

32 Guaiacol Color Culture Screening Guaiacol is one of thesubstrates of laccase and the degradation product of guaiacolis rufous Therefore we used the red brown color to indicatethe laccase production by the strains and the diameterof the colored circle to estimate the quantity of laccaseproduced All plates showed a reddish brown color after oneweek except for the plate with Grifola frondosa which wasreddish brown the second day after inoculation As timewent by the reddish brown circle expanded and eventuallystabilizedHericium erinaceus Lentinus edodes868 Pleurotusnebrodensis and Coprinus comatus had large reddish browncircles Their reddish brown circle diameters were 433 cm

Table 2 The decomposing degree of 10 strains on filter paper

Strains The decomposing degreeLentinus edodes939 ++b

Pholiota nameko +c

Lentinus edodes868 ++

Coprinus comatus ++

Macrolepiota procera + + +

Auricularia auricula ++

Hericium erinaceus + + +a

Grifola frondosa +Pleurotus nebrodensis ++

Shiraia bambusicola 0d

a+ + + stands for the strain decomposed the filter paper in a good manner

b++ stands for the strain decomposed the filter paper in a common manner

c+ stands for the strain faintly decomposed the filter paper

d0 stands for the strain decomposed no filter paperThe data represent the average of three independent experiments

410 cm 335 cm and 320 cm respectively (Figure 1) So thesefour strains were selected to determine the activity of laccase

33 Determination of CMCase Activity Based on the quan-titative analysis of transparent circles to mycelial growthdiameter we were more interested in the numerical valueof enzyme activity The determination showed that CMCaseactivity of Coprinus comatus was obviously higher thanothers As the incubation time increased CMCase activityalso increased and the peak (3392UL) appeared on day9 Hericium erinaceus showed the second highest CMCaseactivity The variation rule of CMCase activity of Hericiumerinaceus seemed similar to Coprinus comatus Howeverthe activity produced two peaks one on day eight and theother on day 10 Furthermore the CMCase activity increaseddramatically on day 10 by nearly threefold more than thaton day nine and 234-fold of the first peak Pholiota namekoshowed no significant CMCase activity increase during thecultivation Although CMCase activity of Grifola frondosaand Macrolepiota procera gradually increased the level ofactivity remained low It seemed that Auricularia auricularwas deficient in CMCase production (Figure 2) CMCaseactivities of other strains which were not shown in Figure 2were not detectedThe CMCase activity is also approximatelyconsistent with of the results of CMC culture and filter paperculture screening experiment

34 Determination of Laccase Activity The laccase activitiesof superior strains that had been selected based on Guaiacolcolor culture screening are shown chronologically in Figure 3Coprinus comatus and Pleurotus nebrodensis displayed muchhigher laccase activities thanHericium erinaceus andLentinusedodes868 Laccase activities of Coprinus comatus and Pleu-rotus nebrodensis reached their peaks with 49667UL and48917UL at day 16 and day 18 respectively while Hericiumerinaceus reached its peak of 4854UL at day 10 and Lentinusedodes868 reached its peak of 3088UL at day 11


Coprinus comatus has not only the highest CMCaseactivity but also the highest laccase activity It is a kind ofdelicious mushroom at the same time soCoprinus comatus isa promising mushroom strain to be used in the developmentof bioenergy

4 Discussion

Cellulose a homopolysaccharide composed of 120573-D-glucopy-ranose units linked by 120573-(1rarr 4)-glycosidic bonds can beconverted to cellobiose and glucose by cellulase [4] Congored can bind to polysaccharides like cellulose but cannotbind to saccharides to form red compounds Therefore whencellulose was hydrolyzed to monosaccharide the unconju-gated Congo red was eluted by saline solution and then thetransparent circles were apparent in culture plates [20] Ifcellulose was not hydrolyzed it could not bound to Congored and therefore could not be eluted and remained redHankin and Anagnostakis [21] found that transparent circleswere produced around the colonies in some cellulose culturemedium while Teather and Wood [22] hold that thereto some extent existed a linear relationship between thetransparent circle size and cellulase activity However somestrains that were tested in this investigation such as Lentinusedodes939 Lentinus edodes868 and Pholiota nameko showedbigger transparent circles in CMC culture screening but indi-cated no or low activity in the following CMCase activity testOn the other hand Coprinus comatus with inferior results inCMC culture screening showed the highest CMCase activityin carbomethyl cellulase activity (CMCase) determinationAbout d1d2 the situation was similar For exampleCoprinuscomatus had the highest CMCase activity and was obviouslyhigher than others but the d1d2 of Coprinus comatus wasinferior Another case is Hericium erinaceus it had thehighest d1d2 while CMCase activity was second but thelatter increased remarkably after 9 d during fermentationIn addition the results of CMC culture and filter paperculture screening experiment are approximately consistentTherefore the transparent circle d1d2 degradative degreeof filter paper or CMCase activity alone cannot determinethe fungirsquos ability to secrete cellulase It is better to combinethe results of four aspects to choose the cellulose degradingsuperior mushroom strains

Although it indicated that Coprinus comatus and Heri-cium erinaceus were preferential candidates of degradingcellulose it is only under our technique of fermentation So itis necessary to optimize the liquid culture technique in orderto increase the activity of cellulose next stage And at thesame time the cellulase activity of the strains when they growon the solid lignocellulose waste is worthy of being studiedbecause the microorganisms grow under such conditionscloser to their natural habitat and can possibly produce largeramounts or higher activity of extracellular enzymes

In fungi laccase is known as a ligninolytic enzyme andappears to be involved in fruiting body formation fungalplant-pathogenhost interaction and stress defense Sincelaccase plays a major role in fungal degradation of lignolyticcompounds its activity to some extent reflects the ability

of the fungus to decompose lignocellulosic materials Inaddition to a remarkably low substrate specificity laccase hasother properties that make this enzyme potentially usefulfor biotechnological application like laccase does not needthe addition or synthesis of low molecule weight cofactorAlthough laccases have been found in many species theinformation from the edible fungi is less reported [23]

In this report we found that Coprinus comatus andHericium erinaceus had good ability of producing CMCasewhile Coprinus comatus and Pleurotus nebrodensis had goodability of producing laccase So Coprinus comatus was ableto produce both enzymes at high levels In recent years Itis found that Hericium erinaceus is capable of biodegradingstraw [24] These results may provide a useful tool forscreening of high laccase-producing and CMCase-producingstrains and offer a fundamental method to study the laccaseproduction capacity of fungi

As the world energy crisis is becoming increasinglysevere it is necessary and urgent to find a new energy tosubstitute or complement petroleum energy Undoubtedlylignocellulose is the optimal candidate Since microbes playa crucial rule in the process of conversion of lignocelluloseto bioenergy this report may offer a promising mushroomstrain to use in the development of bioenergy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by Natural ScienceFunding Proposal of Zhejiang Province (LY12C14005)

References

[1] S J Sun J Z Liu KHHu andH X Zhu ldquoThe level of secretedlaccase activity in the edible fungi and their growing cycles areclosely relatedrdquoCurrentMicrobiology vol 62 no 3 pp 871ndash8752011

[2] S A Furlan L J Virmond D A Miers M Bonatti R M MGern and R Jonas ldquoMushroom strains able to grow at hightemperatures and low pH valuesrdquoWorld Journal ofMicrobiologyand Biotechnology vol 13 no 6 pp 689ndash692 1997

[3] L R Han S X Zhang C S Zhu and X Zhang ldquoScreening andidentification of superior fungus degraded celluloserdquo Journal ofNorthwest AampFUniversity vol 36 pp 169ndash174 2008 (Chinese)

[4] O S Isikhuemhen N A Mikiashvili and V Kelkar ldquoAppli-cation of solid waste from anaerobic digestion of poultrylitter in Agrocybe aegerita cultivation mushroom productionlignocellulolytic enzymes activity and substrate utilizationrdquoBiodegradation vol 20 no 3 pp 351ndash361 2009

[5] H PDo RQ Song andYQWang ldquoComparative study on thelignocellulolytic enzymes produced by fungirdquo Forestry Scienceamp Technology vol 31 pp 20ndash24 2006

[6] M S Chandra B Viswanath and B R Reddy ldquoCellulolyticenzymes on lignocellulosic substrates in solid state fermenta-tion by Aspergillus nigerrdquo Indian Journal of Microbiology vol47 no 4 pp 323ndash328 2007


[7] T Kajisa K Igarashi and M Samejima ldquoThe genes encodingglycoside hydrolase family 6 and 7 cellulases from the brown-rot fungus Coniophora puteanardquo Journal of Wood Science vol55 no 5 pp 376ndash380 2009

[8] R Kumar S Singh and O V Singh ldquoBioconversion of lig-nocellulosic biomass biochemical and molecular perspectivesrdquoJournal of IndustrialMicrobiology and Biotechnology vol 35 no5 pp 377ndash391 2008

[9] Y Liang J Yesuf S Schmitt K Bender and J Bozzola ldquoStudyof cellulases from a newly isolated thermophilic and cellulolyticBrevibacillus sp strain JXLrdquo Journal of Industrial Microbiologyand Biotechnology vol 36 no 7 pp 961ndash970 2009

[10] T K Kirk and D Cullen ldquoEnzymology and molecular geneticsof wood degradation by white-rot fungirdquo in EnvironmentallyFriendly Technologies for the Pulp and Paper Industry R AYoung and M Akhtar Eds pp 273ndash308 John Wiley and SonsNew York NY USA 1998

[11] O V Morozova G P Shumakovich M A Gorbacheva S VShleev and A I Yaropolov ldquoldquoBluerdquo laccasesrdquo Biochemistry vol72 no 10 pp 1136ndash1150 2007

[12] G Elegir D Bussini S Antonsson M E Lindstrom and LZoia ldquoLaccase-initiated cross-linking of lignocellulose fibresusing a ultra-filtered lignin isolated from kraft black liquorrdquoApplied Microbiology and Biotechnology vol 77 no 4 pp 809ndash817 2007

[13] P Rani N Kalyani and K Prathiba ldquoEvaluation of lignocel-lulosic wastes for production of edible mushroomsrdquo AppliedBiochemistry and Biotechnology vol 151 no 2-3 pp 151ndash1592008

[14] X G Wang Q Yang and H Yan ldquoScreening and identificationfor cellulose degradation of strainrdquoHeilongjiang Animal Scienceand Veterinary Medicine vol 8 pp 70ndash71 2007 (Chinese)

[15] S Jahangeer N Khan S Jahangeer et al ldquoScreening andcharacterization of fungal cellulases isolated from the nativeenvironmental sourcerdquo Pakistan Journal of Botany vol 37 no3 pp 739ndash748 2005

[16] H Y Yu G M Zeng G H Huang T J Hu and Y N ChenldquoScreening of lignin- degrading fungi and their enzyme produc-tionrdquo Chinese Journal of Applied amp Environmental Biology vol10 no 5 pp 639ndash642 2004 (Chinese)

[17] Y S Lin ldquoSeparate and screen fungus which can biodegradecellulose and assay its enzyme activityrdquo Acta Scientiarum Nat-uralium Universitatis Sunyatseni vol 43 pp 82ndash85 2004

[18] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959

[19] G K Han H P Sang H K Seong G P Hyuk and M P WonldquoDetection and recovery of hydrolytic enzymes from spentcompost of four mushroom speciesrdquo Folia Microbiologica vol50 no 2 pp 103ndash106 2005

[20] C Gao J Y Meng and F Y Feng ldquoSeparating and screeningon the cellulose-decomposing bacteria and study on its enzymeactivityrdquo Animal Husbandry and Feed Science vol 4 pp 17ndash202008 (Chinese)

[21] L Hankin and S L Anagnostakis ldquoSolid media containingcarboxymethylcellulose to detect Cx cellulase activity of microorganismsrdquo Journal of General Microbiology vol 98 no 1 pp109ndash115 1977

[22] RM Teather and P JWood ldquoUse of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolyticbacteria from the bovine rumenrdquo Applied and EnvironmentalMicrobiology vol 43 no 4 pp 777ndash780 1982

[23] W S Zhang X L Luo L Zhu Y S Xiong and P Zhu ldquoTheresearch review of laccase in the edible fungusrdquo Edible Fungi ofChina vol 28 pp 8ndash10 2009 (Chinese)

[24] Y R Li G Y Zhou Q X Hu and Z S Feng ldquoResearch advanceon biodegradation of lignin by mushroomrdquo Edible Fungi ofChina vol 28 pp 3ndash6 2009 (Chinese)

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Table 1 Ratio of transparent circle to mycelium circle of 10 strains under CMC culture

Strains Transparent circle diameter (1198891)cm Mycelium circle diameter (1198892)cm 11988911198892Lentinus edodes939 387 plusmn 022 349 plusmn 009 111 plusmn 006C

Pholiota nameko 518 plusmn 011 504 plusmn 027 103 plusmn 006C

Lentinus edodes868 478 plusmn 022 384 plusmn 022 124 plusmn 006C

Coprinus comatus 334 plusmn 008 242 plusmn 022 139 plusmn 017C

Macrolepiota procera 432 plusmn 021 232 plusmn 030 188 plusmn 027BC

Auricularia auricula 141 plusmn 010 062 plusmn 017 230 plusmn 030B

Hericium erinaceus 495 plusmn 049 061 plusmn 009 816 plusmn 056A

Grifola frondosa 384 plusmn 019 170 plusmn 011 226 plusmn 005B

Pleurotus nebrodensis 000 plusmn 000 276 plusmn 051 000 plusmn 000D

Shiraia bambusicola 000 plusmn 000 591 plusmn 072 000 plusmn 000D

Different capital letters followed by the data in the table mean the significant difference at 001 levelThe data represent the average of three independent experiments

for cellulose degradation are hydrolytic [7] The cellulose-hydrolysing enzymes (ie cellulases) are divided into threemajor groups endoglucanases cellobiohydrolases (exoglu-canases) and 120573-glucosidases [8 9] Lignin degradationmainly depends on oxidative enzymes [10] including ligninperoxidase manganese-dependent peroxidase and laccasewhich are well studied In wood-destroying white rot fungilaccases are the most important components of the lignolyticcomplex responsible for decomposition of lignin whichis one of the most-difficult-to-be-decomposed polymers innature [11 12]

Many researches focus on the fields of screening oflignocellulose-degrading superior strains while many othersfocus on high production mushrooms all over the world butvery few put the two areas together At present microbesused in cellulose degradation are mainly fungi of which Tri-chodermaAspergillus Rhizopus andMyrothecium have beenstudied the most [3] Since edible and medicinal fungi canconvert lignincellulose to form fruiting bodies it indicatedthey can decompose lignocellulose to micromolecular nutri-ent substance for fungi to be utilizedThus studies on screen-ing of lignocellulose-degrading superior edible fungi play animportant role [13] In this study 10 strains of mushroomswere investigated for their ability to degrade lignocelluloseThe purpose and significance of the research are obtainingsome strains of mushrooms that have high ability to degradelignocellulose So in the process of cultivating them onlignocellulose littersmushroomproduction and rawmaterialto produce bioenergy could be obtained at the same time

2 Materials and Methods

21 Fungal Strains and Culture Media Ten commercialstrains (Lentinus edodes939 ACCC51655 Pholiota namekoACCC51896 Lentinus edodes868 ACCC51717 Coprinus com-atus ACCC51773 Macrolepiota procera ACCC51531 Auric-ularia auriculaACCC51604Hericium erinaceusACCC51322Grifola frondosa ACCC51962 Pleurotus nebrodensisACCC51498 and Shiraia bambusicolaACCC50027 (Agricul-tural Culture Collection of China)) were subjected to screen-ing experiments

All strains were seeded on potato dextrose agar medium(PDA potato 200mgmL glucose 20mgmL and agar 20mgmL) slants and discs and incubated at 26∘C for 7ndash10 days untilcolonies appeared which were prepared for the followingexperiments

Carboxymethylcellulose (CMC)-agar culture mediumcontained 10mgmL sodium CMC 4mgmL (NH

4)2SO4

2mgmL KH2PO4and 20mgmL agar [14] in which sodium

carboxymethylcellulose (CMCNa) was the sole carbon sourcefor the fungus

Filter paper culture medium contained 1mgmL(NH4)2SO4

1mgmL KH2PO4 07mgmL MgSO

4sdot7H2O

05mgmLNaCl and 6 cm times 1 cm filter paper [14 15]CMCase production medium contained 10mgmL

CMCNa 4mgmL (NH4)2SO4 2mgmL KH

2PO4 05mg

mL MgSO4sdot7H2O 10mgmL peptone and 5mgmL beef

extract [3]Guaiacol color culture medium contained 10mgmL

guaiacol 01mgmL C4H12N2O6 26mgmL peptone

05mgmL MgSO4sdot7H2O 1mgmL KH

2PO4 02mgmL

Na2HPO4 and 20mgmL agar [16]

Laccase production medium was equal to PDA withoutagar

All media were autoclaved at 121∘C for 20 minutes

22 CMC Culture Screening Five mm disc diameter wastaken from the PDA plates as inoculated on CMC platesand then grew at 26∘C for about one week until myceliumwas proper to measure Afterwards the plates were dyed byadding 20mL Congo red (1mgmL) and three hours later1molLNaCl was added to decolorize them The diametersof transparent circle and mycelium circle on each plate weremeasured

The ratio of transparent circle to colony diameter wasthen calculated to estimate the ability of the fungi to degradecellulose [17] The experiment was repeated three times Thedata presented in Table 1 represent mean values plusmn standarddeviation

23 Filter PaperCulture Screening Each 15times 150mmtest tubewas filled with 5mL filter paper culture medium so that there


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Acknowledgments


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Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


005

115

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a b c d e f g h i j

Dia

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The kind of strains

Series1

brow

n ci

rcle

(cm

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05

10152025303540

5 6 7 8 9 10

CMCa

se ac

tiviti

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L)

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0

100

200

300

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4 6 8 10 12 14 16 18 20

Lacc

ase a

ctiv

ity (U

L)

Time (day)







3 Results






Pholiota nameko +c


Coprinus comatus ++















4 Discussion









Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


3 Results






Pholiota nameko +c


Coprinus comatus ++















4 Discussion









Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Discussion









Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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