A novel lignin degradation bacterial consortium..


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Anovellignindegradationbacterialconsortiumforefcientpulping
YanxiaWang,QuanLiu,LeiYan,YameiGao,YanjieWang,WeidongWang
CollegeofLifeScienceandTechnology,HeilongjiangBayiAgriculturalUniversity,Daqing163319,PRChina
highlights
Alignindegradationbacterialconsortiumwasscreenedfromsludgeofareedspond.
Approximate60.9%ligninand43.0%hemicelluloses,but2.0%celluloseinreedswasdegraded.
Theconsortiumwasconsistedofculturedsixisolatesandunculturedbacteria.
articleinfo
Articlehistory:
Received23January2013
Receivedinrevisedform8April2013
Accepted8April2013
Availableonline13April2013
AlignindegradationbacterialconsortiumnamedLDCwasscreenedfromthesludgeofareedspondbya
restrictedsubculture.Itcouldbreakdown60.9%lignininreedsat30
Cunderconditionsofstaticculture
within15days.InordertoanalyzethediversityofLDC,plateisolation,16SrDNAclonelibraryand
ARDRA(AmpliedRibosomalDNARestrictionAnalysis)wereperformed.Sixbacterialstrainswereiso-
latedfromLDCandeighteenDNAphylotypeswereidentiedfrom230bacterialanalyzedclones.They
wereclassiedinto
Geovibriothiophilus
sp.(25.2%),
sp.(5.1%),
sp.(5.1%),
sp.(2.2%),
1.Introduction
Ligninisthesecondabundantbiomassthatisassociatedwith
celluloseandhemicelluloseinplantcell.Itscomplicatedaromatic
polymerandhighmolecularweightmakeitresistanttomicroor-
ganisms,resultinginthepreventionofeffectivecelluloseutiliza-
tion(
Ithasbeenshownthatfungihavehighcapabilityoflignindeg-
Correspondingauthor.Tel./fax:+864596819298.
E-mailaddresses:
(Y.Wang),
(W.Wang).
BioresourceTechnology139(2013)113119
Contentslistsavailableat
SciVerseScienceDirect
BioresourceTechnology
journalhomepage:www.elsevi
er.com/locate/biortech
).Thesepreviousstudiesillustratethatbacteriaalsocontrib-
utetolignindegradationaswellasfungus
However,itwasfound
thatbacterialconsortiumsweremoreeffectiveinlignocellulose
degradationthanotherisolates(
InChina,duetotheshortageofwood-pulp,non-woodpulpwas
Inthisstudy,abacterialconsortiumnamedLDCwiththe
capabilityoflignindegradationwasscreenedfromthesludgeof
areedspondinDaqing(China).Ligninandhemicelluloseinreeds
wasdecomposedefcientlybyLDC,however,cellulosewasnotas
efcientlydegradedbyLDC.Themicrobialdiversityofthis
2.1.Ligninmaterials
Guaiacolandalkalilignin(lowsulfonatecontent;Mn10,000;
MW60,000)werepurchasedfromSIGMA(Aldrich).Allthechem-
icalsusedinthisworkwereallanalyticalormolecularbiology
3%NaOHsolutionfor24h,andwashedwithdistilledwateruntil
thepHwas7.0.Thestrawsweredriedat60
Candcutinto
34cminlengthforuse(
2.2.Samplecollection
2.3.Bacterialscreeningandcultivation
Thebasicmediumusedinthisstudywasasfollow:2.5g/L
,1.0g/LKH
,1.0g/LMgSO
,1.0g/LNaCl,0.5g/LCaCl
Traceelementsolution1mL/L(0.16g/LFeCl
6H
O,1.5g/LZnSO
O,0.16g/LCoCl
6H
O,0.15g/LCuSO
5H
O,1.5g/LMnSO
O,0.3g/LH
,0.1g/LNa
2H
O,pH8.0).Soilsamples
weretreatedasfollows.1gofeachsoilsamplewassuspendedin
sterilewaterandshakenat200rpmfor2h.Thesupernatant,
whichcontainedmicroorganisms,wascollectedforuse.The
screeningprocedurewasdividedinto3parts.First,thesamples
treatedintheaforementionedstepsweretreatedwithbasicmed-
iumcontaining9mmol/Lguaiacol.Afterbeingculturedstatically
at30
Cfor15days,thesamplesthatexhibitedacolorchangefrom
transparenttopurplewereregardedashavingpositiveguaiacol
degradationactivity.Thesesamplesweresortedoutforthenext
stepofscreening.Thesesoilsamplesweretransferredtoabasic
mediumcontaining1g/Lalkalilignin(AL).Afterbeingcultured
staticallyat30
FiberAnalyzerinaccordancewith
theprocedureusedby
GoeringandVanSoest(1970)
.Theeffective
samplesweresubsequentlycultured10generationsuntilthedeg-
radationefciencyofligninwasstable.Themosteffectiveenrich-
mentwasselectedastheideallignindegradationcommunityand
analyzed.
2.4.Isolationandidenticationofcultured-bacterialstrainsfromLDC
ThesamplesofLDCforisolationweretakenatstablegrowth
phase.InordertoexplorethebacteriaisolatedfromLDC,weuti-
lizeddifferentmolecularweightsligninfractionsandreeds,took
ten-folddilutionstospreadtheLDConsolidmediumscontaining
thecarbonsourcesguaiacol,AL,andreeds(Thereedstrawswere
pulverized,passedthrough1mmscreens).Afterindividualcolo-
nieswereisolated,theywereidentiedby16SrDNAsequencing.
2.5.DNAextraction,amplicationandcloningof16SrDNA
TotalgenomicDNAwasextractedfromstablegrowthphase
-AGAGTTTGATCCTGGCTCAG-3
);0.5
L50mM
1492R(5
-GGTTACCTTGTTACGACTT-3
);5
L10
PCRbuffer;
LrTaqDNA(5units/
L,TAKARA);and41.5mLddH
O.PCR
wasdoneusingthefollowingprogram:1cycleofinitialdenatur-
ationat94
Cfor10min;30cyclesofdenaturationat94
Cfor
5min,annealingat55
Cfor1min,andextensionat72
Cfor
Table1
CK
0.1
0.2
0.3
Fig.1.
Theresidualweightofcellulose,hemicellulosesandligninin1greedsafter
degradationbyLDC.
2min,andnalelongationat72
Cfor10min.Theamplication
productswereanalyzedona1.5%agarosegel.
PCRproductswerepuriedusingaQIAquickPCRpurication
Kit(Qiagen,UK).TheproductswereligatedintothepGEM-Teasy
vector(Promega,Madison,WI),andclonedinto
Escherichiacoli
TOP10.Thepositivecloneswererandomlypickedbyblue-white
selectionfromtheovernightLBplatescontaining20mg/mL
X-galand200mg/mLIPTG.Therecombinantplasmidswereampli-
edusingthevectoruniversalprimersM1347(5
-CGCCAGGGT
TTTCCCAGTCACGAC-3
)andRV-M(5
-GAGCGGATAACAATT
TCACACAGG-3
).TheprogramforPCRwasperformedusingthe
sameprogramastheonedescribedabove.
ThePCRproductsweredigestedby
Iand
fIandthen
analyzedon2.0%agarosegels.Thetypicalclonesweresequenced
atHUADAGenomicsCompany(China).Allsequencesobtained
2.7.Pulpingandpapermaking
Sincetheligninwasdecomposedsignallywithoutcelluloseand
littlehemicellulosesduringtherst3days,therawpulpingmate-
3.Resultsanddiscussion
3.1.Screeningofbacterialconsortiumwithcapableoflignin
Therewere12outof22samplesthatdisplayedcolorreaction
duringtherststepofscreening.Inthesecondscreeningstep,
therewere3of12sampleswhichhadobviousALdecomposition
ability.Onlyonesamplehadastableandsignicantlignindegra-
dationeffect.Therewas60.9%lignin,43.0%hemicellulose,and2.0%
cellulosedecomposedinreeds(
Fig.1
Previousstudieshaveindicatedthatthecapabilityoflignindeg-
radationbyasinglebacteriumismuchweakerthanLDC.
sp.,whichisknownasanefcientbacteriumforlignin
breakdown,onlydegraded26.75%
L.olgensis
chipsin46days(
3.2.IsolationandidenticationofbacteriafromLDC
Therewere6bacterialstrainsisolatedfromLDCnamedD-1to
D-6thatincludedtwo
Paenibacillus
sp.,two
Pseudomonas
sp.,one
Microbacteriumpumilum
andone
ThemicrobialcompositionofLDCwasanalyzedby16SrDNA
Inaddition,therarefactioncurvereachedsaturation,which
indicatedthatasufcientnumberofbacterialcloneswere
Table2
The16SrDNAsequencesofbacteriaisolatedfromLDCandthesimilarsequencesfromNCBIBLAST.
NameSimilarspeciesAccessionNo.Similarityrate(%)
sp.YIM110206JQ30963898
sp.cloneIAFpp7123GU21413297
sp.RMV1EU57016298
sp.YT0073AB36282497
sp.BSi20387EU33037499
Pseudomonasstutzeri
ATCC17591U2626199
Microbacteriumpumilum
strainHPG1JQ29159499
Microbacteriumpumilum
strainkV-488NR_04133199
Pseudomonasqianpuensis
NR_04353399
sp.H7GQ25427799
analyzedtorepresentthediversityofthemicrobialclonelibrary
Fig.3
3.3.1.Azoarcussp.andThauera
sp.hadahighidentityvaluewith
;theyboth
hadtheabilitytodegradearomaticcompounds.TheOUTsLDC-8
andLDC-9hadsimilarityvaluesof99%with
sp.respectively,bothofwhichwerefoundinadenitrifyingquino-
line-removalbioreactor(
werecapableofbreakingdownlowmolecularweightaromatic
compounds.
Sinceithadanidentityvalueof99%with
Fig.2.
isolatedfromLDCandparallelizedwithD-6,whichcanuseguaia-
colandreedsascarbonsources.EvidencesuggeststhatLDC-18
contributestolowandhigherpolymerizationlignindegradation.
3.3.3.Pseudomonassp.
TheOUTsLDC-13andLDC-17wereassignedto
sp.,whosesimilarityvalueswereall99%.Theywereisolatedfrom
LDCandparallelizedwithstrainsD-3andD-5,whichcanutilize
guaiacol,ALandreedsascarbonsources.Manystudiesonlignin
degradationby
Pseudomonas
sp.Suggestthatitisthemostefcient
lignindegradationbacterium(
TheOUTsLDC-7andLDC-15wereassignedto
,otherwiseknownasSRB(sulfatereducingbacteria).Itwasre-
portedthatSRBcouldmakeuseofsulfate,sulteandthiosulfateas
electronacceptorsandproduceH
TheOUTLDC-16hadasimilarityvalueof99%with
riumpumilum
wasisolatedfromLDCandparallelizedwithD-4,whichcanuse
guaiacolasitscarbonsource.Severalstudiessupportedthat
wasefcientindegradingcelluloseandhemicellulose
maticcompoundsoflignin(
TheOUTsLDC-1,LDC-10andLDC11wereallassignedto
.TheOUTLDC-1hadanidentityvalueof92%with
whichisfoundinthegutofthetermite
TheOUTLDC-6wasclassiedto
withanidentityvalue
of98%.Therewerefewstudiesreportedonthedecompositionof
ligninoraromaticcompoundsby
;however,
wasassignedto
,whichwasreportedtodegradelignin
sp.isolatedfrompulppaperwastecouldde-
grade37%kraftligninbycultureat30
Cfor6days(
EL-Hanafy
TheOUTsLDC-4andLDC-5wereassignedto
Paenibacillus
.LDC-
4hada98%identityvaluewith
sp.YIM110206while
LDC-5hada98%identityvaluewith
sp.RMV1.They
wereisolatedfromLDCandparallelizedwithD-1andD-2which
containedguaiacol,ALandreedsastheircarbonsources.
sp.wasreportedtoproducexylanases(
TheOUTLDC-2istheonlybacteriumwhichwasassignedto
thiophilus
.LDC-2hada98%similaritywith
G.thiophilus
,whichwas
assignedto
Deferribacteres.Deferribacteres
0
2
0
0
1
0
0
4
12
16
OUTs
Fig.3.
RarefactioncurveforthedifferentARDRApattersof16SrDNAclones.
Table3
3.3.10.Unculturedbacteria
TheLDC3,LDC11,LDC12andLDC14representedtheuncul-
turedbacteria;theproportionofthewholeclonewas21.3%.Based
onourdata,theunculturedbacteriamustplaykeyrolesinreed
Basedonaboveresults,LDCiscomposedofvarioustypesof
bacteriawithdifferentresponsibilities.
aremainlypresentforcellulosedegradation,although
theymightalsohavesomeprobablelignindegradationabilities.
ismainlyresponsibleforhemicelluloseandlignin
degradation.Theotherbacteriaareallresponsibleforlignindegra-
dation.Both
Pseudomonas
sp.and
arecapableofdegrading
highpolymerizationligninandaromaticcompounds.
Inadditiontothehighlignindegradationrate,anotherinterest-
ingobservationwasthelowdecompositionofcellulose.Itwasdif-
culttounderstandthatbacteriadidnotprefertousecellulose.
ButafteranalyzingthediversityofLDC,wediscoveredthatthelig-
nindegradingbacteriadominatedthecommunity.Thishasthepo-
tentialtorestrictthegrowthandfunctionofcellulose-degrading
ToinvestigatethefeasibilityofpulpingbyLDCandstudythe
4.Conclusions
AbacteriaconsortiumLDCwasfoundtodegrade60.9%lignin,
43.0%hemicellulosefromreedstraws,andonly2.0%cellulose
basedonvariousscreeningprocedures.Analysisofthediversity
ofLDCrevealedthattherewereatleastninegeneracoexistingin
theconsortiumwhichweremainlyresponsibleforlignindegrada-
investigatedandasimpliedcommunitymodelshouldbecon-
structedtomakeitconvenientforbiopulping.
Acknowledgements
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Science&TechnologyPillarProgramduringtheTwelfthFive-Year
PlanPeriod(GrantNo.2012BAD12B05),ExcellentYouthFounda-
tionofHeilongjiangScienticCommittee(GrantNo.JC201002),
KeyScienceandTechnologyProgramofHeilongjiangProvince
(GrantNo.GA09B504-2),ProgramofInnovationTeaminHeilongji-
angProvinceUniversity(2012TD006),ProgramforNewCentury
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