Changes in N-Transforming Archaea and Bacteria in Soil during the Establishment of Bioenergy ...

12 Pages·2011·1.04 MB·English

Changes in N-Transforming Archaea and Bacteria in Soil
during the Establishment of Bioenergy Crops
Yuejian Mao1,2, Anthony C. Yannarell1,2,3, Roderick I. Mackie1,2,4*
1EnergyBiosciencesInstitute,UniversityofIllinois,Urbana,Illinois,UnitedStatesofAmerica,2InstituteforGenomicBiology,UniversityofIllinois,Urbana,Illinois,United
StatesofAmerica,3DepartmentofNaturalResourcesandEnvironmentalSciences,UniversityofIllinois,Urbana,Illinois,UnitedStatesofAmerica,4DepartmentofAnimal
Sciences,UniversityofIllinois,Urbana,Illinois,UnitedStatesofAmerica
Abstract
Widespread adaptation of biomass production for bioenergy may influence important biogeochemical functions in the
landscape,whicharemainlycarriedoutbysoilmicrobes.Hereweexploretheimpactoffourpotentialbioenergyfeedstock
crops(maize,switchgrass,MiscanthusXgiganteus,andmixedtallgrassprairie)onnitrogencyclingmicroorganismsinthesoil
bymonitoringthechangesinthequantity(real-timePCR)anddiversity(barcodedpyrosequencing)ofkeyfunctionalgenes
(nifH, bacterial/archaeal amoA and nosZ) and 16S rRNA genes over two years after bioenergy crop establishment. The
quantitiesoftheseN-cyclinggeneswererelativelystableinallfourcrops,exceptmaize(theonlyfertilizedcrop),inwhich
the population size of AOB doubled in less than 3 months. The nitrification rate was significantly correlated with the
quantity of ammonia-oxidizing archaea (AOA) not bacteria (AOB), indicating that archaea were the major ammonia
oxidizers.DeepsequencingrevealedhighdiversityofnifH,archaealamoA,bacterialamoA,nosZand16SrRNAgenes,with
229, 309, 330, 331 and 8989 OTUs observed, respectively. Rarefaction analysis revealed the diversity of archaeal amoA in
maizemarkedlydecreasedinthesecondyear.OrdinationanalysisofT-RFLPandpyrosequencingresultsshowedthattheN-
transformingmicrobialcommunitystructuresinthesoilunderthesecropsgraduallydifferentiated.Thusfar,ourtwo-year
studyhasshownthatspecificN-transformingmicrobialcommunitiesdevelopinthesoilinresponsetoplantingdifferent
bioenergycrops,andeachfunctionalgrouprespondedinadifferentway.Ourresultsalsosuggestthatcultivationofmaize
withN-fertilizationincreasestheabundanceofAOBanddenitrifiers,reducesthediversityofAOA,andresultsinsignificant
changesin the structure ofdenitrification community.
Citation:MaoY,YannarellAC,MackieRI(2011)ChangesinN-TransformingArchaeaandBacteriainSoilduringtheEstablishmentofBioenergyCrops.PLoS
ONE6(9):e24750.doi:10.1371/journal.pone.0024750
Editor:JackAnthonyGilbert,ArgonneNationalLaboratory,UnitedStatesofAmerica
ReceivedMay24,2011;AcceptedAugust16,2011;PublishedSeptember14,2011
Copyright:(cid:2)2011Maoetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted
use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwasfundedbytheEnergyBiosciencesInstitute,EnvironmentalImpactandSustainabilityofFeedstockProductionProgramattheUniversity
ofIllinois,Urbana.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction efficiency perennial grasses will result in altered nitrogen-
transformingmicrobialcommunitiesincomparisontothosefound
Bioenergy derived from cellulosic ethanol is a potential underN-fertilized maize.
sustainablealternativetofossilfuel-basedenergy,sincetheenergy The biological nitrogen cycle is one of the most important
from green plants is renewable and largely carbon neutral in nutrient cycles inthe terrestrial ecosystem. It includesfour major
comparison to fossil fuel combustion. Perennial grasses, such as processes: nitrogen fixation, mineralization (decay), nitrification
switchgrass (Panicum virgatum) and Miscanthus6giganteus, with large anddenitrification. Because manyof themicroorganisms respon-
annual biomass production potential, are proposed as biofuel sible for these processes are recalcitrant to laboratory cultivation,
feedstocks that can maximize ethanol production without previous studies of the distribution and diversity of nitrogen-
adversely affecting the market for food crops (e.g. maize). transformingmicroorganismshaveemployedcultivation-indepen-
However, our knowledge of the impacts of various bioenergy dent techniques targeting functional genes: nifH, amoA and nosZ
feedstockproductionsystemsonthesoilmicrobialecosystemisstill genes, which encode the key enzymes in nitrogen fixation,
very limited. The chemistry of perennial crop residues and plant ammonia oxidization and complete denitrification, respectively
root exudates may stimulate or inhibit the growth and activity of [4,5,6,7,8,9].
different fractions of the soil microbial community, and thus the Biologicalnitrogenfixation,whichconvertsatmosphericN into
2
planting of different crops can result in distinct microbial ammoniumthatisavailabletoorganisms,isanimportantnatural
communities [1,2,3]. Differences in management techniques input of available nitrogen in many terrestrial habitats [10].
between traditional row-crop agriculture and perennial biomass Although nitrogen fixation in terrestrial ecosystems is thought to
feedstocks represent different soil disturbance regimes, altered bemainlycarriedoutbythesymbioticbacteriainassociationwith
water use, differing rates of fertilizer application, etc., and these plants,free-livingdiazotrophsinsoilscanplayimportantrolesinN
should have a direct impact on soil microbial dynamics, cycling in a number of ecosystems [11,12]. In average, 2–
subsequently influencing the terrestrial biogeochemical cycles. In 3kgNha21 year21 could be imported by free living N-fixers
particular, we predict that the cultivation of high nitrogen-use [13].Variousfieldexperimentshaveshownthatthebiomassyieldof
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one candidate biofuel feedstock crop, Miscanthus6giganteus, is not It is known that plant species can change the soil microbial
significantlyincreasedbytheadditionofmineralNfertilizer[14]. community [1]. However, while much previous work has
Thelackofresponsetonitrogenfertilizationandthehighbiomass examined the microbial community differences between the
production suggest that biological nitrogen fixation may play an established crops [7,29,34,35,36], less is known about how
important role in supplying the nitrogen needs of Miscanthus [15]. microbial communities in the agricultural soils develop during
Plantspecieshavepreviouslybeenshowntohaveasignificanteffect the transition from one cropping system to another (e.g. annual
on the composition of diazotrophs in the field; for example, row crops to perennial biofuel feedstocks). Thus, to improve our
diazotrophdiversityishigherinsoilunderAcaciatortilisssp.raddiana knowledgeoftheeffectsofbioenergyfeedstockproductiononthe
(aleguminoustree)thanBalanitesaegyptiaca(anon-leguminoustree) complex N-cycling microbial communities of terrestrial ecosys-
[16]. Plant genotype also has a strong effect on the rhizosphere tems, we followed the changes in soil microbial communities
diazotrophsofrice[17].Agronomicpracticescanalsoinfluencesoil during a two-year establishment period of maize, switchgrass,
diazotrophs, e.g. application of N-fertilization can reduce the Miscanthus6giganteus,andmixedtallgrassprairie.Wemonitoredthe
diversity of diazotrophs [17]. Therefore, we hypothesize that the abundanceofkeygenesfornitrogenfixation,ammoniaoxidation
cultivation of maize with inorganic N-fertilizer will reduce the and complete denitrification (nifH, bacterial/archaeal amoA and
abundanceanddiversityofdiazotrophsinthesoilecosystem,while nosZ)aswellasthestructuralchangesoftheseN-cyclinggenesand
biofuelfeedstocksreceivinglittleornoN-fertilizer(e.g.Miscanthus) bacterial/archaeal 16S rRNA genes using real-time PCR and
willencouragethedevelopmentofactivediazotrophiccommunities. barcoded pyrosequencing methods respectively.
Nitrification,whichconvertsammoniumtonitrate,includestwo
steps:ammoniaoxidationtonitrite,andnitriteoxidationtonitrate. Materials and Methods
The production of nitrate in soil not only supplies nutrition for
Study site and sampling
plants, but it can also mobilize nitrogen to groundwater through
nitrate leaching. Ammonia oxidation is the first and rate-limiting The experiment was conducted at the Energy Biosciences
stepofnitrification[18].Itistypicallythoughttobecarriedoutbya Institute’s Energy Farm located southwest of Urbana, Illinois,
few groups in b- and c- Proteobacteria, referred to as ammonia- USA.Miscanthus(Miscanthus6giganteus,MG),switchgrass(Panicum
oxidizing bacteria (AOB) [18]. However, recent environmental virgatum, PV), maize (Zea mays, ZM) and restored tallgrass prairie
metagenomic analyses revealed that ammonia monooxygenase a- (used as control, NP) were planted in the spring of 2008.
subunit (amoA) genes are also present in archaea (AOA) [19], and Miscanthus was replanted in the spring of 2009 due to its poor
archaeal amoA has been shown to be widespread in many growth in 2008. Each crop was planted in a randomized block
environments, e.g. soils, hot springs and marine water design,witha0.7-haplotofeachcroprandomlypositionedwithin
[6,19,20,21,22]. Recent work has found that AOA can be up to four blocks (n=4 for eachcrop). Samples were collected inApril
3000timesmoreabundantinsoilthanAOB[22,23,24],meaning 2008, before planting of these crops, in order to characterize the
thatAOAarethemostabundantammoniaoxidizingorganismsin background soil microbial communities. Bulk soil samples were
soil ecosystems [25]. The soil ammonia oxidizing community is collected at monthly intervals during the growing seasons (June–
known to be influenced by plant types and management, but September2008and2009).10soilcores(0–10cmdepth,1.8cm
differentsegmentsofthiscommunityresponddifferently[24,26,27]. diameter) were collected from each plot and homogenized in an
For example, the abundance and composition of AOB is ethanol-sanitized,plasticbucket.About60gofthewell-mixedsoil
significantlyalteredbylong-termfertilization,butAOAarerarely wasthensubsampledintoa50mLtubeforeachplot,andkepton
affected [24,27]. The nitrification activity in soil ecosystems is iceuntilbroughttothelabandstoredina280uCfreezer.Intotal,
known to be correlated with the abundances and structures of 112 soil samples were collected. Following standard agricultural
ammonia oxidizers [24,28,29]. We therefore hypothesize that practices,onlymaizewasfertilized(17g/m2in2008,20g/m2in
differentbiofuelcroppingsystems,especiallythosethatrelyonN- 2009) with a mixture of urea, ammonia and nitrate (28% UAN).
fertilization,willinfluencethecompositionofammoniumoxidizers Herbicideswereappliedinthesecropsexcepttherestoredtallgrass
prairie:1.56 l/ha ofRoundup (onlyappliedin2008)and4.13l/
insoil,withpotentialconsequencesfornitrificationrates.
ha of Lumax for maize; 1.37l/ha of 2,4-Dichlorophenoxyacetic
Denitrification, which reduces nitrate to N gas, is carried out
2
acidforMiscanthus;1.37l/haof2,4-Dichlorophenoxyaceticacid
byadiversegroupofmicroorganismsbelongingtomorethan60
forswitchgrass in 2008.
generaofbacteria,archaea,andsomeeukaryotes[30].Complete
denitrification involves four steps: NO 2RNO 2RNOR
3 2
N ORN .Theenzymenitrousoxidereductase(encodedbynosZ) DNA extraction and purification
2 2
that reduces N O to N is essential for complete conversion of Soil samples were freeze-dried overnight until completely dry
2 2
NO 2toN .Approximately17TgNisestimatedtobelostfrom andthenmanuallyhomogenizedwithasterilescrewdriver.DNA
3 2
the land surface through denitrification every year [31]. It is was extracted from 0.3g soil using the FastDNA SPIN Kit For
known that the structure and activity of denitrifiers in the Soil (MP Biomedicals) according to manufacturer’s protocol.
terrestrialecosystemcouldbesignificantlyinfluencedbytheplant ExtractedDNAwasthenpurifiedusingCTAB.DNAconcentra-
species[7,29,32].Inastudyofamaize-croppedfield,itwasfound tions were determined using the Qubit quantification platform
thatorganicormineralfertilizerapplicationscouldaffectboththe with Quant-iTTM dsDNA BR Assay Kit (Invitrogen). DNA was
structure and activity of the denitrifying community in the long dilutedto10ng/mLandstoredin280uCfreezerforthefollowing
term, with changes persisting for at least 14 months [33]. The molecular applications.
potential denitrification rate was found to be significantly
correlated to the denitrifier density, as estimated by the Real-time PCR
quantification of nosZ gene copy numbers [34]. Denitrification TheabundancesofnifH,archaealamoA,bacterialamoAandnosZ
releases mineralized nitrogen in the soil ecosystem to the genesinallthesoilsampleswerequantifiedusingreal-timePCR.
atmosphere, and thus, the balance between denitrification and Quantitative real-time PCR was performed according to the
N-fixation, can determine the biologically available N for the methodsmodifiedfrompreviousstudies:nifH(asameasureofN-
biosphere (Arp,2000). fixingbacteria)usedprimersPolF(59-TGCGAYCCSAARGCB
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GACTC-39)andPolR(59-ATSGCCATCATYTCRCCGGA- reverse primer (10 mM), 1.25 ml dNTP Mix (10mM), 2ml DNA
39) [5]; archaeal amoA (as a measure of ammonia-oxidizing template (10 ng/ml). The PCR reaction was performed in a
archaea) used primers Arch-amoAF (59-STA ATG GTC TGG thermocycler(BioRad,Hercules,CA)usinga5-minheatingstep
CTTAGACG-39)andArch-amoAR(59-GCGGCCATCCAT at 94uC followed by 30 cycles of denaturing at 94uC for 1 min,
CTGTATGT-39)[6];bacterialamoA(asameasureofammonia- annealingat60uC(54uCfornifH)for45s,andextensionat72uC
oxidizing bacteria) used primers amoA-1F (59-GGG GTT TCT for1 min,withafinalextensionstepof5 minat72uC.ThePCR
ACT GGT GGT-39) and amoA-2R (59-CCC CTC KGS AAA products were purified by QIAquick PCR Purification Kit
GCCTTCTTC-39)[4];andnosZ(asameasureofdenitrification (QIAGEN) and digested at 37uC overnight in a 20-ml mixture
bacteria) used primers nosZ-F (59-CGY TGT TCM TCG ACA containing 2 ml NEB Buffer (106), 0.5ml AluI/HhaI (20U/ml)
GCC AG-39) [37] and nosZ 1622R (59-CGS ACC TTS TTG and5 mlPCRproduct.5 mlofthedigestedproductwassenttothe
CCS TYG CG-39) [7]. Purified PCR products from a common Core Sequencing Facility (University of Illinois at Urbana-
DNAmixture(equalamountsofDNAfromallsamplescollected Champaign) for fragment analysis. ROX1000 was used as inner
inAugustof2008and2009)wereusedtopreparesample-derived standard.T-RFLPprofileswereanalyzedbyGeneMarker(v1.85)
quantification standards as previously described [38]. The copy accordingtothemanufacturer’sinstruction.Fragmentswithsizes
number of gene in each standard was calculated by DNA between50bpandthelengthofthePCRproductsandpeakarea
concentration(ng/mL,measuredbyQubit)dividedbytheaverage .500 were selected for T-RFLP profile statistical analysis. The
molecularweight(estimatedbasedonthebarcoded-pyrosequenc- profiledatawerenormalizedbycalculatingtherelativeabundance
ingresults)ofthatgene.Incomparisontousingaclone(plasmid) (percentage)ofeachfragment(individualpeakareadividedbythe
as standard, this method avoids the difference of PCR amplifica- total peak area).
tion efficiency between standards and samples caused by the
different sequence composition in the PCR templates (single Barcoded pyrosequencing
sequenceinaplasmidforthestandardvs.mixtureofthousandsof ThesamesamplesusedinT-RFLPwerealsousedinbarcoded
sequencesinasoilsample).The10mLreactionmixturecontained pyrosequencing. The four replicated samples collected from the
5 mLof26PowerSYBRGreenMasterMix(AppliedBiosystems), same crop at the same time were combined in to one composite
0.5 mLofBSA(10mg/mL,NewEnglandBiolabs),0.4mLofeach samplefortheconstructionofsequencelibraries.Altogether,nine
primer(10 mM)and5 ngofDNAtemplate.Real-timeamplifica- samples (one sample for background soil [mixed from the soils
tionwasperformedinanABIPrism7700SequenceDetectorwith fromalltheplotsbefore plantingthese crops],foursamplesfrom
MicroAmpOptical384-WellReactionPlateandOpticalAdhesive eachofthedifferentcropsatAug2008,andfourfromAug2009)
Film (Applied Biosystems) using the following program: 94uC for wereobtained.Furthermore,alloftheseN-transforminggenesof
5 min;40cyclesof94uCfor45s,56uCfor1 min(54uCfornifH the sample collected from MG at the end of the second year
gene),72uCfor1 min.Adissociationstepwasaddedattheendof (MG2)weresequencedtwiceintwodifferentlanestoestimatethe
the qPCR to assess amplification quality. The specificity of the variationofthesequencingmethod.The16SrRNAgeneofZM2
PCR was further evaluated by runningtwenty randomly selected wasalsosequencedtwice.DetailsofprimersandPCRconditions
samples (for each gene) on a 1% (w/v) agarose gel. The usedin thestudyare listed inTableS1.
corresponding real-time PCR efficiency for each of these genes The nifH, archaeal amoA, bacterial amoA, nosZ and 16S rRNA
was estimated based on a two-fold serial dilution of the common genes (V4–V5 region) were amplified using the barcode primers
DNA mixture described above. The qPCR efficiency (E) was PolF/R, Arch-amoAF/R, amoA-1F/2R, nosZ-F/1622R and
calculated according to the equation E=10[21/slope] [39]. U519F/U926R, respectively (primers are shown in Table S1).
TriplicateqPCRrepetitionswereperformedforeachofthegene Theprimers(HPLCpurified,IntegratedDNATechnologies)were
forallthesamples.Thereal-timePCRamplificationefficiencyof designed as 59-Fusion Primer+barcode+gene specific primer-39
nifH,archaealamoA,bacterialamoAandnosZgeneswas1.9060.01, (Fusion Primer A, 59- CGTATCGCCTCCCTCGCGCCAT-
1.9060.06,1.7660.01and1.8260.01,respectively.TheR2ofall CAG-39; Fusion Primer B, 59-CTATGCGCCTTGCCAGC-
thesestandardcurveswashigherthan0.99.Thedetectionlimitof CCGCTCAG-39). The PCR conditions were optimized and
thisreal-time PCR assaywas 10copies/mL. primers with appropriate barcodes (10bp) were selected. The
The copy numbers of these genes per gram of dry soil was barcodesusedforeachprimeraredescribedinNCBISRA,with
calculated by the copy numbers of the gene per ng of DNA accession number SRA023700. The 50mL PCR mixture
multipliedbytheamountofDNAcontainedineachgramofdry contained 10mL of 56Phusion HF Buffer (Phusion GC Buffer
soil.ThequantitiesofthesegeneswerecorrectedassumingaDNA was used for bacterial amoA gene amplification, both buffer
extraction efficiencyof 30%[40,41]. contains 7.5 mM MgCl ), 1 mL of 10mM dNTPs, 2.5mL of
2
10mg/mL BSA, 0.5mL of 2 U/mL Phusion Hot Start DNA
T-RFLP Polymerase (FINNZYMES), 4 mL of 10mM forward/reverse
Thesoilsamplescollectedfromfourreplicated(blocks)plotsof primer mixture and 4 mL of 10ng/mL DNA templates. 1 mL of
thefourcropspriortoplanting,andthenAugustofestablishment 100%DMSOwassupplementedintothePCRmixtureinbacteria
years1and2(2008and2009;48samplesintotal)wereanalyzed amoA gene (GC rich) amplification. The PCR amplification was
by terminal restriction fragment length polymorphism (T-RFLP). performed in a thermal cycler (BioRad, Hercules, CA) using the
ThenifH,archaealamoA,bacterialamoA,nosZgenewereamplified program98uCfor2min;30cyclesof98uCfor10s,60uC(54uC
fromthesesampleswithFAM-labeled(onforwardprimer)primers fornifHgeneand56uCforbacterialamoAgene)for30s,72uCfor
PolF/R, Arch-amoAF/R, amoA-1F/2R and nosZ-F/1622R (see 20s; 72uC for 5 min. The PCR product was first checked on a
TableS1).The16SrRNAgenewasamplified with8F(59-FAM- 1.2% w/v agarose gel, and then purified by QIAquick PCR
AGAGTTTGATCMTGGCTCAG-39) and 1492R (59-GGTT- Purification Kit (QIAGEN). The DNA concentration of the
ACCTTGTTACGACTT-39)[42,43].ThePCRreactionmixture purifiedPCRproductwasmeasuredbyQubitFluorometerusing
(25 ml) contained 5 ml GoTaq Flexi Buffer (56), 2 ml MgCl the Quant-iTTM dsDNA BR Assay Kit (Invitrogen) according to
2
(25 mM), 0.25 ml DNA Polymerase (5 U/ml, Promega, Madison, themanual.PCRproductsofthesamegene,toberuntogetherin
Wis.),1.25 mlBSA(10 mg/ml),1 mlforwardprimer(10 mM),1 ml thesamelane(1/16plate)in454sequencing,weremixedinequal
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moleamountsandrunona2%w/vagarosegel.Thetargetbands withdifferentregionsmatchingthesamesequenceinthedatabase
were cut from the gel and purified by QIAquick Gel Extraction butwithdifferentframepositionswereconsideredtobeframeshifts.
Kit (QIAGEN). The DNA concentrations of the purified PCR Sequences that matched two or more different origin sequences
products were measured by Qubit Fluorometer and adjusted to were classified as chimeras. The nucleotide sequences of nifH,
50nM. The nifH, archaeal amoA, bacterial amoA and nosZ genes archaealamoA,bacterialamoAandnosZgenesweretranslatedinto
PCR products were then mixed in equal mole amounts and aminoacidbyGeneious(http://www.geneious.com/)basedonthe
sequenced on a Genome Sequencer FLX Instrument (Roche) framepositionsobtainedfromBLASTX.Theredundantsequences
usingGSFLXTitaniumseriesreagents.The16SrRNAgenewas (identical sequences) were removed using CD-Hit [49], and the
run ina separate lane. representatives with longest length were selected for following
phylogenetic analysis. Both of the nucleotide and amino acid
Sequence analysis sequencesoftheseN-cyclinggeneswerealignedbyMUSCLE3.7
Sequenceswerefirstextractedfromtherawdataaccordingthe [50]usingprogramdefaultsettings.OperationalTaxonomicUnits
Genome Sequencer Data Analysis Software Manual (Software (OTUs)werethenclassifiedandrarefactioncurveswereconstructed
Version 2.0.00, October 2008) by the sequencing center (Roy J. basedonthedistancematrices(bothofnucleotideandaminoacid
Carver Biotechnology Center, University of Illinois at Urbana- sequences) using DOTUR [46]. Previous studies showed that the
Champaign). The sequences with low quality (length ,50bp, aminoacidsequencesofAmoAandNosZsimilarityaround90%is
which ambiguous base ‘N’, and average base quality score ,20, generally relevant to 97% similarity of 16S rRNA gene [51,52].
for detail see manual) were removed. The sequences that fully Thus,alltheseN-cyclinggenesequenceswereclassifiedintoOTUs
matched with the barcodes were selected and distributed to using a 90% amino acid sequence similarity cutoff, and phyloge-
separate files for each of the different genes, after removal of the netic trees were built in ARB using the neighbor-joining method.
barcode, using RDP Pipeline Initial Process (http://pyro.cme. Sequences of all the samples and genes were also randomly re-
msu.edu/). For each gene, the sequences that didn’t match with sampled to identical sequencing depth (the smallest sequencing
thegenespecificprimersorhadareadlengthshorterthan350bp effort)usingDaisy_chopper(http://www.genomics.ceh.ac.uk/Gen-
were removed. The sequences that matched with the reverse eSwytch/Tools.html) to avoid the potential bias caused by
primer were converted to their reverse complement counterparts sequencingeffortdifference[53].
using BioEdittomakeall thesequences forward-oriented. The 454-pyrosequencing data were deposited in NCBI SRA
The 16S rRNA gene sequences were aligned by NAST underaccession number SRA023700.
(Greengenes). The sequences with significant matched minimum
length,300andidentity,75%wereremoved.Thealigned16S Statistical analysis
rRNA gene sequences were used for chimera check using ANOVA combined with post hoc Tukey B test was used to
Bellerophon method in Mothur [44]. Distance matrices were estimate the difference of archaeal/bacterial amoA, nifH and nosZ
calculatedbyARBusingtheneighborjoiningmethod[45].Alane genesabundancesunderdifferentcropsbasedonthequantitative
mask was used in calculating the 16S rRNA gene sequences to PCRresultsfromthereplicatedplots.TheT-RFLPdatafromthe
filter out the hyper variable regions. Operational Taxonomic replicated plots were used to follow the structural changes of soil
Units (OTUs) were then classified using a 97% nucleotide microbial communities by plant types, and significance tests for
sequencesimilaritycutoffandrarefactioncurveswereconstructed these changes were conducted using Analysis of Similarity
basedonthedistancematrices(bothofnucleotideandaminoacid (ANOSIM) based on Bray–Curtis similarity coefficients. Corre-
sequences) using DOTUR [46]. The phylogenetic affiliation of spondence analysis (CA) and Canonical correspondence analysis
each 16S rRNA gene sequence was analyzed by RDP CLASSI- (CCA) were also used to visualize the predominant microbial
FIER(http://rdp.cme.msu.edu/) using confidence levelof 80%. community changes of archaeal/bacterial amoA, nifH, nosZ and
The16SrRNAgenesequenceswerealsoprocessedbyQIIME 16S rRNA genes after planting bioenergy crops based on the T-
pipelineanddenoisedbyDenoiserV0.91accordingtothemanual RFLP data. These statistical analyses were done using the free
[47,48]. The results were compared to that obtained by RDP software PAST (http://folk.uio.no/ohammer/past/). Based on
pipeline. In total, 26,431 valid sequences were obtained after our extensive pyrosequencing library, the OTUs/genera that
denoisingusingQIIME,whichis12.2%lessthanthatobtainedby showed monotonic (i.e. continuously increasing or decreasing)
RDPpipeline(withoutdenoising).Usingthe97%similaritycutoff, trends for each crop treatment over the two year establishment
8,568 OTUs were obtained, which is 4.7% lower than that were presumed to be particularly noteworthy in terms of crop
observedbyRDPpipeline.Afterrandomre-samplingtothesame impact.Thepopulationswithcontinuouslyincreasedordecreased
sequencedepth(1789sequencespersample)usingDaisy_chopper abundance in the two-year period after planting these bioenergy
(http://www.genomics.ceh.ac.uk/GeneSwytch/Tools.html), the cropswere selected usinga customPerl script.
number of OTUs for each sample obtained by two different
processing methods (QIIME, denoised and RDP, non-denoised) Results
was compared (Fig. S1). The estimated number of OTUs after
denoising was similar to that obtained by RDP pyrosequencing Quantification of nifH, archaeal amoA, bacterial amoA
pipeline (without denoising), showing that the denoising process and nosZ genes
had a very limited influence on our diversity analysis. The data Quantities of AOA in all of crops fluctuated over the two
reportedinthispaperwasanalyzedusingRPDpipelinedescribed growingseasonsinasimilarpattern(Fig.1),buttheabundanceof
in theprevious paragraph. thisgenewasalwayshigherinMGthanNPandPV.Thequantity
ThenifH,archaealamoA,bacterialamoAandnosZgenessequences of bacterial amoA genes in ZM significantly increased from
were blasted against a non-redundant protein sequence database 1.4760.616108 to 3.2660.946108 during the first three months
(download from NCBI) using BLASTX with an E-value cutoff of ofestablishmentandthereafterremainedhigherinZMthaninthe
0.001.Thetop10closestmatchesofeachsequencewereestimated other cropping systems. The nitrification rates under these crops
using a custommadePerlscripttoremovepossiblechimerasand wereanalyzedinthesecondyearbyestimatingtheaccumulation
sequences with sequencing errors causing frameshifts. Sequences of the nitrate in buried soil bags, and linear regression revealed
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Figure1.ChangesinabundanceofnifH,archaealamoA,bacterialamoAandnosZgenesinplotsafterplantingMiscanthus6giganteus
(MG),Panicumvirgatum(PV),restoredprairie(NP)andZeamays(ZM).Thecopynumberofgenesineachgramofdrysoilwasestimated
basedontheresultsofreal-timePCR(copynumberineachngDNA)andtheaverageamountofextractedDNA(6.23mgperdrysoil)andassuming
DNA extraction efficiency was 30% [40]. The R2 of the standard curve of all these genes was higher than 0.99. The real-time PCR amplification
efficiencyof nifH,archaealamoA,bacterialamoA andnosZ geneswas 1.9060.01, 1.9060.06, 1.7660.01and 1.8260.01respectively. *Represents
valuesthataresignificantlydifferent(P,0.01).
doi:10.1371/journal.pone.0024750.g001
that the nitrification rates were significantly related to the the other N-cycling populations; however, the copy number of
quantities (log) of archaeal amoA genes (R2=0.61, P=0.03, nosZ increased in ZM during the second year of the study and
n=12), but not to the quantities of bacterial amoA genes (Fig. 2). remained higherthan inMG(Fig.1).
The abundance of nifH genes remained stable for all the crops,
rangingfrom76107to96107copiespergramofdrysoil(Fig.1). Structural changes of N-cycling genes and microbial
Nosignificantdifferenceswereobservedamongthedifferentcrops communities after planting of bioenergy crops
in the first year. In the second year, the population sizes of The community structural differentiation of nifH, archaeal
diazotrophs in PV and NP had significantly increased in amoA, bacterial amoA, nosZ and 16S rRNA genes under different
comparison to the first year (P=0.0001 and 0.0002). The bioenergy crops were analyzed by T-RFLP. These analyses used
population size of denitrifiers was less variable in comparison to thefullyreplicatedsamplesetfromtherandomizedblockdesign.
Figure2.Relationshipbetweentheconcentrationofammonia-oxidizingarchaea/bacteriaandnitrificationrate.Thenitrificationrate
wasdeterminedoverthesametimeasoursamplecollectionin2009.Nitrificationratewascalculatedbasedontheaccumulationofnitrateinsoil
bagsincubatedinthefield(0–10cmdepth)for15to32days.
doi:10.1371/journal.pone.0024750.g002
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Correspondence analysis (Fig. 3) showed that the soil microbial followedbyZM,andnifHwasequallyseparatedunderallcropping
communitiesintheinitialplotsdidnotshowanyrelationshipwith systems(Fig.S2).
the crop treatments being applied. During the establishment of
thesebioenergycrops,thecommunitycompositionofdenitrification Diversity of nifH, archaeal amoA, bacterial amoA, nosZ
bacteria (nosZ) under ZM was completely separated (ANOSIM, and 16S rRNA genes
P,0.05,TableS2)fromthoseundertheothercropsbytheendof Tofurtherunderstandthecompositionofmicrobialcommunity
thesecondyearalongthefirst-axis,whichexplained51.7%ofthe inthefield,thenifH,archaealamoA,bacterialamoA,nosZand16S
total variance. None of the other groups showed significant rRNA genes were deeply sequenced using the pyrosequencing
clusteringbyplant,althoughthecommunitycompositionofAOB approach. In total, 143,487 reads were obtained for these genes.
(bacteria amoA) under ZM appeared to be separated from that of The numbers and qualities of these sequences are described in
MG (ANOSIM, P=0.17) along the second axis, which explained Table 1, Table S3 and Text S1. The reproducibility of the
14.2%ofthetotalvariance.Inadditiontoplantspecies,thechanges pyrosequencing result was estimated by comparing the observed
ofsoilmicrobialcommunitiesalsocouldbecausedbythevariation microbial composition between repeat sequencing runs for all
of environmental conditions. To compare the magnitude of the these genes (Fig. 4). Linear regression analysis indicated a high
changesofsoilmicrobialcommunitiesrelatedonlytoplantspecies, reproducibility (R2=0.95)of our pyrosequencing.
a constrained ordination method was also used. Canonical High diversity of nifH,archaeal amoA,bacterial amoA and nosZ
Correspondence analysis (CCA, Fig.S2)revealed that, at theend genes were observed with 10899, 3187, 3945 and 11242 unique
ofthesecondyear,themicrobialcommunitiesunderZMweremost nucleotide sequences and 2286, 2246, 3633 and 4208 unique
differentfromthethreecroppingsystemsforbacterialamoA,nosZ, deduced amino acid sequences respectively (Fig. S3). These
and 16S rRNA. Archaeal amoA was most distinct under MG, sequenceswerethentranslatedtoaminoacidsequenceaccording
Figure3.StructuralchangesofarchaealamoA,bacterialamoA,nifH,nosZand16SrRNAgenesafterplantingMiscanthus6giganteus
(MG),Panicumvirgatum(PV),restoredprairie(NP)andZeamays(ZM)revealedbyT-RFLPandCorrespondenceanalysis(CA).The
numberoneachaxisshowsthepercentageoftotalvariationexplained.Thesoilsampleswerecollectedfromfourreplicateplotsforeachplantat
eachtimepoint.
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Table1. Qualityofbarcoded pyrosequencing reads.
Numberofsequences
Genes Correctbarcodeandprimer Length.350bp aValid Eachsample(range)
nifH 28,334 27,781 21,111 1,312–2,956
ArchaealamoA 16,978 16,226 14,025 697–1,792
BacterialamoA 28,254 27,874 21,817 1,726–2,569
nosZ 33,838 28,819 22,590 1,600–2,951
16SrRNA 30,487 30,175 30,101 2,034–3,488
Total 137,891(96.1%) 130,875(91.2%) 109,644(76.4%) 697–3,488
Totalnumberofrawreadswas143,487.
aValidsequencesofnifH,archaealamoA,bacterialamoAandnosZgenesweredefinedashighqualitysequenceswithcorrectbarcodeandprimer(at59-end),length
.350bpandthatdidnothaveframeshiftsandchimericstructure.Thepossiblesequencingerrorscausingframeshiftsandchimeraswereremovedbasedonthe
BLASTXresult.Sequenceswithdifferentregionsmatchingthesamesequenceinthedatabasebutwithdifferentframepositionswereconsideredtobeframeshifts.
Sequencesthatmatchedtwoormoredifferentoriginsequenceswereclassifiedaschimeras.Validsequencesof16SrRNAgeneweresequenceswithcorrectbarcode
andprimer,length.350bpandpassedthechimericcheckprograminGreengeneswiththeBellerophonmethod.Thesequencenumbersforeachsamplearelistedin
TableS3.
doi:10.1371/journal.pone.0024750.t001
to the BLASTX report. The amino acid sequences of nifH, sequences belonging to Proteobacteria were distributed over 86
archaealamoA,bacterialamoAandnosZgeneswereclassifiedinto different genera, while 94.1% of the sequences in Acidobacteria
229, 309, 330 and 331 OTUs, respectively, with a similarity belonged to Family Gemmatimonadaceae, with GP1 as the most
cutoffof90%.Afterrandomre-samplingtothesamesequencing predominant genus (accounted 35.9%oftheAcidobacteria).
depth(697sequencesforeachsample),theadjustedtotalnumber To understand which phylotypes were impacted by vegetation
ofOTUsforthesegeneswere217,303,319and278,respectively type, the OTUs of nifH, archaeal amoA, bacterial amoA, nosZ and
(Table S4). Rarefaction analysis of these genes showed that the 16S rRNA (genus for 16S rRNA) genes that continuously
diversity of archaeal amoA gene in ZM2 (second year ZM) and increased or decreased over the two-year establishment of these
MG1 (first year MG) was markedly lower than the others (Fig. bioenergy crops were identified (Fig. 5 and Fig. S6, S7, S8, S9).
S4). The diversity of nifH and nosZ genes slightly decreased in Afterplantingofthesebioenergycrops,27.5%,15.4%,22.7%and
ZM2. 14.5% of the total archaeal amoA, bacterial amoA, nifH, and nosZ
The diversity of bacterial and archaeal 16S rRNA genes was phylotypes,respectively,werefoundtobecontinuouslyincreasing
much higher than these N-cycling genes. In total, 19,824 unique ordecreasing(Table2).DetailsofthesecontinuouslychangedN-
16S rRNA gene sequences and 8,989 species (OTUs classified at cyclingOTUsaredescribedinTextS1andFigureS6,S7,S8,S9.
97% similarity cutoff) were observed. RDP classification showed Pyrosequencing of 16S rRNA gene revealed 19.9% of the
that these sequences covered 16 bacterial and 1 archaeal phyla, bacterial genera (39), spanning six phyla, continuously changed
including 201 genera (Fig. S5). Proteobacteria and Acidobacteria after planting of these bioenergy crops (Fig. 5). Only genus
were the most predominant phyla in the soil (.20%). The Methylibium was changed in all the crops, with decreased
abundance in MG and increased abundance in the other crops.
RhodanobacteronlyappearedafterplantingofZM(7sequencesfor
both of ZM1 and ZM2), and it was undetectable either in the
background soil or in the soil under other crops. Consistent with
thechangesofNitrosospira-likebacterialamoAOTU(seeabove),the
abundance of genus Nitrosospira in the 16S rRNA library also
increased in ZM. The abundance of genus Nitrospira, which is
knownasanitrite-oxidizingbacteria,alsoincreasedinZM.Most
of the changed genera in MG decreased or even disappeared,
exceptTerrabacterandHerbaspirillum.Allofthemwerefoundatlow
abundance(,1%).AlthoughmanygenerainProteobacteriawere
changed, the total abundance of this most predominant phylum
was quitestable underall of thecrops(Fig.S5).
Discussion
In this study, we monitored the structural and quantitative
changes of the key genes involved in N-cycling as well as the
overallbacterial/archaealcommunityduringtwo-yearestablish-
ment of four different bioenergy feedstock crops, and analyzed
Figure 4. Reproducibility of the pyrosequencing replicates. the shifts of specific soil microbial populations in response to
OTUs of nifH, archaeal amoA, bacterial amoA and nosZ genes were
different types of crops. We were able to detect significant
classified at a nucleotide similarity cutoff 90%. The 16S rRNA gene
changesintheabundanceofmanyofthesemicrobialfunctional
sequenceswereclassifiedtogenuslevelbyRDPclassifier.Oneofthe
sampleswasduplicatedforeachgene. groups within 2 years of initial crop establishment. We also
doi:10.1371/journal.pone.0024750.g004 foundthattraditionalrow-cropagricultureofmaizehasalarger
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Figure5.MicrobialgenerathatchangedafterplantingofMiscanthus6giganteus(MG),Panicumvirgatum(PV),restoredprairie(NP)
andZeamays(ZM).SequenceswereclassifiedbyRDPClassifierproject.2/+representsthegenuscontinuouslydecreased/increasedafterplanting
thecrops;22/++representsthegenusdisappeared/appearedafterplantingthecrops.*sequencesbelongingtoCrenarchaeota,whichcouldnotbe
classifiedtogenuslevel.
doi:10.1371/journal.pone.0024750.g005
impact on the soil N-cycling community than any of the Traditional maize cultivation significantly increased the total
perennial bioenergy feedstock crops (Figs. 1, S2), while the abundance of ammonia-oxidizing bacteria and denitrifying
perennialcropswereassociatedwithoverallcommunityshiftsin bacteria (Fig. 1), altered the community composition of
thephyla Planctomyces,Firmicutes,andActinobacteria(Fig. 5). denitrifying bacteria (Fig. 3) and decreased the diversity of
ammonia-oxidizing archaea, denitrifying bacteria, and diazo-
trophs (Fig. S4). This may be due to the application of N-
Table2. NumberofOTUsorgenera thatchanged fertilizer, which occurred only in ZM plots. Ammonia oxidizers
continuouslyafterplanting Miscanthus6giganteus (MG), are sensitive to N-fertilizer [24,27], and these responses were
Panicumvirgatum (PV),restored prairie(NP) and Zeamays manifestedintheincreasedpopulationsizeofAOBandthehigh
(ZM). number of markedly changed AOA species. The nitrification
rate was significantly correlated with the quantity of archaeal
amoA, but not bacterial amoA, indicating AOA was the major
Genes MG PV ZM NP *Total ammonia-oxidizer.
Deep understanding of the structural shifts of key functional
ArchaealamoA 4 16 61 23 85
genes can help us to better understand changes in microbial
BacterialamoA 18 18 7 20 51 activityintheenvironment.Fromthepresentdatabase,weknow
nifH 8 21 14 23 52 thattheglobaldiversityofthenifH,archaealamoA,bacterialamoA
nosZ 12 18 24 16 48 and nosZ genes, as well as the other functional genes of
microorganisms, is high. The traditional approaches (e.g. clone
16SrRNA 17 15 14 21 40
library, DGGE and T-RFLP) used in previous studies may
OTUsofN-cyclinggeneswereclassifiedbasedonacutoffof90%aminoacid largely underestimate the diversity of microbial communities
sequencessimilarity.16SrRNAgeneswereclassifiedintogenuslevelbyRDP involved in soil nitrogen cycling. Mounier et al. (2004) revealed
Classifier.DetailsoftheabundancechangesoftheOTUsorgeneraareshownin
that even a large library with 713 clones was insufficient to
Fig.5andS6,S7,S8,S9.
*TotalnumberofchangeduniqueOTUsorgenera. enumeratethediversitynosZgeneinmaizerhizosphere,showing
doi:10.1371/journal.pone.0024750.t002 the high complexity of N-cycling genes [54]. Thus, high-
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N-TransformingArchaeaandBacteriainSoil
throughputdeepsequencingapproachesareessentialtoimprove found that, while the population size of AOA was relatively
our knowledge of the diversity of these functional genes. In the stable, the structure of the AOA community was sensitive to the
presentstudy,usingbarcoded454-pyrosequencingapproach,we different cropping systems. The diversity of AOA markedly
found high diversity (ranging from ,3100 to ,11200 unique decreasedafterplantingofmaize,with41oftheAOAphylotypes
nucleotidesequences)ofnifH,archaealamoA,bacterial amoAand disappearing (Fig. S6). In contrast, the population size of AOB
nosZgenesinthesoilecosystem,whichfarsurpassesthediversity significantly increased after planting of maize in both the qPCR
of the N-cycling genes observed in previous studies of bacterial amoA and the 16S rRNA pyrosequencing results. In
[54,55,56,57,58,59,60,61]. The rarefaction curvesof thesegenes addition to the increase of genus Nitrosospira (AOB) [69], the
wereclosetosaturationaftersequencing,1000foreachsample, abundance of Nitrospira (nitrite-oxidizing bacteria) [70] also
indicating that such a sequencing effort is sufficient to elucidate increased in N-fertilized maize (Fig. 5). However, the number
the diversity and structure of the complex soil N-cycling of changedbacterialamoA phylotypes inZMwasmuchlessthan
communities. The high similarity between repeat runs of these the other crops. Therefore, these two different groups of
genes (Fig. 4) demonstrates the high reproducibility and ammonia-oxidizers respond to the N-fertilization in a very
reliability of this barcoded pyrosequencing method. This result differentway.ThepopulationsizeofAOBincreasedimmediately
alsoindicatesthatthevariationofpyrosequencing,resultingfrom in the first growing season, thus, we hypothesize that the
randomsamplingofgenetargetsduringemulsionPCR[62],can increased AOB abundance in maize was likely due not to the
be greatly reduced by increasing the sequencing depth and growth of maize plants, but to the application of N-fertilizer,
library coverage. which increased the ammonia content in the soil [24,27]. It has
During the establishment of these bioenergy crops, about been found that AOB population size increased in seven days
15%–30% of N-cycling genes and the detected bacterial/ after applying of N-fertilizer, and it was still significantly greater
archaeal genera were continuously changed, indicating that a than unfertilized soil 8 months after the last application of
large proportion of soil microbes were affected by the transition ammonia[41].Consistently,wefoundthatthepopulationsizeof
to different bioenergy feedstock systems. Most of these AOB doubled in less than three months and maintained a
phylotypes changed uniquely in one of the crops, indicating relatively high level over the two-year study even though
that the changes were mainly caused by the particular measurable NH + in the soil declined over this time period to
4
experimental crop treatment (specific plant species or manage- levelsclosetothatoftheunfertilizedplots(Fig.1andS10).These
ment, such as fertilization) and not due to the environmental resultsindicatetheAOBpopulationsizecanbequicklyincreased
conditionsthatfluctuatedinalltreatments(e.g.temperatureand byN-fertilizationandcanremainforalongperiodevenafterthe
moisture). Contrary changes of certain populations in different measurable ammonia has been consumed.
crops also support this conclusion; for example, the abundance The nitrification rate was found to be significantly correlated
of Methylibium (belonging to b-Proteobacteria) decreased in MG with the quantity of archaeal amoA rather than bacterial amoA,
but increased in the other crops. The abundance of Bacter- indicating AOA rather than AOB may be the major active
oidetes was previously found to be lower in the soil of ammonia-oxidizer in these soils. Contradictory conclusions on
Miscanthus-dominated grasslands (4%) in comparison to forest the relative importance of AOA and AOB in soil nitrification
soil (6%) [63]. We found that the decrease of Bacteroidetes in havebeenpreviouslyreportedwherenitrificationwasfoundtobe
MG was mainly due to the disappearance of the genus associated with the changes of archaeal amoA abundance or
Ferruginibacter (Fig. 5 and S5). higher archaeal transcriptional activity in some of the soils
ThestructureofnifHgenewascompletelyseparatedaccording [58,71,72].Incontrast,thenitrificationkineticsintheothersoils
to vegetation by the end of the second year (Fig. S2), which were correlated with the growth of AOB [73,74]. It has been
suggests that the structure of N-fixation bacterial population was found that the ammonia affinity of ‘‘Candidatus Nitrosopumilus
particularly sensitive to plant genotype. Tan et al. (2003) has maritimus’’ (a marine AOA) is much higher than AOBs, and its
revealed that the structure of diazotrophs was not only different growth may be enhanced by relatively low ammonia concentra-
amongricespecies,butalsochangedrapidlywithfertilization.The tion [75]. Thus, thecontradictory conclusions from thesestudies
diversity of the nifH gene was obviously reduced within 15 days may be due to the different soil ammonia concentration used in
afterfertilization[17].Thus,thedecreaseddiversityofnifHinZM these experiments [58,73]. These results hint that AOB may be
maybealsoduetotheapplicationoffertilizernotthepresenceof more active in soils amended with ammonia, while AOA are
maize. However, the population size of N-fixing bacteria did not more active in soils with low ammonia concentration [58]. The
change under ZM, indicating N-fertilization may not change the nitrificationrateoutlier(ZMAug;Fig.2)hadhighestpopulation
quantity of soil diazotroph [64]. The N-fixing activity was sizeofAOB,suggestingthattheactivityofAOBwasenhancedby
expected to increase in MG [14,15]. Although the population N-fertilization and also supported the above speculation. In
size of total free-living soil N-fixing bacteria was not significantly support of this speculation, a recent publication shows that
increasedbygrowthofMGinthetwoyearperiod,theabundance recoveryofnitrification potential afterdisruptionwasdominated
of genus Herbaspirillum increased. Herbaspirillum species are known by AOB in cropped soils while AOA were responsible RNP in
as endophytic diazotrophs that are enriched by C4-prennial pasture soils [76].
grasses including Miscanthus [65,66,67]. Thus, we speculate that It is known that nitrogen fertilization can change the structure
theabundanceincreaseofHerbaspirilluminthebulksoilwaslikely andactivityofdenitrifyingcommunity,andsubsequentlyaffectthe
due to the root exudates (e.g. organic carbons) released by N Oemission[33,34,77,78].Largeamounts(1.3%)oftheapplied
2
Miscanthus, which favored the growth of this population. Our N-fertilizerinmaizefields(northColorado)areconvertedtoN O
2
results also suggest that Miscanthus may only selectively enhance by the combination of nitrification and denitrification [79].
the activity of specific diazotrophs, not the whole N-fixing However,itisstillunclearhowfertilizationchangesthemicrobial
microbial community. community, since most of the previous studies are based on the
TheAOAarethoughttobemorestableandlessresponsiveto already established fields [7,29,33]. Our study revealed that the
environmental differences than AOB, as revealed by previous structure of denitrifying bacteria in maize soil was significantly
quantitative studies [6,68]. However, in the present study we differentiated from the other crops at the second year (Fig. 3).
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N-TransformingArchaeaandBacteriainSoil
However,thepopulationsizeofdenitrifierswasrelativelystablein mays(ZM).OTUswereclassifiedbasedonacutoffof90%amino
all the crops in comparison to other N-cycling microbial acidsequence similarity.
communities,whichonlyslightlyincreasedinmaizeatthesecond (TIF)
year. The high stability of denitrifying population abundance
FigureS7 (a)Phylogenetictreeofand(b)abundanceofbacterial
could be explained by the high diversity and functional
amoAOTUsthatcontinuouslychangedafterplantingMiscanthus6
redundancy of denitrificationcommunity [30,80].
giganteus(MG),Panicumvirgatum(PV),restoredprairie(NP)andZea
In conclusion, our two-year study of transitional agriculture
mays(ZM).OTUswereclassifiedbasedonacutoffof90%amino
showsthatspecificN-transformingmicrobialcommunitiesdevelop
acidsequence similarity.
inthesoilinresponsetodifferentbioenergycrops.EachN-cycling
(TIF)
microbial group responded in a different way after planting with
different bioenergy crops. In general, planting of maize has a Figure S8 (a) Phylogenetic tree of and (b) abundance of nifH
larger impact on the soil N-cycling community than the other OTUsthat continuouslychangedafterplantingMiscanthus6gigan-
bioenergy crops. Our results also indicate that application of N- teus(MG),Panicumvirgatum(PV),restoredprairie(NP)andZeamays
fertilizer maynot onlycauseshort-term environmentalproblems, (ZM).OTUswereclassifiedbasedonacutoffof90%aminoacid
e.g.watercontamination,butalsocanhavelong-terminfluenceon sequence similarity.
(TIF)
the global biogeochemical cycles through changing the soil
microbial community structure and abundance. Since soil types Figure S9 (a) Phylogenetic tree of and (b) abundance of nosZ
and other environmental factors may also impact the N-cycling OTUsthat continuouslychangedafterplantingMiscanthus6gigan-
microbialcommunity,theuniversalityofourfindingsneedstobe teus(MG),Panicumvirgatum(PV),restoredprairie(NP)andZeamays
confirmed by additionalstudy at different sites. (ZM). OTUs of were classified based on a cutoff of 90% amino
acidsequence similarity.
Supporting Information (TIF)
Figure S1 Number of OTUs obtained by two different FigureS10 Nitrateandammoniaconcentrationinbulksoil.Soil
processingmethods:QIIME(denoised)andRDPpyrosequencing samples for chemical and microbiological analysis were collected
pipeline (non-denoised). inthesameweekforeachtimepoint,exceptSep2008whenthe
(TIF) nitrate and ammonia concentrations were not measured. MG,
Miscanthus6giganteus; PV, Panicum virgatum; NP, restored prairie;
FigureS2 Structural changes of archaeal amoA, bacterial amoA, ZM,Zeamays.Thesedataweremeasuredoverthesameperiodof
nifH,nosZand16SrRNAgenesafterplantingMiscanthus6giganteus oursamplecollectionbytheBiogeochemistrylaboratory(C.Smith
(MG), Panicum virgatum (PV), restored prairie (NP) and Zea mays andM.David).
(ZM)revealedbyT-RFLPandCanonicalcorrespondenceanalysis (TIF)
(CCA). The number on each axis shows the explained total
Figure S11 Pyrosequencing read length based on the raw
variation. The soil samples were collected from four replicated
sequence reads.
plotsforeachplantateachtimepoint.*Correspondenceanalysis
(TIF)
wasusedforthesamplescollectedbeforeplantingbioenergycrops.
(TIF) TableS1 PrimersandannealingtemperaturefornifH,archaeal
amoA,bacterial amoA, nosZ and16SrRNA genes.
Figure S3 OTU classification of valid sequences at different
(DOC)
distance levels based on nucleotide and deduced amino acid
sequences. Table S2 Comparsion of the microbial community structures
(TIF) between different crops.
(DOC)
FigureS4 RarefactionanalysisofthediversitiesofnifH,archaeal
amoA, bacterial amoA, nosZ and 16S rRNA genes in the soil Table S3 Number of valid sequences for each gene in each
underneathdifferentbioenergycrops.TheOTUsofnifH,archaeal sample.
amoA,bacterialamoAandnosZgeneswereclassifiedat90%similarity (DOCX)
cutoff based on amino acid sequences, and 16S rRNA gene was
TableS4 NumberofOTUsobservedafterrandomre-sampling
classified at 97% similarity cutoff on nucleotide sequences. BG0
totheidentical sequencing depth(697 sequences/sample.
represents the samples collected before planting bioenergy crops.
(DOCX)
MG, PV, NP, and ZM represent Miscanthus6giganteus, Panicum
virgatum,restoredprairieandZeamaysrespectively.1and2represent TextS1
(DOC)
samplescollectedinthefirstandsecondgrowingseasons.
(TIF)
Acknowledgments
Figure S5 Phylum level microbial community composition in
the soil under different plants before and for two years after TheauthorsgratefullyacknowledgehelpfromA.Duong,D.Fieckert,A.
Groll,N.Peld,andM.Masterswithfieldsamplingandlaboratorywork.C.
transitiontobioenergycropping.*representsignificantlychanged
Smith and M. David kindly provided denitrification rate estimates for
phylum. MG, Miscanthus6giganteus; PV, Panicum virgatum; NP,
sampledplots.P.Y.Hongprovidedhelpfulcommentsonthismanuscript.
restored prairie;ZM, Zeamays.
(TIF)
Author Contributions
FigureS6 (a)Phylogenetictreeofand(b)abundanceofarchaeal
Conceivedanddesignedtheexperiments:YMACYRIM.Performedthe
amoAOTUsthatcontinuouslychangedafterplantingMiscanthus6 experiments: YM ACY. Analyzed the data: YM. Contributed reagents/
giganteus(MG),Panicumvirgatum(PV),restoredprairie(NP)andZea materials/analysistools:YM.Wrotethepaper:YMACYRIM.
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