Using genomics in mine reclamation

Abstract

For the majority of mines, closure succeeds when healthy, self‐sustaining ecosystems develop on previously mined lands. In British Columbia, Canada, the regulations require reclamation of ecosystems; however, there are few specified targets, and those that are presented are vague. Genomics technologies may provide the key to both understanding the elements necessary to recreate functional ecosystems and provide sufficient benchmarks for success. In this review, we highlight the use of genomics to meet mine closure goals, enhance ecosystem development and optimise ecosystem services inherent in self‐sustaining reclaimed ecosystems. We outline practical steps for applying genomics technologies to characterise the composition and activity of microbial communities in soils and treatment substrates. From this framework, we address the state of the science and how recently developed techniques have transferable value to mine reclamation. We then define three areas in which genomics technologies have already proven effective at informing management and reclamation of mine sites in the form of bioreactors, passive treatment systems and novel gene discovery. Finally, we speculate on the future applications of genomics technologies and the necessary steps to integrate these data into comprehensive management of mined sites.

Full text

MineClosure2015–A.B.Fourie,M.Tibbett,L.SawatskyandD.vanZyl(eds)
©2015InfoMineInc.,Canada,978‐0‐9917905‐9‐3
MineClosure2015,Vancouver,Canada1
Usinggenomicsinminereclamation
L.H.FraserNaturalResourceSciences,ThompsonRiversUniversity,Canada
H.W.GarrisNaturalResourceSciences,ThompsonRiversUniversity,Canada
S.A.BaldwinChemicalandBiologicalEngineering,UniversityofBritishColumbia,Canada
J.D.VanHammeBiologicalSciences,ThompsonRiversUniversity,Canada
W.C.GardnerNaturalResourceSciences,ThompsonRiversUniversity,Canada

Abstract
Forthemajorityofmines,closuresucceedswhenhealthy,self‐sustainingecosystemsdevelopon
previouslyminedlands.InBritishColumbia,Canada,theregulationsrequirereclamationof
ecosystems;however,therearefewspecifiedtargets,andthosethatarepresentedarevague.
Genomicstechnologiesmayprovidethekeytobothunderstandingtheelementsnecessaryto
recreatefunctionalecosystemsandprovidesufficientbenchmarksforsuccess.Inthisreview,we
highlighttheuseofgenomicstomeetmineclosuregoals,enhanceecosystemdevelopmentand
optimiseecosystemservicesinherentinself‐sustainingreclaimedecosystems.Weoutlinepractical
stepsforapplyinggenomicstechnologiestocharacterisethecompositionandactivityofmicrobial
communitiesinsoilsandtreatmentsubstrates.Fromthisframework,weaddressthestateofthe
scienceandhowrecentlydevelopedtechniqueshavetransferablevaluetominereclamation.We
thendefinethreeareasinwhichgenomicstechnologieshavealreadyproveneffectiveatinforming
managementandreclamationofminesitesintheformofbioreactors,passivetreatmentsystems
andnovelgenediscovery.Finally,wespeculateonthefutureapplicationsofgenomicstechnologies
andthenecessarystepstointegratethesedataintocomprehensivemanagementofminedsites.
1Introduction
Inanidealsystem,minesoperatewithminimalenvironmentalimpactwithinandoutsideoftheoperational
footprint.Aftertheperiodofprofitableextractionisreached,adesirableconditionisrecreated,beitforest,
pastureorsuburbanneighbourhood,sothatthebiota(soilmicrobestrees,andconcernedcitizensalike)can
functionwithinanatural,self‐sustainingecosystem.Inreality,minesitesoftenleavealegacy,including
perpetuallyalteredplantcommunities(Holl,2002);elevatedcontaminantsinsurfaceandgroundwater(Cidu
etal.,2001);thin,compactsoils(Skousenetal.,2009);alteredsoilfunction(Mummeyetal.,2002);and
magnificationofcontaminantswithinthefoodchain(Allan,1995;MuscatelloandJanz,2009).
InCanada,governmentregulationsonminingoperationssetthestandardsforreclamationsuccess,andasa
result,directthefateofminedlands.TheBritishColumbiaMinesActandHealth,SafetyandReclamation
CodeforMines(theCode)specifythatreclamationmustsatisfytherequirementsofthechiefinspector
(GovernmentofBritishColumbia,2008).Thelegislationisvagueandsubjecttointerpretation(byboththe
miningcompaniesandtheinspectors),sotherehasbeenvariabilityingoalsandmeasuresofsuccess.
Historically,goalsweresetforplantproductivity,likelybecauseproductivityisrelativelyeasytomeasureand
canbeassociatedwithecosystemfunction(Britton,1998).ThecurrentCode(10.7.5)expressesagoalfor
equivalentlandcapability,whichisachallengetoachievebecausetherearenosetpredefinedtargets.There
hasbeenarecenttrendtoconsidermineclosureintermsofthewholeecosystem,withallofitsfunctions
andservices.
Ecosystemservicescanbedefinedas“theaspectsofecosystemsutilised(activelyorpassively)toproduce
humanwell‐being“(Fisheretal.,2009).Theecosystemservicesforreclaimedminedlandsmayincludeforage

UsinggenomicsinminereclamationL.H.Fraser,H.W.Garris,S.A.Baldwin,J.D.VanHammeandW.C.Gardner
2MineClosure2015,Vancouver,Canada
forcattleandwildlife,timber,cleanairandwater,carbonsequestration,biodiversityandculturally
significantnaturalproductsrelatedtotraditionalpracticesandmedicines.Canadianregulationsexistto
protecttheseservices,orensuretheircontinuation,duringandafterminingactivitiestakeplace.Despite
pervasiveeffortstomeetandimproveuponregulatoryguidelinesforecosystemservicesatpreviouslymined
sites,thereisalargedegreeofuncertaintyinminelandrestoration.Consistentapplicationofrevegetation,
soilamendmentsandregradingtreatmentscanleadtoverydifferentresults,evenonthesamesite
(Martínez‐RuizandMarrs,2007).Whysuchinconsistency?Manyauthorsattributeittovariabilityinstarting
conditions(theprevailingmineralogicalsubstrate),slope,aspectandmyriadunmeasuredfactors(Martínez‐
RuizandMarrs,2007),withoneemergingfrequently―thesoilmicrobiota(Harris,2003).
Microorganismsarecatalystsforsoilformation.Theyareresponsibleforcreatingcomplex
microenvironmentsthatleadtonutrientuptakeinplants,semi‐homeostaticwaterandchemicalregulations,
andtooverallresiliencetoerosion,contaminationandinvasionbyexoticplantspecies.Newsoilsthatform
onexposedbedrockoftenfollowapredictablesuccessionalsequence.Microbialcommunitiesformon
weatheredinorganicsubstrates,fixatmosphericnitrogenandCO2andcontributethekeyelementsoforganic
life.Multi‐cellularplantsandanimalsarerelativelatecomerstothedevelopingcommunity,arrivingwhen
soilformationprocesseshaveprovidedsufficientorganicresourcestosustainthem(seeFrouzetal.,2008,
foradiscussionofthisprocessonpreviouslyminedsites).Chronologically,thisnaturalapproachcanbe
consideredbottom‐up,withsoilorganismsandprocessesestablishingandpreparingthesubstratebefore
largerplantsandanimalsarrive.Thescienceofrestorationhasnotdevelopedamechanismforrecreating
(muchlessaccelerating)thisbottom‐upprocess.Rather,practitionersoftenattempttorecreatethedesired
ecosystemirrespectiveofthenaturalchronologicalsequencebysuperimposingsoilamendments,andwith
seedingorplantingpluggedvascularplants(Tordoffetal.,2000).Thispracticeisnotundertakenoutof
ignoranceoftherolesoilmicrobialcommunitiesplay,butbecausetoolstoobserve,quantifyandmanipulate
thesecommunitiesarenotwithinthetypicalorganisation’stoolkit.
Untilrecently,techniquesforobservingandquantifyingmicrobiotahavebeenextremelyrestrictive(Ficetola
etal.,2008).Lessthanonepercentofallsoilmicrobialorganismshavebeenculturedinalaboratorysetting
(Hugenholtzetal.,1998;Harris,2009).Asonealternative,microbiologistshavedevelopedtechniquesfor
quantifyingthemass‐actionofmicrobesinsoils,includingtherespirationandproductionratesofmetabolic
products.Genomicstechnologiesofferusanopportunitytoobservethecomplexityofmicrobial
communitiesastheyformonminedsites,andtoapplyecologicaltheorytosoilcommunityformationand
structureinawaythathasuntilveryrecentlybeenimpossible.Inrecentdecades,microbialgenomicshas
beenappliedtominesites,aswillbediscussed,butithasyettobeincorporatedintoacomprehensive
monitoringandtroubleshootingparadigmformineclosureandminesiterestoration.Inthispaper,we
provideacontextforgenomicsintheminingcommunityandhighlightspecificapplicationsofgenomicsand
metagenomicstomineclosure.
2Genomicstechniques:Fromsampletomanagementdecision
2.1Sampling
Microbialgenomicsbeginswithasource,eitheranindividualorganismorsamplematerialcomprisingmany
organisms.Soilcontainsacombinationofliving,deadanddormantorganisms,allofwhichcontributetothe
soil’sgeneticsignature.Representativesamplingofsoilsonminesitesfortheirmicrobialcommunity
structuresisachallenge,asminesitesarelarge,oftenincorporatingentirelandscapeswithvarying
geophysicalproperties.Furthermore,soilcommunitiesarenotoriouslypatchyindistribution(amonghabitat
typesandbothhorizontallyandverticallywithineachsoilstratum)(Foster,1988;RanjardandRichaume,
2001).Therefore,itisnotfeasibletosampleeveryhabitattypeandsoilstratumwithinaminedlandscape.
Rather,samplingshouldtargethabitatsandsoilstratathatmayprovidesignsofimminentdegradationof
waterquality(streamsandassociatedwetlandareaswithinandadjacenttotheminedlandcatchment),
contaminantsequestration(bioreactorsubstrates,ororganic‐richsoilsreceivingrunofffromtheminesite)
orreclamationsuccess(processedparentmaterial,organicamendmentsandunimpactedreferencesites).

KeynotesandPlenaries
MineClosure2015,Vancouver,Canada3
Thereareafewmajorconcernstoconsiderwhendesigningasamplingprotocolforgenomicanalysis.First,
thesoilchemistryandwatercontentofsampleschangerapidlyuponcollection,possiblyleadingtoshiftsin
theactivityandabundanceofmicrobeswithinthesoil,especiallywhencollectedfromanoxicsubstrates.
Biologicalactivitymustthereforebesuspendedasquicklyaspossiblefollowingcollection,whichgenerally
involvesflashfreezingofsampleswithliquidnitrogen(−195°C)oronblocksofdryice(−79°C)whenliquid
nitrogenisnotavailableoristoodangeroustouse(e.g.onaboat).Ethanol(95%)canbesprayedonblocks
ofdryicetoaccelerateheattransfer.Thisfreezingprocesssuspendsmetabolicactivityinthesoiland
preservesinsitubacterialabundance.Second,replicationshouldbelargeenoughtoaccountforthehigh
levelofvariationinsoilmicrobialcommunitycompositionandstructure,witheachreplicaterepresentinga
homogeneousmixtureofmultiplesamples(oftensoilcores)takeninthefield.Biogeochemicalgradients
acrossphysicalinterfacesandwithinsedimentscanbeverysteep.Therefore,preservationandassessment
oflayersispreferable(seedeGruijteretal.,2006,foracomprehensivediscussionofsamplingdesignsspecific
tonaturalresourcemonitoring).Asmicrobialcommunitiesdependverystronglyonlocalconditions
(water/soilchemistryandbiologicalinteractions),sufficientmetadatashouldberecordedalongwitheach
sampletoenablethisvariationtobedescribedforaparticularcommunity.Asaminimum,thisshouldinclude
pH,temperature,dissolvedoxygen,conductivity,nitrate,nitriteandphosphorus.Finally,becauseoftheir
abilitytobringoxygenandcarbonresourcesintootherwiseanaerobicsoils,plantrootingzonescanplaya
pivotalroleindeterminingmicrobialcommunitycomposition(Marschneretal.,2001;Marschneretal.,
2004),andsamplestakenfromvegetatedsubstratesshouldaccountforthecompositionandcharacteristics
ofco‐occurringplantcommunities.
2.2Extraction
DNAextractioninvolvestwosteps:(1)breakingapartcellwallsandmembranes(celllysis)throughsome
combinationofheating,sonicationand/orchemicaltreatment,and(2)isolation/concentrationofDNAvia
filtration(Picardetal.,1992;Zhouetal.,1996).Althoughconceptuallysimilar,differentlysismethodscan
produceconflictingresults(Martin‐Laurentetal.,2001;Carriggetal.,2007).
Therearethreemajorchallengestoconsistencyintheextractionprocess.First,manymicroorganismsform
coloniesorcrustsonsubstratecomponents(sandgrainsandsmallstones).Thesecoloniescanbedifficultto
breakapart,andfundamentallyprotectmanycellsfromthelysisprocedure.Thiscanleadtodifferencesin
perceivedcommunitycompositionwhencomparingsampleswithdifferentsubstrategrainsizes.Pre‐
washingprocedureshavebeendevelopedtosuspendadheredcommunitiesinsolutionbeforebreakingapart
cells,leadingtomorerepresentativeextractions(Fortinetal.,2004;Heetal.,2005).
Second,thethicknessandmaterialpropertiesofcellwallsandmembranesarenotuniformforall
microorganisms.Developinglysisproceduresthereforeinvolvesidentifyingthemajorgroupsof
microorganismsofinteresttothestudy,andconsideringperformingparallelextractionstoisolatedistinct
groupsforcomparison.Sulphate‐reducingbacteria(SRB)andmethanogenicarchaeaareofparticularinterest
totheminingindustryfortheirprevalenceandactivityinminedsubstrates.Bacterialandarchaealcellwalls
comprisedifferentmaterials(seeKandlerandKönig,2014,forachemicalandfunctionaldescriptionofthese
differences).Asaresult,usingasinglelysisprocedurewillinvariablyunderrepresentoneofthesegroups,
andmultiplelysisproceduresshouldbeevaluatedifacomprehensiveunderstandingofmicrobialcommunity
compositionandprevalenceisdesired.
Finally,highlydegradedorganicmaterialincarbon‐richsoils(humicacids)aredifficulttoseparatefromDNA,
oftenleadingtocontaminationofDNAextractsandpoorsequencereads(Yeatesetal.,1998).Procedures
havebeendevelopedtoaccommodatesamplesrichinorganicacids;theseinvolveremovalofhumicacids
withadditionalfiltrationsteps(TsaiandOlson,1992)orionexchangechromatography(TebbeandVahjen,
1993).

UsinggenomicsinminereclamationL.H.Fraser,H.W.Garris,S.A.Baldwin,J.D.VanHammeandW.C.Gardner
4MineClosure2015,Vancouver,Canada
2.3Amplificationandsequencing
DNAextractedfromthemineenvironmentsampleprovidesinformationaboutthemicrobialcommunityand
metabolicprocessesatthatsite.Onemethodtodescribethemicrobialcommunityusesspecificcomponents
oftheDNA,suchasgenesknowntovaryamongspecies,thatareamplifiedviapolymerasechainreaction
(PCR)(Valentinietal.,2009).Thesereferencegenesfoundintheminesamplearecomparedtocurated
databasesofknownannotatedsequences(BOLD,GenBank,MG‐RAST)(RatnasinghamandHebert,2007;
Meyeretal.,2008;Bensonetal.,2013),therebyidentifyingwhichspeciesarepresentattheminesite.Early
usesofgenomicstechnologyforbacterialcommunitycharacterisationfocusedonthe16SrRNAgene,a
segmentofDNAcriticalfortheproductionofproteinsbyprokaryoticcells,andthatisgenerallyuniqueto
eachspecies(Weisburgetal.,1991).Sequencingof16SrRNAgeneshasbeenusedtoquantifymicrobial
diversitywithsufficientresolutiontodetectshiftsalongmajorecologicalgradients(Schmidtetal.,2014).
However,thisapproachislimitedtodeterminingwhichmicrobialspeciesarepresentandprovideslittle
informationabouttheactualmetabolicprocessestakingplace(Eisen,2007).Stableisotope(with13C)
probinghasbeenusedinrecentyearsincombinationwithenvironmentalDNAsequencingtoisolate
microbialcommunityfractionsinvolvedinspecificmetabolicprocesses(DumontandMurrell,2005;
Verasteguietal.,2014),whichmakestheimportantlinkbetweenbacterialcommunitycompositionand
specificmetabolicfunctions.
Withtheadventofhighthroughputsequencingtechnologies,targetingonlyspecificregionswithinDNAhas
givenwayto“shotgun”andwhole‐genome/metagenomesequencingusingalloftheDNAinthesample
(Tringeetal.,2005).Thetermmetagenomeisusedwhenthesamplecontainsmanyorganisms(andthus
genomes),asisthecaseinsoils.Shotgunsequencinginvolvesbreakingthemetagenomeintomanysmall
fragmentsviaphysicalshearingorenzymaticprocesses(Sharpton,2014).Aftersequencing,thesefragments
canbeusedtoreconstructentiregenomesfortheorganismspresentatthesitebyaligningthesequences
wheretheyoverlap.Themetagenomeincludesbothfunctionalinformation(genemarkerslinkedtospecific
metabolicproducts)andcompositionalinformation(e.g.16Sandotherspecies‐specificreferencesequences)
(Xiaetal.,2011).Thecomputationaltechniquesrequiredtocompile,alignandinterpretthesemillionsof
basesofgeneticcodearrangedintoshortfragmentsistermedbioinformatics(discussedinthenextsection).
AnadditionaluseofenvironmentalDNAfragmentsisfunctionalscreening.Cloningofthesefragmentsinto
livehostssuchasthebacteriumE.coliortheyeastP.pastorisenablesexpressionoftheproteinproducts
andtheirfunctionalscreening.Forexample,growingofE.colicellscontainingenvironmentalDNAon
mediumcontainingcelluloseallowedfordirectidentificationofnovelcellulaseenzymes(Mewisetal.,2013)
fromamineremediationenvironment.
2.4Bioinformatics
Bioinformaticsreferstothecomputationalproceduresusedtoextractmeaningfulinformationfromthevery
largedatasetsproducedinmetagenomicstudies.Bioinformaticspipelinesrequireconsiderable
computationalpoweranduniquealgorithmstocarryoutthestepsfromqualitycontrolofthesequencesto
assemblingfunctionalcomponents(i.e.sequencescodingforproteinsusedincellularfunctionscalledopen
readingframesorORFs)thatyieldinformationaboutthemetabolicpotentialoftheorganismspresentatthe
site.Bioinformaticsformetagenomicsarecomplex,ascompositionalaswellasfunctionalreconstructionis
required.Toidentifycompositionalstructure,themarkergenes(i.e.16SrRNA)arebinned,basedontheir
similarity,intooperationaltaxonomicunits(OTUs)thatarelikelytobederivedfromthesamespecies.
Assembledfunctionalcomponents(theORFs)andOTUsareinterrogatedagainstproteinandgenedatabases
inbioinformaticspipelinesusingtheblasttools(Altschuletal.,1990)inordertoassignputativefunctions
andidentitiestothemicrobialcommunityfoundintheminesitesample(seeSharpton,2014,fora
comprehensivediscussionofprocessingandinterpretationofshotgun‐derivedsequences).

KeynotesandPlenaries
MineClosure2015,Vancouver,Canada5
3Applications
3.1DNAbarcoding
Giventhemuchlarger,morecomplexgenomesfoundinplantandanimalspecies,themoleculartoolsfor
identifyingthemhavedevelopedmoreslowlythanthoseformicroorganisms.Whereasthe16SrRNAgeneis
foundinallbacteriaandarchaea,thecorresponding18SrRNAgeneinhighereukarya,andevenfungi,isnot
asreliableasaspeciesidentifier.Assuch,mucheffortisbeingputintofindingsignaturegenes,called“DNA
barcodes,”fordifferentgroupsofplants,invertebratesandvertebrates(Hebertetal.,2003).Eachspecies
collectedcanberapidlyandcost‐effectivelyidentified,andwiththepropersamplingprotocols,quantifiedin
termsofrelativeabundanceanddiversitybysite.
NewGold’sNewAftonMinenearKamloops,BritishColumbia,Canada,beganoperationin2012.Although
themineisinitsearlydaysofoperation,ithastakenaproactiveapproachtofuturemineclosureplansby
implementingapartnershipwiththeBiodiversityInstituteofOntariotoimplementDNAbarcodingfor
environmentalimpactassessments.Thepilotprograminvolvedfoursites:twograsslandsites(disturbedand
undisturbed)andtwowetlandsites(disturbedandundisturbed).Invertebratesamplesfromthesefoursites
werecollectedinthesummerof2013,andDNAbarcodeanalyseswerecompletedinAugust2014.Between
294and5,560individualinvertebrateswerecapturedinMalaisetrapseachweek,and3,956specieswere
identified(D.WilsonandS.Davidson,personalcommunication8May,2015).Differenceswereobserved
betweenhabitattypessuchthatwetlandscontainedmorespeciesthangrasslands,andthenaturalgrassland
hadmorespeciesthanthedisturbedgrassland.Theintentionistocontinuemonitoringonafour‐tofive‐
yeartimescale.Suchbaselinedataprovideinvaluableinformationforfuturemineclosureandsite
reclamation.
3.2Meta‐omics
Thankstotheconservednatureof,andrelativelylonghistoryofcollectingsequencedatafor,the16SrRNA
geneinbacteriaandarchaeaand,toalesserextent,the18SrRNAgeneinfungi,researchersinterestedin
microbialcommunities(e.g.environment,health,industry)havereachedastagewhereusingthese
referencegenestodescribemicrobialcommunitycompositionanddiversityisnowroutine(Schmidtetal.,
2014).Ashortnumberofyearsago,publicationswouldbebasedsolelyon16SrRNAgenesurveysof
microbialcommunities,whereastodaythesesurveysareconsideredoneofmanystandardanalyticaltools
forscanningmicrobiallandscapes.While16Sand18SrRNAgenesurveyswillcontinuetobepowerfultools,
microbiologistshaverealisedthatthesereferencegenesdonotuniversallyreflectthemetabolicpotential
andbiochemicalactivitiesofindividualmicroorganisms,letalonecomplexmicrobialcommunities.This
realisationcontinuestomotivatethedevelopmentof“meta”toolstoqualifyandquantifyalloftheDNA,
mRNAandproteinsinmicrobialcommunities(metagenomics,metatranscriptomicsandmetaproteomics)
andtorelatethesedatatobiochemicalfluxes(metabolomics)and,ultimately,ecosystemfunctions(Krause
etal.,2014).
Atthepresenttime,therearegoodgenomicdatabasesforpurebacterialculturesgrownunderlaboratory
conditions.Generatinggooddraftsofmicrobialgenomescannowbedoneindaysratherthanyears,by
individualsratherthanteams,forhundredsratherthanmillionsofdollars.Thisisamajorshiftfromlessthan
adecadeago(Kyrpidesetal.,2014).Highthroughputtranscriptomicandproteomictoolshavecomeonline
andarebeingincreasinglyusedforpureculturablemicroorganismsaswell.Thetoolsareavailableto
characterisenaturalmicrobialcommunities,butanalysingdatafromevenafewsamplesinameaningfulway
stillrequirestheuseofsupercomputersorpowerfulcomputerclusters,andisbasedonimperfectdatabases
establishedintheearlydaysofgenomics(Howeetal.,2014).A“meta‐omics”studyisnotforthefaintof
heart,butthefieldisshiftingrapidly,andthesetypesofstudiesarebecomingmorecommondespitethe
needforlargemonetaryandpersonnelinvestments.Majorbreakthroughsarecurrentlybeingmadeto
advanceourunderstandingofthedominantrolesmicroorganismsplayinthemetabolismandlifestylesofall
macroorganisms.Further,biotechnologicalapplicationsarebeingrealisedinanarrayoffields,including

UsinggenomicsinminereclamationL.H.Fraser,H.W.Garris,S.A.Baldwin,J.D.VanHammeandW.C.Gardner
6MineClosure2015,Vancouver,Canada
forestry,agriculture,animalhusbandry,humanhealth,andfood,beverageandfuelproduction(Ekkersetal.,
2012).
Giventherichliteratureonmetal‐microbeinteractions(Gadd,2010),thereistremendousscopeforapplying
meta‐omicstominereclamation,particularlygiventhesmallbutsolidfoundationofgenomicworkbeing
doneinphytoremediation(Baietal.,2014),metal‐plantinteractions(HanikenneandNouet,2011;Bhargava
etal.,2012)andsoilecology(Howeetal.,2014)onwhichtobuild.Lookingtothefuture,oncecurrentmeta‐
omicstoolshavesufficientlymaturedformicrobialcommunities,thegene,transcript,proteinandmetabolic
signaturesfromplantsandothermacroorganismsmustbeintegratedinholisticmodelsinordertobetter
appreciatetheholobionts(macroorganismsandtheirassociatedmicrobialandviralcommunities)essential
tohealthyecosystems.
3.3Stateofthescience
3.3.1 Example 1: Bioreactors
Seepagefromminetailingsstoragefacilities,wasterockpiles,openpitsandundergroundworkings,aswell
asexcessprocesswater,containsmetalssuchasselenium,copper,molybdenum,zincandarsenic,often
alongwithsulphateandnitrate(McDonaldandStrosher,1998;WangandMulligan,2006).High‐density
sludgetreatmentisusedatmanyminesitestotreatthismine‐influencedwater,butthischemicalprocess
consumeslargequantitiesofreagentsandproduceshighvolumesoftoxicsludgerequiringlong‐termsafe
storage(ZinckandGriffith,2013).Bioreactorsofferasustainable,cost‐effectivealternativetoreagent‐based
watertreatmenttechnologies.
Biologicalprocessesusenaturalmicroorganismstotreatmine‐influencedwater.Somebioreactorsreduce
sulphatetoproducesulphide,whichchemicallybindswithmetalions,causingthemtoprecipitateoutof
solutionasstablemetalsulphides(Barnesetal.,1994).Additionally,bioreactorshavebeendesignedto
selectivelyremovespecificvaluablemetals,suchascopperandzinc,thatcanberecycledtothemetal
extractionfacility(ZinckandGriffith,2013).Bioreactorsareusedtoremovenitrate,whichishighinsome
mine‐influencedwaterowingtotheuseofexplosivesonminesites(Korenetal.,2000).Seleniumandarsenic
aremetalsthatoccurasanions,andtherearenaturalmicroorganismsthattransformthesecompoundsin
ordertogainenergyforgrowth.Biologicalreactorsusingtheseorganismssuccessfullyremoveseleniumand
arsenicfrommine‐influencedwater(Moritaetal.,2007).
Giventhebenefitsineconomicsandeffectivenesspromisedbybiologicaltreatment,itissurprisingthatuse
ofbioreactorsisnotwidespreadonminesites.Afewreasonsforthishavebeenrevealedthroughthe
applicationofmetagenomics.Bioreactorscontainconsortiaofmicroorganisms,ratherthanonesingletype
(Baldwinetal.,2012),andtheperformanceofthesebioreactorsdependsonthetypesofmicroorganisms
usedtoinoculatethem(Prudenetal.,2007).Microbialcommunitiesaredynamic,andtheirmembers
fluctuateinabundanceandactivityinresponsetochangesintheinfluentwateroroperatingconditions(Dar
etal.,2008).Shiftsinmicrobialcommunitycompositioncanleadtofailuretomeetwaterquality
specifications,ultimatelyputtingthereceivingenvironmentatrisk(MirjafariandBaldwin,2011).To
overcomethislimitationofbiologicaltreatment,itisnecessarytomonitormicrobialcommunitycomposition
inbioreactorsandcorrelatethistooperationalsettingsandperformancemetrics.Themicrobialcommunity
inmine‐basedbioreactorscanbemonitoredusingthemetagenomicstechniques,suchasthosetargetingthe
16SrRNA,describedinSection2.3(SchmidtovaandBaldwin,2011;Baldwinetal.,2015).
High‐throughputsequencingcangenerateenoughinformationtocharacterisetheentiremicrobial
communityincludingveryraremembers.Oftentheimportantfunctionalgroupsformetalremovalin
bioreactorsarerare,eventhoughtheiractionachievessuccessfultreatment(Rezadehbashietal.,2012).
Someothermicrobespresentfacilitatetheactivityofthesedesiredgroups,orsomemightcompetefor
nutrientsandhindertheiractivity.Nutrientconsumptionisoneofthemajorcostsinrunningabioreactor,
andwhenundesiredmicrobescompetesuccessfullyformostofthenutrients,thedesiredorganismsdecline
innumberandbioreactorperformancefails(Silvaetal.,2012).

KeynotesandPlenaries
MineClosure2015,Vancouver,Canada7
Muchoftheworkdonetodateonmicrobialcommunitiesinbioreactors,especiallythoserelatedtomine
remediation,hasfocusedonmicrobialcommunitycompositionbasedonsurveysofthe16SrRNAgene.
Thesesurveyshaverevealedthatmicrobialgroupsassociatedwithmetal‐richenvironmentsareunknown
anduncharacterised,suchthatnewtaxonomicgroupshavebeeninventedtoclassifythem(Khoshnoodiet
al.,2013).Thus,metagenomicstudiesareneededtodiscoverthepotentialfunctionsofthesenovel
organisms(Ellisetal.,2012).
SequencingofDNAprovidesinformationonmetabolicpotentialonly,anddoesnotrevealwhichofthegenes
arebeingactivelyexpressed.Activemetabolicprocessescanbedetectedbysequencingthetranscribed
genesusingatechniquereferredtoastranscriptomics(Luoetal.,2014).Althoughveryrecentlydeveloped
formicrobialcommunitiesinbioreactors(Luoetal.,2014),microbialtranscriptomicsispossiblebutrequires
carefulsamplingtopreservetheactivestateofthemicrobes.
Giventheaffordabilityofhigh‐throughputsequencing,useofmetagenomicsandtranscriptomicsfor
bioreactormonitoringisboundtoincrease.Sincethesedatasetsarenew,correlationofmicrobial
communitycomposition,metagenomicsandtranscriptomicswithbioreactoroperationandperformanceis
stillbeinginvestigated.Asthebioinformaticsimproves,thesemetagenomicstoolsshowpromiseforfuture
processcontrolofbioreactors.
3.3.2 Example 2: Passive treatment systems
Thebenefitofusingbioreactorsformineremediationisthattheycanbecontrolledusingtanks,pumps,
valves,instrumentationanddefinednutrientstoachievejusttherightconditionsandrapidkineticsfor
treatinglargeflowrates(ZinckandGriffith,2013).However,theircapitalandoperatingcostsaddtothe
expenseofmineoperationandclosure.Inaddition,althoughtheyarehighlyautomated,operatorsare
needed.
Thevastarrayofnaturalprocessesforbiogeochemicalcyclingofmetalsandnutrientscanbeharnessedin
so‐calledpassiveorsemi‐passivetreatmentsystems(Ziemkiewiczetal.,2003).Thesetypicallytaketheform
ofconstructedwetlands,eithersub‐surfaceflowanaerobic,surfaceflowaerobicor,mostcommonly,
combinationsthereof.Insteadofdefinednutrients,mixturesofwasteorganicmaterialssuchaswooddebris,
hay,compost,pulpandpapermillbiosolidsareused.Thesecomplexorganicmaterialsaredecomposedinto
thesmallercarboncompoundsneededforthesulphate‐andmetal‐reducingbacteria.
Ifsuccessful,passivetreatmentcanremovemetalsatseepsdistributedacrosstheminesiteforafractionof
thecostofactivebioreactortreatment(ZinckandGriffith,2013,p.14).Themetalresidualsarecapturedand
securedinsidetheorganicmatrix,mostoftenassparinglysolublemetalsulphides(Khoshnoodietal.,2013).
Theecosystemofapassiveremediationsystemisascomplicatedasthatofsoils(Baldwinetal.,2015).Like
bioreactors,theyareconsortiaofinteractingspeciesthatshiftincompositionwithgeochemicalgradients,
seasonsandtheageofthesystem(vanderLelieetal.,2012).Theirperformancemaydeclineastheorganic
materialdecomposes,evolvingtowardsmicrobialcommunitieswithcompletelydifferentmetabolic
potentialthanatthestart(Mirjafari,2014).Preliminarystudiesofthemicrobialcommunitiesinthese
systemsrevealthatthemetabolicpotentialformetalremovalinthemismuchwiderthanpreviouslythought
(Baldwinetal.,2015).Theycontainspeciesthattoleratehighmetalconcentrations,manynovelunclassified
candidatedivisiongroupsfoundinothermetal‐contaminatedenvironmentsandspeciescapableofusing
usuallyrecalcitrantaromaticcompoundsforgrowth.
SuccessfulmetalremovaloccurseveninplaceswhereSRBareextremelyrare,meaningeitherthatmany
othergroupsoforganismswedonotknowaboutarecapableofsulphatereductionand/ormetal
precipitation,orthatonlyafewsulphatereducersareneededforsuccessfultreatment(Khoshnoodietal.,
2013;Baldwinetal.,2015).Chartingofmicrobialcommunitiesinpassiveremediationbioreactorshas
revealedthattheyarenotstatic,butfluctuatecyclically(Baldwin,unpublisheddata).Usingmetagenomics
andmetatranscriptomics,wecanlearnmoreaboutthedynamicsoftheseecosystemsastheyrespondto
changingconditionsandusethisknowledgetodesignbettersystemsordiagnoseperformanceissues.

UsinggenomicsinminereclamationL.H.Fraser,H.W.Garris,S.A.Baldwin,J.D.VanHammeandW.C.Gardner
8MineClosure2015,Vancouver,Canada
3.3.3 Example 3: Novel gene discovery
Metal‐richecosystemsareconsideredextremeenvironments.Theyharbourhighlyspecialised
microorganismsthathaveevolveduniquemetabolismstotransform,sequesterordetoxifymetalsinorder
tosurvive.Examplesincludesulphatereducers’overproductionofextracellularpolymericmaterialtobind
upcopperions,therebycreatingnucleationsitesforprecipitation(JalaliandBaldwin,2000),intracellular
mineralisationoftelluriumtosequesterthishighlytoxicmetal(AmoozegarandKhoshnoodi,2012),and
methylationandvolatilisationofarsenicbyMethanocorpusculumlabreanum,suggestingthatitmaybea
significantcontributortometalcyclinginanaerobicenvironments(Khoshnoodietal.,2012).
Enzymesinvolvedinmetalcycling,orbiochemicalcompoundswiththeabilitytosequesterspecificmetal
ions,canbeusedinfuturebiotechnologiestoimprovebioremediation,andevendevelopmethodsforinsitu
mining.Thefieldoffunctionalmetagenomics,firstmentionedinSection2.3,isbeingusedtoscreenlarge
DNAfragmentsfromminesites(Mewisetal.,2011).TheselargefragmentsofenvironmentalDNAmay
containnovelgenesformetalcycling,and,usingselectivemediainthelaboratory,wecanscreentheE.coli
clonesformetalresistance.Itmaybepossibleinthefuturetoconstructbiochemicalpathwaysformetal
removalusingsimpleandeasy‐to‐groworganismsandsyntheticbiology.
3.4Futureofthescience
3.4.1 Site-specific pre-assessments for closure targets
Theminingindustryhasrecognisedforsometimethatplanningformineclosurebeginsevenbefore
overburdenisremovedfromthesite(Thirgood,1986inPolster1989).Untilrecently,thishasnotincludeda
significantconsiderationofthepre‐impactcommunityofplants,animalsandmicroorganismsresidinginthe
unalteredsubstrate,butseeMorrisonetal.(2005)andJasper(2007).Inthefuture,suchassessmentsshould
includeevaluationofbothunimpactedoverburdensoilcommunitiesandstockpilesofsuchmaterialfor
eventualrecoveringofthesite.Microbialgenomicsmayplayakeyroleinexpeditingminelandrestoration
byprovidinginformationonsoilcommunitydynamicsinoverburdenstockpiles,allowingmanagersto
maintainthesecommunitiesinawaythatexpeditesrecoveryoncethesesubstratesareusedtorecoverthe
minedsite.
Microbialgenomicsmayalsoserveasanindicatorforsuccessinthesesystems,providingadditionalevidence
ofsoilcommunityformationandecosystemtrajectory.Tobeabletosetobjectivesforeffectivemine
reclamationandevaluatewhatissuccessful,themeaningof“equivalentlandcapacity”needstobedefined
foreveryminesite.
Barcodingandmetagenomicscanbeincorporatedintomethodsforevaluatingecosystemservices.Microbial
communityanalysisprovidesvaluableinformationaboutnutrient(carbon,nitrogenandsulphur)cycling,
greenhousegasemissionsandmetaltransformationsthatcanbefedintodeterminingpre‐miningland
capacityassessment.Thiswillallowtargetstobespecifiedforpost‐minereclamation,andthesesametools
canbeusedtoevaluatewhetherremediationstrategiesareworking.
3.4.2 Whole-ecosystem modelling
Microbialgenomicsfillsacriticalgapineffortstosimulatetheformationofmetabolicnetworksin
ecosystems.Thepresenceorabsenceofkeymicrobialcommunitiesmaymeanthedifferencebetweena
successfullyremediatedsiteandpersistentdegradationofwaterquality(e.g.acidificationandmetal
leaching).Effortsareunderwaytodevelopcomprehensivepredictivenetworksforminesites,inwhich
environmentalgenomicsaretiedtoenvironmentalmonitoringdatatogenerateacomprehensive
understandingofecosystemfunction.Probabilisticmodellingframeworksmayverywellprovideearly
warningsignsofacidgenerationinminedsubstrates,andgenomicsdatamayyieldthenecessaryevidence
todeterminewhatremediationoptionswillbemostlikelytosucceedinboththeshortandlongterm.

KeynotesandPlenaries
MineClosure2015,Vancouver,Canada9
3.4.4 Defining benchmarks
Definingbenchmarksmayverywellbethekeycomponentnecessarytoleveragegenomicstoolsformine‐
sitemanagement.Bioinformaticsdataprocessingworkflows(pipelines)exist(sometimesincorporated
directlyintosequencinghardware)thatprovideforrapidprocessingandinterpretationofsequencingruns.
Thegreatquestionsfortheminingindustrycanbeputquitesimply:(1)Whatdowesequence?,and(2)How
doweusetheresultingdatatoimprovemanagement,reclamationandcontainment?
Section2.1providesguidelinesforsamplingofthesesites,buttheindustryrequiresawealthofcontext‐
dependentvalidationoftheseapproachestobecomewidespread.Therefore,researchanddevelopment
resources(bothacademicandindustrial)shouldbeleveragedinsuchawaythatsamplingprocedurescanbe
standardisedtothesortsofquestionsofinterestatminesites(e.g.Howdosoilmicrobialcommunitiesform
onminedsubstrate?Whatsoilconditionspromoterecoveryofnativespecies?).
Ultimately,withstandardsamplingprocedures,bioinformaticspipelinescanbetailoredtoindustry‐specific
questions.Forexample,asamplingprotocolmightbedevelopedtoevaluatediffuseleachateexposurein
ripariansubstrates,withthequestion:Doesexposuretoleachateaffectmicrobialcommunitycomposition
anddynamicsinawaythatcouldlimitcontainmentinthefuture?Fieldsamplingandsequencingfollowing
establishedprotocolswouldbeprocessedthroughatailoredbioinformaticspipelinethatyieldsthefollowing
outputs:
1. Communitycompositionmatricesandplannedcomparisons(diversity,similarity,etc.)
2. Compositionaldifferencesandknownassociations(i.e.variationsingroupsoforganismsknown
tobeinvolvedincertaingeochemicalpathways)
3. Managementrecommendationsandreferencestosimilarscenarios/responses.
4Conclusions
Wehaveoutlinedarangeofgenomicsapplicationstomineclosure,fromcharacterisingnaturalsubstrates
beforeoverburdenremoval,towatertreatment,tobioremediationandmonitoringofhealthyreclaimed
ecosystems.Inperformingthisreview,weidentifiedtwokeyconstraintsonthewidespreadapplicationof
genomicsformineclosure.First,industry‐widestandardoperatingprotocolsneedtobedevelopedformine
closure,includingsamplingproceduresdesignedforrepresentativenessandcomparability(spatialextent,
replication,temporalfrequency).Second,sequencinganddatainterpretationpipelinesmustbeestablished
inparallelwiththedevelopmentofthesestandards,allowingminemanagerstomoreeasilydiscoverwhat
worksinagivensystemandtoestablishbenchmarksforreclamationsuccess.Miningisakeyglobalindustry
fordevelopmentandforqualityoflife.Theintegrationofgenomicstechnologiesintomineclosureplanning
andimplementationmaydrasticallyimprovethestabilityandreliabilityofecosystemreclamation.
Acknowledgements
WewouldliketothankJonTaylor(UniversityofBritishColumbia)forhelpfultroubleshootingandadvisement
onsamplingdesignformine‐siteapplications.WewouldliketothankGenomeBC,GenomeCanada,NSERC
EngageandMitacsAccelerateforfundingtosupportpreliminaryR&Donmetagenomicstechnologies.We
wouldalsoliketothankDennisWilsonandScottDavidsonofNewGoldforprovidingdetailsabouttheir
project.Finally,wewouldliketothankMountPolleyMineCorporationandparticularlyKatieMcMahon,
ColleenHughes,LukeMogerandArtFryefortheirpartnershipinprojectsaimedataddressingmanyofthe
questionsaddressedinthisreview.


UsinggenomicsinminereclamationL.H.Fraser,H.W.Garris,S.A.Baldwin,J.D.VanHammeandW.C.Gardner
10MineClosure2015,Vancouver,Canada
References
Allan,R.(1995)Impactofminingactivitiesontheterrestrialandaquaticenvironmentwithemphasisonmitigationandremedial
measures,inHeavymetals:problemsandsolutions,U.Förstner,W.SalomonsandP.Mader(eds),SpringerBerlinHeidelberg,
Berlin/Heidelberg,Germany,pp.119–140.
Altschul,S.F.,Gish,W.,Miller,W.,Myers,E.W.&Lipman,D.J.(1990)Basiclocalalignmentsearchtool,JournalofMolecularBiology,
Vol.215,pp.403–410.
Amoozegar,M.andKhoshnoodi,M.(2012)Telluriteremovalbyatellurium‐toleranthalophilicbacterialstrain,Thermoactinomyces
sp.QS‐2006,Annalesdel’InstitutPasteurMicrobiologie,Vol.62,pp.1031–1037.
Bai,Y.,Liang,J.,Liu,R.,Hu,C.andQu,J.(2014)Metagenomicanalysisrevealsmicrobialdiversityandfunctionintherhizospheresoil
ofaconstructedwetland,EnvironmentalTechnology,Vol.35,pp.2521–2527.
Baldwin,S.A.,Resadehbashi,M.,Taupp,M.,Mattes,A.,andHallam.S.J.(2012)Thegeneticdiversityofmicrobesinananaerobic
biologicalreactortreatingmetal‐richlandfillleachate:whatthistellsusabouttreatmentmechanisms,Proceedingsofthe9th
InternationalConferenceonAcidRockDrainage.20‐26May,Ottawa,Canada.Vol.1.pp.128–139.
Baldwin,S.A.,Khoshnoodi,M.,Rezadehbashi,M.,Taupp,M.,Hallam,S.,Mattes,A.andSanei,H.(2015)Themicrobialcommunityof
apassivebiochemicalreactortreatingarsenic,zincandsulfate‐richseepage,FrontiersinBioengineeringandBiotechnology,
Vol.3(27),pp.1–13.
Barnes,L.J.,Scheeren,P.J.M.andBuisman,C.J.N.(1994)Microbialremovalofheavymetalsandsulfatefromcontaminated
groundwaters,inEmergingtechnologyforbioremediationofmetals,Volume2,J.L.MeansandR.E.Hinchee(eds),CRCPress,
BocaRaton,USA,pp.38–49.
Benson,D.A.,Cavanaugh,M.,Clark,K.,Karsch‐Mizrachi,I.,Lipman,D.J.,Ostell,J.andSayers,E.W.(2013)GenBank,NucleicAcids
Research,Vol.41,pp.D36–42.
Bhargava,A.,Carmona,F.F.,Bhargava,M.andSrivastava,S.(2012)Approachesforenhancedphytoextractionofheavymetals,
JournalofEnvironmentalManagement,Vol.105,pp.103–120.
Britton,J.M.(1998)AnevaluationofpublicinvolvementinreclamationdecisionmakingatthreemetalminesinBritishColumbia,MA
Thesis,UniversityofBritishColumbia,Canada.
Carrigg,C.,Rice,O.,Kavanagh,S.,Collins,G.andO’Flaherty,V.(2007)DNAextractionmethodaffectsmicrobialcommunityprofiles
fromsoilsandsediment,AppliedMicrobiologyandBiotechnology,Vol.77,pp.955–964.
Cidu,R.,Biagini,C.,Fanfani,L.,LaRuffa,G.andMarras,I.(2001)MineclosureatMonteponi(Italy):effectofthecessationof
dewateringonthequalityofshallowgroundwater,AppliedGeochemistry,Vol.16,pp.489–502.
Dar,S.A.,Kleerebezem,R.,Stams,A.J.M.,Kuenen,J.G.andMuyzer,G.(2008)Competitionandcoexistenceofsulfate‐reducing
bacteria,acetogensandmethanogensinalab‐scaleanaerobicbioreactorasaffectedbychangingsubstratetosulfateratio,
AppliedMicrobiologyandBiotechnology,Vol.78,pp.1045–1055.
deGruijter,J.,Brus,D.J.,Bierkens,M.F.P.andKnotters,M.(2006)Samplingfornaturalresourcemonitoring,Springer‐VerlagBerlin
Heidelberg,NewYork,USA.
Dumont,M.G.andMurrell,J.C.(2005)Stableisotopeprobing–linkingmicrobialidentitytofunction,NatureReviews:Microbiology,
Vol.3,pp.499–504.
Eisen,J.A.(2007)Environmentalshotgunsequencing:Itspotentialandchallengesforstudyingthehiddenworldofmicrobes,PLoS
Biology,Vol.5,pp.384–388.
Ekkers,D.M.,Cretoiu,M.S.,Kielak,A.M.andvanElsas,J.D.(2012)Thegreatscreenanomaly–anewfrontierinproductdiscovery
throughfunctionalmetagenomics,AppliedMicrobiologyandBiotechnology,Vol.93,pp.1005–1020.
Ellis,J.T.,Sims,R.C.andMiller,C.D.(2012)Monitoringmicrobialdiversityofbioreactorsusingmetagenomicapproaches,Sub‐Cellular
Biochemistry,Vol.64,pp.73–94.
Ficetola,G.F.,Miaud,C.,Pompanon,F.andTaberlet,P.(2008)SpeciesdetectionusingenvironmentalDNAfromwatersamples,
BiologyLetters,Vol.4,pp.423–425.
Fisher,B.,Turner,R.K.andMorling,P.(2009)Definingandclassifyingecosystemservicesfordecisionmaking,EcologicalEconomics,
Vol.68,pp.643–653.
Fortin,N.,Beaumier,D.,Lee,K.andGreer,C.W.(2004)SoilwashingimprovestherecoveryoftotalcommunityDNAfrompolluted
andhighorganiccontentsediments,JournalofMicrobiologicalMethods,Vol.56,pp.181–191.
Foster,R.C.(1988)Microenvironmentsofsoilmicroorganisms,BiologyandFertilityofSoils,Vol.6,pp.189–203.
Frouz,J.,Prach,K.,Pižl,V.,Háněl,L.,Starý,J.,Tajovský,K.,Materna,J.,Balík,V.,Kalčík,J.,andŘehounková,K.(2008)Interactions
betweensoildevelopment,vegetationandsoilfaunaduringspontaneoussuccessioninpostminingsites,EuropeanJournal
ofSoilBiology,Vol.44,pp.109–121.
Gadd,G.M.(2010)Metals,mineralsandmicrobes:geomicrobiologyandbioremediation,Microbiology,Vol.156,pp.609–643.
GovernmentofBritishColumbia(2008)Health,safetyandreclamationcodeforminesinBritishColumbia,viewed10February2015,
http://www.empr.gov.bc.ca/Mining/HealthandSafety/Documents/HSRC2008.pdf.
Hanikenne,M.andNouet,C.(2011)Metalhyperaccumulationandhypertolerance:amodelforplantevolutionarygenomics,Current
OpinioninPlantBiology,Vol.14,pp.252–259.
Harris,J.A.(2003)Measurementsofthesoilmicrobialcommunityforestimatingthesuccessofrestoration,EuropeanJournalofSoil
Science,Vol.54,pp.801–808.
Harris,J.(2009)Soilmicrobialcommunitiesandrestorationecology:facilitatorsorfollowers?Science,Vol.325,pp.573–574.
He,J.,Xu,Z.andHughes,J.(2005)Pre‐lysiswashingimprovesDNAextractionfromaforestsoil,SoilBiologyandBiochemistry,Vol.
37,pp.2337–2341.

KeynotesandPlenaries
MineClosure2015,Vancouver,Canada11
Hebert,P.D.N.,Cywinska,A.,Ball,S.L.anddeWaard,J.R.(2003)BiologicalidentificationsthroughDNAbarcodes,ProceedingsofThe
RoyalSocietyB:Biologicalsciences,Vol.270,pp.313–321.
Holl,K.D.(2002)Long‐termvegetationrecoveryonreclaimedcoalsurfaceminesintheeasternUSA,JournalofAppliedEcology,
Vol.39,pp.960–970.
Howe,A.C.,Jansson,J.K.,Malfatti,S.A.,Tringe,S.G.,Tiedje,J.M.andBrown,C.T.(2014)Tacklingsoildiversitywiththeassemblyof
large,complexmetagenomes,ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica,Vol.111,
pp.4904–4909.
Hugenholtz,P.,Goebel,B.M.andPace,N.R.(1998)Impactofculture‐independentstudiesontheemergingphylogeneticviewof
bacterialdiversity,JournalofBacteriology,Vol.180,pp.4765–4774.
Jalali,K.andBaldwin,S.A.(2000)Theroleofsulphatereducingbacteriaincopperremovalfromaqueoussulphatesolutions,Water
Research,Vol.34,pp.797–806.
Jasper,D.(2007)BeneficialsoilmicroorganismsoftheJarrahForestandtheirrecoveryinbauxiteminerestorationinsouthwestern
Australia,RestorationEcology,Vol.15,pp.S74‐S84.
Kandler,O.andKönig,H.(2014)CellwallpolymersinArchaea(Archaebacteria),CellularandMolecularLifeSciencesCMLS,Vol.54,
pp.305–308.
Khoshnoodi,M.,Rezadehbashi,M.,Taupp,M.,Hallam,S.andBaldwin,S.A.(2012)CorrelationofthemicrobialdiversityIna
biochemicalreactortreatingmetal‐richwaterwithenvironmentalfactors,CanadianSocietyofMicrobiology,Vancouver,
Canada.
Khoshnoodi,M.,Dipple,G.,andBaldwin,S.A.(2013)Mineralogicalstudyofabiologically‐basedtreatmentsystemthatremoves
arsenic,zincandcopperfromlandfillleachate,Minerals,Vol.3,pp.427‐449.
Koren,D.W.,Gould,W.D.andBédard,P.(2000)Biologicalremovalofammoniaandnitratefromsimulatedmineandmilleffluents,
Hydrometallurgy,Vol.56,pp.127–144.
Krause,S.,LeRoux,X.,Niklaus,P.A.,VanBodegom,P.M.,Lennon,J.T.,Bertilsson,S.,Grossart,H.‐P....Bodelier,P.L.E.(2014)Trait‐
basedapproachesforunderstandingmicrobialbiodiversityandecosystemfunctioning,FrontiersinMicrobiology,Vol.5,pp.
1–10.
Kyrpides,N.,Woyke,T.andEisen,J.(2014)Genomicencyclopediaoftypestrains,phaseI:Theonethousandmicrobialgenomes
(KMG‐I)project,StandardsinGenomicSciences,Vol.9,pp.628–634.
Luo,F.,Gitiafroz,R.,Devine,C.E.,Gong,Y.,Hug,L.A.,Raskin,L.andEdwards,E.A.(2014)Metatranscriptomeofananaerobicbenzene‐
degrading,nitrate‐reducingenrichmentculturerevealsinvolvementofcarboxylationinbenzeneringactivation,Appliedand
EnvironmentalMicrobiology,Vol.80,pp.4095–4107.
Marschner,P.,Crowley,D.andYang,C.H.(2004)Developmentofspecificrhizospherebacterialcommunitiesinrelationtoplant
species,nutritionandsoiltype,PlantandSoil,Vol.261,pp.199–208.
Marschner,P.,Yang,C.‐H.,Lieberei,R.andCrowley,D.(2001)Soilandplantspecificeffectsonbacterialcommunitycompositionin
therhizosphere,SoilBiologyandBiochemistry,Vol.33,pp.1437–1445.
Martínez‐Ruiz,C.andMarrs,R.H.(2007)Somefactorsaffectingsuccessionalchangeonuraniumminewastes:Insightsforecological
restoration,AppliedVegetationScience,Vol.10,pp.333–342.
Martin‐Laurent,F.,Philippot,L.,Hallet,S.,Chaussod,R.,Germon,J.C.,Soulas,G.andCatroux,G.(2001)DNAextractionfromsoils:
Oldbiasfornewmicrobialdiversityanalysismethods,AppliedandEnvironmentalMicrobiology,Vol.67,pp.2354–2359.
McDonald,L.andStrosher,M.(1998)SeleniummobilizationfromsurfacecoalminingintheElkRiverBasin,BritishColumbia:Asurvey
ofwater,sedimentandiota,MinistryofEnvironment,LandsandParks:KootenayRegion,Cranbrook,Canada.
Mewis,K.,Armstrong,Z.,Song,Y.C.,Baldwin,S.A.,Withers,S.G.andHallam,S.J.(2013)Biominingactivecellulasesfromamining
bioremediationsystem,JournalofBiotechnology,Vol.167,pp.462–471.
Mewis,K.,Taupp,M.andHallam,S.J.(2011)Ahighthroughputscreenforbiominingcellulaseactivityfrommetagenomiclibraries,
JournalofVisualizedExperiments:JoVE,Vol.48(2461),pp.1‐4.
MeyerF,PaarmannD,D’SouzaM,OlsonR,GlassEM,KubalM,PaczianT,RodriguezA,StevensR,WilkeA,WilkeningJ,andEdwards
RA.(2008)ThemetagenomicsRASTserver–apublicresourcefortheautomaticphylogeneticandfunctionalanalysisof
metagenomes,BMCBioinformatics,Vol.9(386),pp.1‐8.
Mirjafari,P.andBaldwin,S.A.(2011)Factorsaffectingthestart‐up,operationanddeclineofalaboratory‐basedpassivetreatment
systemforseleniumandsulfateremoval,inProceedingsConferenceofMetallurgy,2–5October2011,Montreal,Canada.
Mirjafari,P.(2014)Complexbiochemicalreactorsforseleniumandsulphatereduction:organicmaterialbiodegradationand
microbialcommunityshifts.UBCDissertation,pp.1‐181.http://hdl.handle.net/2429/46013.
Morita,M.,Uemoto,H.andWatanabe,A.(2007)Reductionofseleniumoxyanionsinwastewaterusingtwobacterialstrains,
EngineeringinLifeSciences,Vol.7,pp.235–240.
Morrison,B.,Lamb,D.andHundloe,T.(2005)Assessingthelikelihoodofminesiterevegetationsuccess:AQueenslandcasestudy,
AustralianJournalofEnvironmentalManagement,Vol.12,pp.165–182.
Mummey,D.L.,Stahl,P.D.andBuyer,J.S.(2002)Soilmicrobiologicalproperties20yearsaftersurfaceminereclamation:Spatial
analysisofreclaimedandundisturbedsites,SoilBiologyandBiochemistry,Vol.34,pp.1717–1725.
Muscatello,J.R.andJanz,D.M.(2009)Seleniumaccumulationinaquaticbiotadownstreamofauraniumminingandmillingoperation,
TheScienceoftheTotalEnvironment,Vol.407,pp.1318–1325.
Picard,C.,Ponsonnet,C.,Paget,E.,Nesme,X.andSimonet,P.(1992)DetectionandenumerationofbacteriainsoilbydirectDNA
extractionandpolymerasechainreaction,AppliedEnvironmentalMicrobiology,Vol.58,pp.2717–2722.


UsinggenomicsinminereclamationL.H.Fraser,H.W.Garris,S.A.Baldwin,J.D.VanHammeandW.C.Gardner
12MineClosure2015,Vancouver,Canada
Polster,D.(1989)SuccessionalreclamationinwesternCanada:newlightonanoldsubject.CanadianLandReclamationAssociation
andAmericanSocietyforSurfaceMiningandReclamationConference,27‐31August,Alberta,Canada.pp.333‐337.
PrudenA,MessnerN,PereyraL,HansonRE,HiibelSR,ReardonKF(2007)Theeffectofinoculumontheperformanceofsulfate‐
reducingcolumnstreatingheavymetalcontaminatedwater.Waterresearch,Vol.41,pp.904–914.
Ranjard,L.andRichaume,A.(2001)Quantitativeandqualitativemicroscaledistributionofbacteriainsoil,ResearchinMicrobiology,
Vol.152,pp.707–716.
Ratnasingham,S.andHebert,P.D.N.(2007)BarcodingBOLD:Thebarcodeoflifedatasystem(www.barcodinglife.org),Molecular
EcologyNotes,Vol.7,pp.355–364.
Rezadehbashi,M.,Khoshnoodi,M.,Taupp,M.,Hallam,S.andBaldwin,S.A.(2012)Microbialdiversityofabiochemicalreactor
removingmetalsfromsmelterwasteleachate,CanadianSocietyofMicrobiology,Vancouver,Canada.
Schmidt,T.S.B.,MatiasRodrigues,J.F.andvonMering,C.(2014)EcologicalconsistencyofSSUrRNA‐basedoperationaltaxonomic
unitsataglobalscale,PLoSComputationalBiology,Vol.10,pp.1–10.
Schmidtova,J.andBaldwin,S.(2011)Correlationofbacterialcommunitiessupportedbydifferentorganicmaterialswithsulfate
reductioninmetal‐richlandfillleachate,WaterResearch,Vol.45,pp.1115–1128.
Sharpton,T.J.(2014)Anintroductiontotheanalysisofshotgunmetagenomicdata,FrontiersinPlantScience,Vol.5(209),pp.1‐14.
Silva,A.F.,Carvalho,G.,Oehmen,A.,Lousada‐Ferreira,M.,vanNieuwenhuijzen,A.,Reis,M.A.M.,Crespo,M.T.B(2011)Microbial
populationanalysisofnutrientremoval‐relatedorganismsinmembranebioreactors.AppliedMicrobiologyand
Biotechnology,Vol.93(5),pp.2171‐2180.
Skousen,J.,Ziemkiewicz,P.andVenable,C.(2009)Treerecruitmentandgrowthon20‐year‐old,unreclaimedsurfaceminedlandsin
WestVirginia,InternationalJournalofMining,ReclamationandEnvironment,Vol.20,pp.142–154.
Tebbe,C.C.andVahjen,W.(1993)InterferenceofhumicacidsandDNAextracteddirectlyfromsoilindetectionandtransformation
ofrecombinantDNAfrombacteriaandayeast,AppliedEnvironmentalMicrobiology,Vol.59,pp.2657–2665.
Thirgood,J.(1986)Reclamationpast,presentandfuture,inProceedings11thAnnualMeetingCanadianLandReclamation
Association,3‐6June,Vancouver,Canada.
Tordoff,G.,Baker,A.J.andWillis,A.(2000)Currentapproachestotherevegetationandreclamationofmetalliferousminewastes,
Chemosphere,Vol.41,pp.219–228.
TringeS.G.,vonMeringC,KobayashiA,SalamovAA,ChenK,ChangHW,PodarM,ShortJM,MathurEJ,DetterJC,BorkP,Hugenholtz
P,andRubinEM.(2005)Comparativemetagenomicsofmicrobialcommunities,Science,Vol.308,pp.554–557.
Tsai,Y.L.andOlson,B.H.(1992)RapidmethodforseparationofbacterialDNAfromhumicsubstancesinsedimentsforpolymerase
chainreaction,AppliedEnvironmentalMicrobiology,Vol.58,pp.2292–2295.
Valentini,A.,Pompanon,F.andTaberlet,P.(2009)DNAbarcodingforecologists,TrendsinEcologyandEvolution,Vol.24,pp.110–
117.
vanderLelie,D.,Taghavi,S.andMcCorkle,S.(2012)Themetagenomeofananaerobicmicrobialcommunitydecomposingpoplar
woodchips,PloSOne,Vol.7(5),No.e36740,pp.1‐–6.
Verastegui,Y.,Cheng,J.,Engel,K.,Kolczynski,D.,Mortimer,S.,Lavigne,J.,Montalibet.J.…andNeufeld,J.D.(2014)Multisubstrate
isotopelabelingandmetagenomicanalysisofactivesoilbacterialcommunities,mBio,Vol.5(4),No.e01157‐14,pp.1‐12.
Wang,S.andMulligan,C.N.(2006)OccurrenceofarseniccontaminationinCanada:sources,behavioranddistribution,Scienceof
TheTotalEnvironment,Vol.366,pp.701–721.
Weisburg,W.G.,Barns,S.M.,Pelletier,D.A.andLane,D.J.(1991)16SribosomalDNAamplificationforphylogeneticstudy,Journalof
Bacteriology,Vol.173,pp.697–703.
Xia,L.C.,Cram,J.A.,Chen,T.,Fuhrman,J.A.andSun,F.(2011)Accurategenomerelativeabundanceestimationbasedonshotgun
metagenomicreads,E.Dias‐Neto(ed),PloSOne,Vol.6,No.e27992,pp.1–13.
Yeates,C.,Gillings,M.R.,Davison,A.D.,Altavilla,N.andVeal,D.A.(1998)MethodsformicrobialDNAextractionfromsoilforPCR
amplification,BiologicalProceduresOnline,Vol.1,pp.40–47.
Zhou,J.,Bruns,M.andTiedje,J.(1996)DNArecoveryfromsoilsofdiversecomposition,AppliedEnvironmentalMicrobiology,Vol.
62,pp.316–322.
Ziemkiewicz,P.F.,Skousen,J.G.andSimmons,J.(2003)Long‐termperformanceofpassiveacidminedrainagetreatmentsystems,
MineWaterandtheEnvironment,Vol.22,pp.118–129.
Zinck,J.andGriffith,W.(2013)Reviewofminedrainagetreatmentandsludgemanagementoperations,MENDreport3.43.1,viewed
10February2015,http://mend‐nedem.org/wp‐content/uploads/MEND3.43.1ReviewofMineDrainageTreatmentSludge
ManagementOperations.pdf.
