TheEffectofDifferentSolutionsHighinNitrogen

onMicrobeInducedOilDegradation

NilesNorthHighSchool
20142016
ZacharyLuna

Luna

TableofContents:
Acknowledgments................................................................................................................................................2
Purpose.....................................................................................................................................................................3
HypothesisandRationale................................................................................................................................4
ReviewofLiterature.....................................................................................................................................514
Materials................................................................................................................................................................15
Procedure.......................................................................................................................................................1617
Variables................................................................................................................................................................18
Results.............................................................................................................................................................1921
DataAnalysis.................................................................................................................................................22
StatisticalAnalysis....................................................................................................................................2324
ExperimentalError...........................................................................................................................................24
Conclusion......................................................................................................................................................26
20122014ProjectOutline....................................................................................................................2737
References......................................................................................................................................................3840

Luna

Acknowledgments
IwouldliketothankMrs.PosnockandMrs.Camelforkeepingmeontaskand
givingmeeffectiveguidanceasmyprojectprogressedforthepasttwoyears.In
additionIwouldliketothankmymentorswhoareresearchersinmyspecifictopicof
study,SteveRockandAnnWhelan.WithoutthemIwouldbelackingasignificant
amountoftheinformationIhadbeforeevengoingintotesttrials.Finally,Iwouldliketo
thankmymotherforkeepingmemotivatedandencouragingmetotakeextrameasures
toensuretheprojectwouldbesuccessful.

Luna

Purpose
Thepurposeofthisexperimentistofindoutwhichnitrogenrichsolutioncause
microbestothriveandconsumeoilatthemostefficientratepossible.Atthesametime
theexperimentalsoseekstofindouttheeasiestandmosteffectivewaytoapplythese
solutionswhetheritinvolvessimplyapplyingthemtothesurfaceofthegroundor
mixingtheminwiththesoil.

Luna

Hypothesis
IfdifferentsolutionshighinNitrogensuchasUrea,StrontiumNitrateandAmmonium
Hydroxideareintroducedtooileatingmicrobestoincreaseoilconsumption,Ammonium
Hydroxidewillbethemostfavorableforthemicrobes,environmentandthemost
financiallybeneficial.

Rationale:
Thehypothesisisvalidbecausecurrentresearchindicatesthatthese
conditionsarefavorableformultiplyingoileatingbacteriacoloniesthathavebeen
studiedinotherterrainsandgeographicregionsofthecountry.Bycombiningthese
favorableconditionsitwillincreasethethebacteriasabilityandefficiencyatingesting
theoilinoillacedsoilsamples.AmmoniumHydroxideisalso82%Nitrogen.WhileUrea
is42%andStrontiumNitrateisabout80%.

Luna

ReviewofLiterature
InSeptemberof2014astringofaccidentsinaremotegulleyinWyoming
resultedinalmost220,000gallonsofoilcontaminatingtheravine.Thisspillwaslocated
inthePowderRiverBasinwheremultiplespillshaveoccurredinthelastfewyears
becauseofariseinoilproduction.Wyomingisnowinamultiyearandmultimillion
dollarcleanupprocesswhichhasmadethestatesixthplaceindebtpercapita(Gruver,
2014).Whenanoilspilloccurs,itendangerstheaffectedplantandanimalspeciesand
disruptstheecosystem.Oilcanpollutedifferenttypesofterrainincludingbodiesof
water,coastline,andsoil.Althoughoilisanaturaloccurringsubstance,itisstill
hazardoustomostspeciesofplantsandanimalsinlargequantities.Furthermore,large
oilspillscantakeyearstoremovefromanenvironment.Althoughtherearemany
methodstocleanupoilspills,mostareinefficientandultimatelycauseaharmfulimpact
ontheenvironment.Methodssuchasburning,skimming,andreleasingchemicalsto
depletetheoilhavebeenshowntobeeffectiveinonlyveryspecificenvironmentsbut
donotthoroughlyremovetheoil.Thesemethodsarenotonlyharmfulbutcostlyaswell.
Scientistshavediscoveredthattheearthhaswaystohealitselffromoilspills
duetonaturaloilseepagefromtheearthscrust.Specializedmicroorganismshave
evolvedwhichingestanddegradeoil.Thesenaturallyoccurringmicrobesfeedon
hydrocarbonsintheoilwhichthenbreakitdown.Oilcontainshydrocarbonswhich
consistsofdifferenttraceamountsofhydrogenandcarbon.Thebacterianeedsoxygen
tocombinewiththehydrocarbonswhichthencreatewaterandcarbondioxideasan
excrete.Theseoileatingbacteriacaneasilybedispersedbyagentssuchascurrents,

Luna

winds,anddeteriorationfromthesoil.Bypropagatingthesenaturallyoccurring
bacterias,orbycreatinglargeamountsofsyntheticoileatingbacteria,amoreefficient
strategymaybefoundtoaddressoilspillswhichmayhavealessharmfulimpactonthe
environment(Hazen,2013).
Thesespecializedbacteriaarebeingstudiedtolearnhowtheybreakdowntheoil
andunderwhatconditionstheythrive.Amyriadoffactorsmustbeconsidered,which
mayaffectthegrowthoftheseoileatingbacteriacoloniessuchastemperature,
nutrients,adhesiontotheoil,carbon/nitrogenlevels,waterandwatercurrents.Certain
oileatingbacteriastrainsmaythrivebestunderveryspecificconditions.Forexample,
oilspillsintheantarcticmayattractdifferentoileatingbacteriastrainsthanoilspillsin
theGulfofMexico.
IthasbeendiscoveredthatbacteriaonthebeachesoftheGulfofMexico
consumedtheoilfromtheDeepWaterHorizonoilleakbyusingthelargeamountsof
nitrogenpresentintheGulfduetofertilizerrunofffromtheMississippiriver.Astatement
wasmadebyaleadresearcherinwhichdifferenteffectivecleanuptechniquesusingthe
oilwereexplored."Becauseoilislowinnutrientssuchasnitrogen,thiscanlimithow
fastthebacteriagrowandhowquicklytheyareabletobreakdowntheoil,ouranalysis
showedthatsomebacteriaareabletosolvethisproblemthemselvesbygettingtheir
ownnitrogenfromtheair(Kostka,2013).Scientiststhenstudiedspecificgenesthat
wereresponsiblefordifferentbehaviors,includingnitrogenfixing,inordertofigureout
howthebacteriawerebreakingdowntheoil."Byunderstandinghowtheoilis
degradedbymicrobes,whichmicrobesdothework,andtheimpactofthesurrounding

Luna

environmentalconditions,wecandevelopwaystointervenetosupportthenatural
cleanupprocess(Kostka,2013)."Forinstance,increasingthelevelsofnitrogenforthe
bacteriatotakeinwillspeedupthebreakdownoftheoilandthismethodofcleansing.
Itisalsoimportanttofindhowtoapplythisnitrogentothesoilbyexperimenting
withnitrogenrichsolutionssuchasUrea.Ureaisveryhighinnitrogenandisalso
inexpensive.Ifitwaspossibletosimplysprinkleureaonoillacedsoilinorderforthe
microbestothriveatanacceleratedrate,ithasthepotentialtobesignificantin
progressingoilspillcleanups,botheconomicallyandenvironmentally.Ureacontains
46.6%nitrogenwhileothersolutionssuchasStrontiumNitrateandAmmonium
Hydroxide,contain8082%.ThismakesUreathecheapestproduct,costingabout257$
perton.StrontiumNitratecostsabout687$pertonandAmmoniumHydroxidecosts
717$perton.Theamountofeachproductwouldbeappliedtothemicrobesaccording
topricecorrelation.
Thesehighnitrogensolutionscouldbeeasierforthemicrobestocollectthe
nitrogentheyutilizefromnutrientssuchasStrontiumNitrateorAmmoniumHydroxide
duetothembeingahigherpercentagethanUrea.Althoughthismaybeavery
beneficialcatalystforspeedinguptheoilremovalprocess,anykindofhuman
interruptioninthenaturalcleanupshouldbehandledwithcaresoastonotcausemore
unintendeddamagetothedelicateecosystem.Regardless,aneffectiveandnatural
cleanupprocessmustbefoundquicklyasmassiveoilspillscontinuetooccurand
furtherdepletetheearth'spreciousenvironment.
Similarly,intermsofenvironmentaldamagecreatedbyoilspills,TheDeepwater

Luna

Horizonoilspillpumpednearly5millionbarrelsofoilinatimeframeofthreemonths.
However,withinseveralweeksoftheoilleakbeingfixed,multipleareasoftheGulf
werefreeofoil.Aleadresearcherstatedavalidexplanationandconclusionfromthese
results:

ThebottomlinefromthisresearchmaybethattheGulfofMexicoismore
resilientandbetterabletorecoverfromoilspillsthananyonethought.Itshows
thatwemaynotneedthekindsofheroicmeasuresproposedafterthe
DeepwaterHorizonspill,likeaddingnutrientstospeedupthegrowthofbacteria
thatbreakdownoil,orusinggeneticallyengineeredbacteria.TheGulfhasa
broadbaseofnaturalbacteria,andtheyrespondtothepresenceofoilby
multiplyingquiterapidly(Hazen,2013)".

ThereasontheGulfissoresilienttooilspillsisbecauseeachyear,asmanyas
1.4millionbarrelsofoilenterthegulffromnaturalseepsintheearth'scrust.Because
ofthis,overtheyearsevolutionhascreatedahugepopulationofoileatingbacteriain
theGulf(Koebler,2013).AlthoughtheGulfmaybebetteroffthaninitiallythought,not
allecosystemsarelikethisandsomestillneedhumaninfluencesforthebacteriato
reallykeepuptospeedagainstthedetrimentaleffectsofoil.However,Insome
locations,ittookonlyonedayforthebacteriatoreduceagallonofoiltoahalfgallon
(Hazen,2013).

Luna

AlthoughTheDeepWaterHorizonoilspillwaseasilyassistedbythehelpof
microbes,manyotherspillslackthisgiftofnaturalcleansingduetotheenvironment.
TheLakeMichiganregionhasexperiencedtroublewithoilspills.BPrecentlymadea
falsestatementabouttheamountofoilspilledinthelake.Inastatement,thecompany
saidamalfunctioninanewdistillationunitforcedupto39barrelsor1,638gallonsofoil
intothelakejustacrosstheIllinoisborder.Adayearlier,thecompanyhadestimated
that18barrelsatmosthadbeenspilled.(Hawthorne,2014).Oilcompaniesoften
intentionallyfibabouttheamountofoilspilledduetothecleanupbeingsocostly.BPis
currentlyusingvacuumtrucksandothermechanicalcleansingdevicesinordertokeep
theoilclearfromthedrinkingwaterofthe7millionpeoplelivinginChicago.Although
thisisagoodtemporarymethodofcleansing,itisnotveryefficientandoftenleaves
residueoilbehind.Evenaftercleanup,oilstillremainsembeddedinshorelines,rocks
andsoil.ResearcherAngieJacksonhasstatedthat,Crewsdeterminedthattrace
amountsofoilremainalongtherockyshorelineofBP'srefineryinWhitingfollowingthe
March24thoilspill.Upto1,638gallonsofoilweredischargedwhenadistillationunit
malfunctioned.Thisthusindicatesthattheoilcleansingmethodsoftodayarenot
efficientlyoreffectivelycleaningspills.Machineryandothermethodsoftentakealong
spanoftimeandarenotthorough.Oilspillsnearlargecitiesneedtobecleansedas
quicklyaspossiblesoastonotaffectthedrinkingwater.
Inanevenmorerecentincident,theOlympicOilspillresultedinthousandsof
gallonsofoillostintoLakeMichiganandthesurroundingarea.Thecontaminationhas
nowmigratedtoaChicagoareacanalandtheEPAhasgotteninvolvedwithacourt

Luna

orderrequiringtheCicerocompanytocleanupthemess.The
EPA
saidTuesdaythat
itsaskedAttorneyGeneralLisaMadigans
office
toobtainanorderrequiringOlympic
OilLtdtostopthespill,determineitssizeandcleanupcontaminatedsoilandwater.
Thefacilityblendsandpackagespetroleumlubricatingoilandantifreezeproducts
(Madigan,2014).SincethegeographyandclimatesurroundingtheLakeMichiganarea
differsgreatlyfromtheGulfofMexicoregionwheretheDeepWaterHorizonspill
occurred,itisintegraltolearnaboutthecharacteristicsoftheoileatingmicrobesnative
tothisspecificenvironment.Sincetheselifeformsareverysensitivetoconditionsin
theirecosystem,propertiesthatwouldspeeduptheefficiencyofoilconsumptionmay
varyduetothedifferenceintheecosystem.Ifafavorablemethodisfoundforthe
environmentofLakeMichigan,spillssuchasthesecanbemoreeasilycleanedina
naturalwaysthatdonotfurthernegativelyimpacttheenvironmentorharmthedrinking
watersupplygoingtotheChicagolandarea.
Someoftheoilspillresearchhasfocusedonfertilizinghydrocarbonoxidising
bacteriasotheycanbetterbreaktheoildownintohydrogenandcarbondioxide.This
strategyiseffectiveinmarshlikewatersthatarefairlystagnantanddonothavemuch
ofacurrent.However,amainlimitationisthecontinuousdilutionofthebacteriainopen
andmovingwaters.Anewmethodwasdevelopedwhichinvolvedaddingthinmineral
film,suchascertaintypesofearthclay,withthebacteriatotheoilspill.Bydoingthis,
thebacteriacanbetteradheretotheoilandnotdriftoffanddisperseinmovingwaters:

10

Luna

TogetherwithadsorbedNandPfertilizers,filmingadditives,andorganoclay,
clayflakescanbeengineeredtofloatonseawater,attachtotheoil,andslowly
releasecontainednutrients.Ourlaboratoryexperimentsofmicrobialactivityon
weatheredsourceoilfromtheDeepwaterHorizonspillintheGulfofMexico
showfertilizedclaytreatmentsignificantlyenhancedbacterialrespirationand
consumptionofalkanescomparedtountreatedoilinwaterconditionsand
reactedfasterthanstraightfertilization(Warr,2013).

Ifthisfertilizerlikesubstanceisaddeddirectlytotheoilspill,thenthiscould
easilyreducethedurationoflargebodiedwateroilspills.However,beforethiscanbe
appliedtoopenwaters,aneffectivemethodmustbefoundforkeepingthesupplements
stucktotheoilforlongdurationsoftimesothebacteriacangrowlocally.
Anotherwaytoincreasetheeffectivenessofoileatingbacteriaisbyboostingthe
exportsoftheenzymethatmultipliesthebacteria."WhatwedoisaddFyreZymeto
existingbacteriaineffect,forcefeedingthemsotheymultiplyandconsumethe
contaminationatamuchmorerapidpace.Asaresult,wecandointhreetoseven
weekswhatwouldtakenatureanywherefrom30to70yearstocomplete(Condy,
2013)."Thiswouldbehelpfulinwaterssuchastheantarcticoceanbecauseduetothe
coldtemperaturesthebacteriamovemuchslowerthantheywouldinawarmplace
suchastheGulfofMexico.TheFyreZymeispumpedintothegroundwithoxygenand
watersothattheFyreZymecanefficientlygettothecontaminatedareaofsoil.Although
thereexistothermethodstodecontaminateoildrenchedsoil,mostworkmuchslower

11

Luna

andhaveaftereffects.Theseincludevaporextraction,whereholesarepunchedinto
theground,airispumpedin,andcontaminantsareextractedthroughacarbonfilter.
Whileeffective,thisprocesscantakefrom7to15years.Anothercommontreatment
widelyutilizedhasbeen
thermaldesorption,
aprocedurewherethesoilisburnedto
eliminatethecontaminants.However,thisprocessalsosterilizesthesoil,renderingit
uselessforsustainingplantlife(Cultice,2013).
Bacteriahasalsoshowntobeabletomovethroughsoilandcoastlines.Howthe
bacteriacandothisandseekouttheoilisstillunknowntothescientificcommunity.
However,becausethisprocessissobeneficial,itisnowakeyareaofresearch.How
theoildispersantsandbacteriamovethroughporousmateriallikesandandmudwould
beahugehelpforcleaninguponshoreoilcontaminations.Oncetheoilreaches
sedimentsandbeachesandbayous,howdoesittransportitselfthroughtheporous
mediumthatthosebeachesandbayousconstitute.Buttheotherveryimportant
questionthatwecananswerthroughthisishowwecaninterveneinorderfortheoilto
beremoved(Papadopoulos,2011).Onlandoilspillshavebeenahotspotforcleanup
methodslikeburningandskimmingthatareharmfultotheenvironment.Byfiguringout
howthebacteriagetaroundandhowhumanscanintervenetoincreasethisfunction,
wouldhelpthecausetremendously.Differentenvironmentscallfordifferentcleanup
methodssoasnottodisturbtheland.Wearelookingatallthesefactorstooptimize
thewindowofreagentsavailablefortakingcareofoilspills.Whywindow?Because
dependingonwherethespillisintheGulf,orinAlaska,thetemperatureisdifferent,
thesalinityisdifferent(Somasundaran,2011).

12

Luna

Testingcontaminatedsoilusingdifferentstrainsofoileatingbacteriawouldallow
ustoseewhatconditionscausethebacteriatothriveandremovetheoilfromthe
environment.Puttingthedifferentstrainsunderavarietyofphysicalconditions,and
testingwithdifferentsoilsamples,willgiveawidevarietyofenvironmentalscenarios
thatwouldyielddataonhowbestthesebacteriascanthriveinourGreatLakes
Midwesternenvironment.Collectingsamplesfromrealcontaminatedoilsites,and
creatingcontaminations,willstretchallpossibleareasofresearch.
Overall,enlistingmicroorganismstoassistwiththecleaningupofoilspillsisthe
mostecofriendlywaytogo.Itisaprocessalreadyoccurringinnatureandispartofthe
earthsnaturalhealingprocess.Howeverfactorssuchastemperature,nutrients,
adhesiontotheoil,carbon/nitrogenlevels,waterandwatermovementplayahugerole
inthisoileatingbacteriacleansingmethod.Thesefactorsmustbetakenintoaccount
andalteringsomeofthesefactorscouldspeeduphowfastthebacteriadoesitsjob.If
humansaretointerferewiththisnaturalcleansing,thenitshouldbehandledwithgreat
cautionsonounintentionalharmiscausedtotheenvironment.
Differentformsofcontaminationfromoilisalsoahugeresearcharea.Soil
decontaminationwouldofcourserequiredifferentmethodsthanwater.Findingouthow
thebacteriaworkincleaningthesoilwouldbeahugehelp.Thiscouldleadtofiguring
outhowhumanscaninterveneandincreasethebacteria'seffectiveness.Byadding
nitrogenoroxygenandincreasingthetemperatureforthebacteria,thiswouldspeedup
theprocessofbreakingdowntheoilandcauselessenvironmentaldamage.Thishas
beenfoundtobethecaseinregionsotherthantheMidwestasdescribedabove.

13

Luna

InconclusionthemicrobeswhichwillbetestedarenativetotheLakeMichigan
region.Therefore,thehypothesisthatifoileatingbacterianativetoLakeMichiganare
exposedtoconditionssuchaswarmtemperature,increasednitrogenandincreased
oxygen,theywillmultiplycausingtheoilinasampletobeingestedatamoreefficient
rateisapredictiononemightnaturallyconcludefromthecurrentresearch.Itisalso
relevant,worthwhileresearchduetoproblemswithoilspillswhichhaveoccurredin
recentyearsinthisregion.TheGreatLakesregionisanareawhichprovidesdrinking
watertoalargepopulationofpeople.Understandingandstudyingtheoileating
bacteriainthesoil(andalsothewater)intheLakeMichiganregioncouldhavegreat
benefitstoaddresscurrentoilspillsinthisregionandoilspillsthatmayoccurinthe
future.Furthermore,discoveringthathighnitrogencontentsisthemostfavorable
sourceofnutrientsforthesemicrobes,introducingdifferentnitrogenrichsolutionstothe
soilsamples.Willhelpfindthemosteffectivesolutionthroughtesting.Thesolutions
includeStrontiumNitrate,UreaandAmmoniumHydroxide.Byvaryinghowtoapply
thesesolutionssuchassprinklingonthetopofthesoilormixingitinwiththesoilwill
alsoplayalargecomponentonconvertingthisexperimenttouseatawiderscalethan
insideofalaboratory.Thesefindingscanhelptheoilcleaningmethodseconomically
andenvironmentally.

14

Luna

Materials
Urea(2gramspersample)
AmmoniumHydroxide(1mlpersample)
StrontiumNitrate(.009gramspersample)
Scoopula
Oileatingmicrobes
Individualcontainersformicrobestobetested
Metricscale
Isolatedandcontainedareaformicrobestoculture
Motoroil
Gravel
Spectrophotometer
Strainer
BoxofCuvettes
Multiplepipettesrangingfrom60microlitersto3millileters

15

Luna

Procedure
1. Create21oillacedsamplesusingthegravelanddesiredamountofoil.Thiswill
beforallthreetrialsoftesting.UseaTablespoonofoilpersample.
2. Createabaselineofoilpercentagessocomparisonscanbemadeonceresults
arerecorded.
3. SettheSpectrophotometerto455nm.Thisworksbestwiththeoilduetoitbeing
inthewavelengthrangeofvioletlight.
4. Startbysettingablankcuvettejustwithdistilledwatertopreparethemachinefor
oilbaseline.
5. Usingapipette,add.06mlofoiltotheblankandrecordresults..06%mlis2%
ofthe3mlcuvetteandtherefore,whateverabsorbancecompiledfromthat
cuvettewillmeanthattheiris2%oilinit.
6. Continuetodothisgoingupby.04mlofoileachtime.Thiswillcontinuetoraise
thepercentageofoilinthecuvetteandcreateabaselinetogooffononce
resultsfromsamplesarecollected.
7. Apply2gramsofoileatingmicrobestoeachsampleandstiraccordingly.
8. Apply2gramsUreato3ofthesamplesbysimplysprinklingonthetopofeach
sample.
9. Apply2gramsofUreatoanother3samplesbymixingintothesamplesusinga
scoopula.
10. Apply1milliliterofAmmoniumHydroxideto3othersamplesbysimplypouring
ontopofeachsample.

16

Luna

11. Apply1milliliterofAmmoniato3othersamplesbymixingintothesamplesusing
scoopula.
12. Apply.0098gramsofStrontiumNitrateto3othersamplesbysimplysprinkling
ontopofeachsample.
13. Apply3gramsofStrontiumNitrateto3othersamplesbymixingintothesamples
usingascoopula.
14. Leave3ofthesamplesalonewithmicrobesaloneandnootherintroduced
variablestosample.Thiswillactasacontrolgroup.
15. Collectdataafter1weekofmicrobesandvariablesbeingapplied.
16. Strainalltrialsamplesusingstrainerand10mlofdistilledwatertoseparate
gravelfromoil.
17. Thenusea15mlpipettetofill3cuvetteswiththeexcesswaterandplaceinto
spectrophotometersetat455nm.Thiswaythespectrophotometercancalculate
theamountofoilinsample.
18. Recorddatacollected.
19. Collectdataagainafter2and3weeks.
20. Recordcollecteddataandcomparewithoilpercentagebaselinetoachieve
generalpercentageofoilineachsample.
21. Observedatathenplaceintographtoseecorrelationsanddifferences.

17

Luna

Variables
Theindependentvariableistheintroductionofdifferentsolutionshighinnitrogentothe
microbes.Thenitrogenrichsolutionswhichwillbeaddedtothemicrobesinclude
StrontiumNitrate,UreaandAmmoniumHydroxide.Thewaythesolutionswillbe
appliedtothemicrobeswillbevariedbyeithersprinklingthesolutiononthesurfaceof
thesoilsampleorbymixingthesolutionintothesoilsample.

Thedependentvariableishowefficientlythemicrobesconsumetheoilinthesamples
oncetheapplicationofnitrogenhasbeenintroduced.Theoilsamplesmayreact
differentlybasedonthetypeofnitrogensolutionthathasbeenappliedtothesample
andthemethoditwasapplied.Aspectrophotometerwillmeasurethequantityofoilin
thesamplebeforeandaftertheintroductionoftheindependentvariable.
The
control
groupisthemicrobesaloneintroducedtoasamplewithnooutside
variablesinvolved.

18

Luna

RawData
Trial1
Weeks:

StrontiumNitrate

.012a

.010a

.005a

Urea

.036a

.036a

.014a

Ammonium
Hydoxide

.009a

.009a

.003a

Ammonium
Hydoxide
Sprinkled

.007a

.007a

.005a

StrontiumNitrate
Sprinkled

.022a

.011a

.006a

UreaSprinkled

.073a

.048a

.038a

Control

.044a

.027a

.027a

19

Luna

Trial2
Weeks:

StrontiumNitrate

.008a

.005a

.005a

Urea

.086a

.040a

.027a

AmmoniumHydroxide

.007a

.003a

.003a

AmmoniumHydroxide
Sprinkled

.016a

.008a

.005a

StrontiumNitrate
Sprinkled

.020a

.006a

.005a

UreaSprinkled

.063a

.046a

.039a

Control

.027a

.024a

.014a

20

Luna

Trial3
Weeks:

StrontiumNitrate

.006a

.005a

.002al

Urea

.040a

.038a

.010a

Ammonium
Hydroxide

.006a

.003a

.003a

Ammonium
Hydroxide
Sprinkled

.007a

.007a

.004a

StrontiumNitrate
Sprinkled

.021a

.005a

.004a

UreaSprinkled

.065a

.045a

.038a

Control

.031a

.029a

.021a

21

Luna

DataAnalysis
Theresultsweresuccessfulinsupportingthehypothesis.AmmoniumHydroxide
wasshowntobethemosteffectivesolutionusingacorrelatedamountbasedonprice
ofproductwiththeothersolutions.AmmoniumHydroxidebyweek3hadanaverage
absorbanceof0.003a.Basedoffoftheoilpercentagebaselinecreatedbeforetest
trialsbegan,thismeansthatsamplewasonlyabout4%oil.Ammoniumhydroxidewhen
appliedonlytothetopofthesamplewasalsothesecondmosteffectivesubstancewith
oilconsumptionforthemicrobeswithanaverageafter3weeksof0.00466.Thismeant
thesamplewasalittlelessthan6%oil.Theoneunusualpieceofdatarecordedwas
thatthecontrolactuallyrecordedtobemoreeffectiveinremovingoilthenthesamples
withUreasprinkledontop.ThiswasmostlikelybasedonanerrorwiththeUrea.
However,withmicrobesalonetheoilcontentscameouttoabout9%.Whilethe
sprinkledUreasamples(US),cameouttoabout9.5%.

22

Luna

StatisticalAnalysis

23

Luna

24

Luna

ExperimentalError
Thelargestpotentialexperimentalerrorwouldhavetodowiththetrials
conductedusingUrea.Ureawhenappliedsimplybysprinklingontopofthesample,
wasshowntobeevenlesseffectivethanthecontrolthatincludedmicrobesalone.This
couldbebecausewhentheUreasampleswerestrainedfortestingoilcontents,the
Ureapelletsdissolvedinthewatermakingtheexcesswaterslightlycloudierthanthe
othersamples.Makingthewatercloudierwouldmakeitmoredifficultforlighttotravel
throughthesampleonceplacedintheSpectrophotometer.Therefore,appearingto
maketheabsorbancelevelslesseffectivethanthecontrol.

25

Luna

Conclusion
Overall,thedatadidsupportthehypothesis.AmmoniumHydroxidewasseenas
themosteffectiveagentforaidingthemicrobesoilconsumption.Thissolutionwasthe
mosteffectivewhenitwasappliedbybeingmixedintothegravelorsimplyplacedon
thetop.TheamountofAmmoniumHydroxideusedwasalsocorrelatedtotheamount
orUreaandStrontiumnitratebypriceondosage.Eachdoseaddedtothesamples
fromeachsolutioncosted.0078cents.ThismeansthatalthoughAmmoniumHydroxide
isthemostexpensiveproductbeingabout717$perton,itisstillthemoreeffective
solutionforthemicrobesefficiencyevenifitisusedinlesseramounts.

26

Luna

TheEffectofNitrogenandOxygenonMicrobeInducedOilDegradationinLake
Michigan:20122014

Purpose:
Thepurposeofthislaboratoryexperimentistolearnabouttheconditionsthatwould
causeoileatingbacterialocaltoLakeMichigantomultiply.Thegreatertheabundance
oftheoileatingbacteriathemoreoilthebacteriawillbeabletoingest.Sincethese
microbesareverysensitivetotheenvironmenttheylivein,testswouldbeusedtoseeif
conditionslikewarmtemperaturesandincreasednitrogencancausetheseoileating
bacteriatomultiply.Testswillbeperformedonoillacedsoilsamplesderivedfromthe
MidwestnearLakeMichigan.
Hypothesis:

IfoileatingbacterianativetoLakeMichiganareexposedtoconditionssuchaswarm
temperature,increasednitrogenandincreasedoxygen,theywillmultiplycausingtheoil
inasampletobeingestedatamoreefficientrate.
Rationale:
Thehypothesisisvalidbecausecurrentresearchindicatesthattheseconditionsare
favorableformultiplyingoileatingbacteriacoloniesthathavebeenstudiedinother
terrainsandgeographicregionsofthecountry.Bycombiningthesefavorableconditions
itwillincreasethethebacteriasabilityandefficiencyatingestingtheoilinoillacedsoil
samplesfromtheLakeMichiganMidwesternregion.

27

Luna

Materials:
3gramsNitrogenfertilizer(Urea)
Oxygenstones(1percontainer)
Oildegradingmicrobes(3grams)
Samplescollectedfromlocalsites(LakeMichiganshore)
Scale
Graduatedcylinder
Scoopula
Isolatedandcontainedareaformicrobestoculture
Incubator
Individualcontainersforsamplesbeingtested.
Procedure:
1. Acquiresamplesofoillacedsoilfromlegitimateoilspills.Thesespillsmustbe
nativetoLakeMichiganbecausethatiswhattheexperimentwillbespecifiedas.
2. Placeeachsampleinsideofgraduatedcylinderandmeasureoutthevolumein
milliliters.
3. Weigheachsamplewithscaletofindmassingrams.
4. Calculateoriginaldensityofcontaminatedsoilsamples.
5. After3gramsofthemicrobeshavebeenintroducedtothesamplesortheyare
alreadypresent,theymustbeplacedincontainedareasotheycancontinueto
cultivateandvariablescannowbeadded.

28

Luna

6. Fortrialone,therewillbefourdifferentvariablesandonecontrolwherejust
microbesarepresentinthesample.
7. Eachvariablewillbeanalyzed2timestoseeprogressinoildegradation.There
willbeasecondtrialandthirdofnewsamplesandtheprocesswillthenwe
repeated.
8. Thefirstsamplewillbeusedasthecontrolwherenovariableswillbeintroduced.
9. Thesecondsamplewillhavethevariablepresenceof3gramsNitrogen
introducedtothemicrobes.
10. ThethirdsamplewillhavethevariablepresenceofincreasedNitrogenand
increasedtemperaturetothemicrobes.
11. ThefourthsamplewillhavethevariablepresenceofincreasedOxygentothe
microbes.
12. ThefifthsamplewillhavethevariablesofincreasedOxygenlevelsand
increasedtemperature.
13. IncreasednitrogencanbeaddedwithUrea(3grams)
14. Increasedoxygencanbeaddedbyintroducingwatertoanoxygenstone
15. Temperaturemustbeincreasedinanincubator(32.6Celsius).
16. Oncemicrobesandvariableshavebeenapplied,allowmicrobestobeginoil
degradation.
17. Thedensityofeachsamplewillthenbecalculatedtwicebydividingthemassto
thevolume.
18. Calculatedensityofeachsample3daysaftermicrobeshavebeenapplied.

29

Luna

19. Calculatedensityofeachsample7daysaftermicrobeshavebeenapplied.
20. Thesestepswillthenberepeatedfortrials2and3.
21. Alldatashouldbedocumentedwithdetailforeachtrial.
22. Chartalldataandcomparenewdensitytooriginalthatwascollectedbeforethe
microbesandvariableswereapplied.
Variables:
The
independentvariable
isthephysicalfactorsthatwillbeintroducedtothe
oileatingbacteriasuchashigherlevelsofNitrogen,Oxygenandwarmertemperatures.
The
dependentvariable
ishowefficientthebacteriacleansedthecontaminatedsoil
duetotheintroducedphysicalfactors.Thiswillbemeasuredbycalculatingthenew
densityandcomparingittotheoriginalofthesamplesoncethetestshavebeen
conducted.
The
control
groupisthemicrobesaloneintroducedtoasamplewithnooutside
variablesinvolved.

30

Luna

Trial1:

Days:

60

Nitrogen
increase

1.677g/ml

1.648g/ml

1.339g/ml

1.259
g/ml

Nitrogenand
tempincrease

1.477g/ml

1.405g/ml

1.122g/ml

.9943
g/ml

Oxygenincrease

1.447g/ml

1.438g/ml

1.217g/ml

1.127
g/ml

Oxygenand
tempincrease

1.9765g/ml

1.893g/ml

1.663g/ml

1.589
g/ml

Control

1.682g/ml

1.672g/ml

1.574g/ml

1.537
g/ml

31

Luna

Trial2:

Days:

60

Nitrogen
increase

1.8085g/ml

1.745g/ml

1.611g/ml

1.39g/ml

Nitrogenand
tempincrease

1.643g/ml

1.5225g/ml

1.1684g/ml

1.085g/ml

Oxygenincrease

1.5425g/ml

1.4475g/ml

1.3725g/ml

1.294g/ml

Oxygenand
tempincrease

1.9625g/ml

1.8485g/ml

1.3455g/ml

1.2415g/ml

Control

1.546g/ml

1.537g/ml

1.4297g/ml

1.358g/ml

32

Luna

Trial3:

Days:

60

Nitrogen
increase

2.2205g/ml

2.138g/ml

1.6945g/ml

1.475g/ml

Nitrogenand
tempincrease

1.5775g/ml

1.537g/ml

1.4297g/ml

1.135g/ml

Oxygenincrease

2.086g/ml

2.008g/ml

1.7265g/ml

1.6445
g/ml

Oxygenand
tempincrease

2.241g/ml

2.112g/ml

1.4715g/ml

1.379g/ml

Control

2.2835g/ml

2.2445g/ml

2.0885g/ml

2.0115

33

Luna

AverageDensityGraph:

34

Luna

DataAnalysis:
Aweekwasgiveninordertofindsignificantresultsinthedecreaseofdensity
peroillacedsoilsample.Thiswasshowntobesuccessfulbecausebythe7thday
somesampleswereabletodecreaseinsuchasubstantialamountthatitwasvisible
whenactuallyviewingthesamplethatsomeoftheoilhadmostdefinitelybeen
consumedbythemicrobes.Althoughallsamplesshowedadecreaseindensity,the
controlwithmicrobesalonehadamuchlowerdecreaseinthecourseofthe7days.
Thisfurtherbacksupthefactthatoildegradingmicrobescanbemanipulatedby
outsidevariables.
Thedataprovedtosupportthehypothesisandgainsuccessfulresultsbecause
themicrobeswereabletoincreasetheirrateofoilconsumptionbybeingintroducedto
numerousdifferentvariablessuchasincreaseinOxygen,Nitrogenandtemperature.
Thisresultedinthemicrobesdramaticallyaffectingthedensityofthesoilinaslittleas7
days.Basedonthedatacollected,theaveragedensityof7daysfortheNitrogenand
temperatureincreasesampleswas1.18975g/ml.Sincethiswasthelowestdensity
averagecomparedtothecontrolaverageof1.6974g/ml,itisportrayedasthemost
efficientvariablegroupforoilconsumption.Thecontrolactuallyhadthelowestdensity
decreasecomparedtotheother4variables.After60daysIreviewedtheresultsand
trialsagaintoseeiftheywereconsistentwiththepriordata.Unfortunatelyalthough
therewasadecreaseindensityduringthe60daytrialitwassubstantiallylowerthan
theprior07dayspan.

35

Luna

Conclusion:
Overall,thedatasupportedthehypothesis.Anidealvariableofincreased
Nitrogenandtemperaturewerefoundtobethemostefficientforspeedingupthe
processofoildegradation.Thisvariablewasproventobethedominantamongthe
othersamplesbecauseinall3trialsthedensitywasreducedatafasterrateinthe7
dayspanthananyothervariable.Thus,provingthatthemicrobeswereableto
consumeandbreakdownoilfasterbecauseofthesespecificconditions.Inlocations
suchastheGulfofMexico,themicrobesfavorthewarmtemperatureandworkbetter
withincreasedNitrogen.InanexperimentwithsoilandmicrobesnativetoLake
Michiganwheretheclimateisverydifferentandcanvarydramaticallyasfaras
temperature,themicrobesshowadesireforincreasednitrogenwithwarmer
temperaturesaswell.However,Oildegradingmicrobescanvaryinhowtheyreactto
certainvariablesbasedontheclimatetheysurvivein.
Thistopicandprojectcanhelpimprovethecleanupofmanmadeoilspillsina
natural,nonharmfulway.Bylearningmoreaboutthesespecialmicrobes,societycan
findtheidealvariablesbasedontheclimateforthebacteriatoworkatfullpotential.An
oilspillwhichwasonceaverydifficult,longanddamagingprocesstocleanup,cannow
beanissueofthepast.Manipulatingthisnaturalformofcleanupcanhelphumansfix
messyoilmistakeswithoutruiningtheearthintheprocess.

36

Luna

ExperimentalError:
Thenumberoneexperimentalerrorintheprojectwouldmostlikelybethelossof
soilwhencalculatingthedensity.Thesoilmustbeweighedandplacedinaseparate
containerforvolume.Thesoilisveryadhesiveandnoteverysinglegrainofsoilcanbe
putbackintotheoriginalsamplecontainer.Thiscanslightlyaffecttheoverallresultsbut
notonalargeenoughscalefortheexperimenttobeinaccurate.Anothererrornoticed
inthetestingprocesswastheoxygenstonesandthefactthattheyneededwaterin
ordertogiveofoxygen.Oncethewaterwasappliedtothestonesatthebottomofthe
samples,thedensitywasmostlikelyslightlyaffectedsincethewaterwasabsorbedby
thesoileventually.Anothererrorwouldbeaddingincorrectmeasurementsofthe
variablesaddedsothattheywerenotkeptconstantinalltrialsandsamples.Thiscould
havehappenedwhenweighingthenitrogenfertilizeronthescaleortheamountof
wateraddedtoeachoxygenstone.

37

Luna

ReferenceList
BiotechnologyResearchInternational.(2013,May27).BioremediatingOilSpillsinNutrient
PoorOceanWatersUsingFertilizedClayMineralFlakes:SomeExperimentalConstraints.
RetrievedDecember13,2013,from
http://www.hindawi.com/journals/btri/2013/704806/

Cultice,C.K.(n.d.).FyreZyme.In
Let'sdolunchCommerceDepartmentgranthelpsfight
pollutionbyboostingexportsofenzymethatmultiplesoileatingbacteria.
. RetrievedDecember
14,2013,fromEBSCOHOST(510390439).
Faeber,F.(2011,April5).OilEatingBacteriaEngineering.In
NationalGeographic
.Retrieved
December14,2013,from
http://news.nationalgeographic.com/news/2011/04/110405nsfoilsomasundaranvideo/

Gruver,M.(2014,September19).WyomingOilSpillsTotal220,000GallonsIn2014,Already
DoubleAmountSpilledIn2013.In
HUFFPOSTGREEN
.RetrievedFebruary28,2016,from
http://www.huffingtonpost.com/2014/09/19/wyomingoilspills_n_5850060.html
Hawthorne,Michael."BPraisesestimateofLakeMichiganoilspill."
ChicagoTribune
.N.p.,28
Mar.2014.Web.1Nov.2014.
<
http://articles.chicagotribune.com/20140328/news/chibpoilspilllakemichigan20140327_1
_londonbasedoilcompanyoilspillwhiting
Joyce,S.(2015,October1).TheRisingCostOfCleaningUpAfterOilAndGas.In
Inside
Energy
.RetrievedFebruary28,2016,from

38

Luna

http://insideenergy.org/2015/10/01/therisingcostofcleaningupafteroilandgas/
Jackson,Angie."CrewsfinishcleanupafterBPoilspillonLakeMichigan,traceamountsofoil
remain."
MichiganLive
.N.p.,4Apr.2014.Web.8Nov.2014.
<http://www.mlive.com/news/grandrapids/index.ssf/2014/04/crews_finish_cleanup_of_bp_oil.h
tml>.
Kemsley,T.(2013,August30).BacteriaNaturally'EatUp'OilSpillContaminationby
SupplementingDietwithNitrogen.In
NatureWorldNews
.RetrievedDecember13,2013,from
http://www.natureworldnews.com/articles/3757/20130830/bacterianaturallyeatupoilspillcon
taminationsupplementingdietnitrogen.htm
KubeM,ChernikovaTN,AlRamahiY,BeloquiA,LopezCortezN,GuazzaroniME,Heipieper
HJ,KlagesS,KotsyurbenkoOR,LangerI,NechitayloTY,LnsdorfH,FernndezM,JurezS,
CiordiaS,SingerA,KaganO,EgorovaO,PetitPA,StogiosP,KimY,TchigvintsevA,FlickR,
DenaroR,GenoveseM,AlbarJP,RevaON,MartnezGomarizM,TranH,FerrerM,
SavchenkoA,YakuninAF,YakimovMM,GolyshinaOV,ReinhardtR,&GolyshinPN(2013).
GenomesequenceandfunctionalgenomicanalysisoftheoildegradingbacteriumOleispira
antarctica.
Naturecommunications,4
PMID:
23877221
Microbes&OilSpillsFAQ(2011).In
FisheriesandOceansCanada
.Retrievedfrom
http://www.dfompo.gc.ca/science/publications/microbes/indexeng.html
NaetherDJ,SlawtschewS,StasikS,EngelM,OlzogM,WickLY,TimmisKN,&HeipieperHJ
(2013).AdaptationofthehydrocarbonoclasticbacteriumAlcanivoraxborkumensisSK2to
alkanesandtoxicorganiccompounds:aphysiologicalandtranscriptomicapproach.
Appliedand
environmentalmicrobiology,79
(14),428293PMID:
23645199
OilTankerSpillStatistics2014(2015,January).In
ITOPF
.RetrievedFebruary28,2016,from
http://www.itopf.com/fileadmin/data/Documents/Company_Lit/Oil_Spill_Stats_2014FINALlow

39

Luna

res.pdf
OileatingBacteria.(n.d.).In
BiotechnologyOnline
.RetrievedDecember13,2013,from
http://www.biotechnologyonline.gov.au/pdf/enviro/oileating_bacteria.pdf
Schwartz,A.(2013,April8).OilEatingBacteriaFixedTheDeepwaterHorizonDisaster,But
TheyMayNotHelpNextTime.In
Co.Exist
.RetrievedDecember13,2013,from
http://www.fastcoexist.com/1681764/oileatingbacteriafixedthedeepwaterhorizondisasterbu
ttheymaynothelpnexttime
Shelton,LeeAnn."Cicerocompanyorderedtocleanupoilspillsnearcanal."
Newsfeed
.N.p.,10
Oct.2014.Web.6Nov.2014.
<
http://chicagosuntimes.com/news/cicerocompanyorderedtocleanupoilspillsnearcanal/
>.

40