The Journal of Biological Sciences

R u t g e r s C a m d e n - J o u r n a l o f B i o l o g i c a l S c i e n c e s | V O L 5 | M a y 2 0 1 9 | 1

Burned and unburned oil-contaminated media impede lateral growth of Neurospora crassa

regardless of hydrophobin expression Julia A. DeFeo and William J. Myers

Department of Biology, Rutgers University, Camden, N.J. 08102

Abstract Bioremediation, or the use of organisms to degradeenvironmental pollutants, is an increasingly prevalentconcern. Due to surging global oil production and thecorrelating increase of improper burned and unburned oildisposal, more information about organisms capable ofbioremediation is needed. Fungi are commonly consideredfor bioremediation due to their production of variousexoenzymes anddocumented capacity for degrading crudeoils.Hydrophobins,anamphipathicproteinutilizedbysomefilamentous fungi to decrease the surface tension ofsurrounding liquid media, may be an importantconsideration in the growth of fungi in the context ofbioremediation.ThisstudyaimstocharacterizethegrowthofNeuropsoracrassainburnedandunburnedoil-contaminatedmediawithandwithouttheproductionofhydrophobins inorder to determine ifN. crassa can tolerate an oil-pollutedenvironment.Hereweshowthatbothburnedandunburnedoil-contaminatedmedia lead to a significant impairment oflateral growth. There was no significant impact on lateralgrowth due to the presence of hydrophobins. ThisexperimentthereforedemonstratesaninihibtedcapacityforN.crassatogrowinoil-contaminatedenvironments.Futureresearch should aim to further define specific growthcharacteristicsofN.crassainoil-contaminatedenvironmentssuch as effects on reproductive structures and hyphalbranching.

Introduction Oil is a potent pollutant, and the threats of oil spills andimproper oil disposal have increased alongside oilproduction. Crude oil spills can contaminate and causemassiveharmtolargeecosystems.Burnedmotoroil,whichcontainsheavymetalsandaromatichydrocarbonsfromtheburning process, is often not disposed of in the properfacilities,andthuscanbetoxicfororganismsthatareexposedtotheoil(Akintundeetal.,2012).Bettertechnologiesneedtobedeveloped for oil spill prevention, containment, and theremediationofoil-contaminatedenvironments.

Bioremediation efforts, or theuseof organisms todegradepollutants,encounterarangeofissuesbasedonthedegreeof

pollution, which microorganisms are used, and whatbyproductsareproduced(Ekundayo,F.O.etal.,2012).Theamount of pollutants present can lead to adverse growtheffects even on organisms that can degrade the pollutants.Determiningifthechosenorganismcantoleratetheseverityof the given pollutant is an important consideration in itscapacity for bioremediation. More information on thepollution tolerance of bioremediative microorganisms isthereforeneeded.

Fungiarecommonlyconsideredforbioremediationbecausetheypossess awide range of enzymes that canmetabolizevarioushighlyrecalcitrantsubstrates.Fungiarealsoknowntobeverytolerantofgrowthconditionsthatareadversetoothereukaryotes,buttheexacttoxicityofthecontaminantsinburnedoilonfungiisnotwell-documented.

However,manyfungalspecieshavebeenreportedtodegradecrudeoil (Ekundayo,F.O.etal.,2012).Fungiare thereforedesirable candidates for the bioremediation of burned oilspills,duetotheirtolerancefortoxicconditionsandtheiroil-degrading capabilities. Further experimentation willdetermine the viability of specific fungal species for oilremediation.

Neurospora crassa is a filamentous fungi that has beenisolated from oil-contaminated environments and has adocumentedabilitytodegradecrudeoil,aswellasphenoliccompounds commonly found in oils, but its efficiencywithregard to degrading crudeoil is generally less than that ofothersimilarlycapablefungalspecies(Ekundayo,F.O.etal.,2012; Luke et al., 2001). However,N. crassa does possesscertainadvantagesthatcouldmakeitadesirableselectionforbioremediation. Not only is N. crassa relativelynonpathogenic,butitisalsoeasilyavailableanditsgenomeisentirelymappedduetoitsstapleuseingeneticresearch.Itis therefore feasible to genetically engineer strains of N.crassa so that they are adapted to best perform in specificecosystemsforparticularbioremediationneeds.Thisgeneticconsideration in particular makes N. crassa an excellentmodel organism for studying the possibility of genetically

The Journal of Biological Sciences

R u t g e r s C a m d e n - J o u r n a l o f B i o l o g i c a l S c i e n c e s | V O L 5 | M a y 2 0 1 9 | 2

adapting microorganisms for specific bioremediationcontexts.

Onegeneticconsiderationistheproductionofamphipathicproteinsknownas“hydrophobins”(Bayryetal.,2012).Beingamphipathicproteins,theyareknowntoself-assembleintosheetswithahydrophilicfaceandahydrophobicface.Thisallowsthemtoadheretosurfaces,changingtheiraffinityforwater. In fungi, hydrophobins coat spores to make themdifficultto“wet,”orbecomesaturatedwithwater.N.crassaproduces the hydrophobin EAS, which is a Type Ihydrophobin(Bayryetal.,2012).HydrophobinssuchasEASarealsoknowntoreducethesurfacetensionofliquid(Bayryetal.,2012)andformassembliesattheinterfacesofoilandwater (Linder et al., 2005). Hydrophobins thereby affectfungal growth by reducing the surface tension of thesurrounding liquid medium so that the fungi can push itssporangia andhyphae through the fluid.This study is thusconcernedwiththeimpactthepresenceofhydrophobinswillhaveonhyphalgrowthinanoil-contaminatedmedium.

Thescientificrecordstillneedstoaddresstheeffectofanoil-contaminated environment on the growth of filamentousfungi.N.crassa’stoleranceforburnedandunburnedoil,andthenatureofitsgrowththroughthesecontaminants,needsto be characterized. The production of hydrophobins is animportant factor for fungal growth through any liquidmedium,buttheimpactoftheirpresenceonfungalgrowthinburnedandunburnedoil-contaminatedmedia isunknown.ForthepurposeofinformingthebioremediativecapabilitiesofN.crassa,thisstudyseekstodemonstratethecapabilityofN. crassa to grow in an oil-contaminated environment. ByexposingN. crassa to an oil-contaminatedmedia, itwill bedeterminedifthereisincreasedgrowththroughthemediumwhen the hydrophobin gene is expressed. This is expectedbecause oil has a lower surface tension than water andhydrophobinswillacttofurtherlowerthisminimalsurfacetension. It is also predicted that there will be decreasedgrowthinburnedoiltreatments,possiblyduetothepresenceoftoxicheavymetalsandaromatichydrocarbons.Therefore,the hypothesis of this study is that N. crassa producinghydrophobins in unburned oil-contaminated media willexhibit the most growth, while N. crassa not producinghydrophobinsinburnedoil-contaminatedmediawillexhibittheleastamountofgrowth.

Materials & Methods Thisstudyused36replicatesevenlydistributedacross thefollowingtreatments:WildType–DistilledWater;WildType–BurnedOil;WildType–UnburnedOil;Mutant–DistilledWater; Mutant – Burned Oil; Mutant – Unburned Oil. (6replicatespertreatment)

StrainsandSuspensions

Twostrainsareusedinthisstudy:FGSC2489(wild-type)andFGSC13319 (mutant lackinghydrophobinproduction; alsocalledeasily-wettable(eas)orclock-controlled-gene-2(ccg-2).ThesestrainswereprovidedbyDr.KwangwonLee.

A conidial suspension was made for eachN. crassa strainusing48-houroldcultures.Sterilizedwaterwaspipettedintothe culture tubes, and the tubes were then vortexed. Theresulting solution was poured through autoclavedcheesecloth, isolating the conidia, into autoclavedmicrocentrifugetubes.UsingaBioRadTC20™AutomatedCellCounter,theconcentrationofconidiaineachsuspensionwasdetermined.Thesuspensionswerethendilutedto2.50x106conidia/mL.

Media3LofVogelMediumNAgarwasmadeusing60mLof50XVogelSalts,2.94Lofdeionizedwater,45gofsucrose,and45gofagar,mixedandheatedto90ºCandstirredat350rpmfor20minutes.Theagarmixturewasthenautoclaved.Afterautoclaving,150mLoftheVogelAgarwaspouredintoeachof18,12”x3”containers.Agarwaslefttosettleandcoolfor30minutes.

Figure 1. Experimental Design. Each large rectangle with thickoutline represents a container. Within each container are twomediumrectanglesrepresentingtreatedfilterpaper(lightbluefordistilledwater,peachforunburnedoil,brownforburnedoil)thatmeetandslightlyoverlapinthemiddleofthecontainer.Ateitherend of each container are small rectangles representing theuncoveredVogelAgarthatservesastheinoculationsite. Thetoprow of containers was inoculated with wild-type FGSC 2489 N.crassaandthebottomrowwasinoculatedwithmutantFGSC13319N.crassa.

ContainersContainers(largerectanglesdepictedinFig.1)wereassignedtheir treatments.Halfof the18containerswere inoculatedwithwildtypeN.crassaFGSC2489,andtheotherhalfwereinoculatedwithFGSC13319.Withineachofthesehalves,3containers were assigned to a distilled water treatment, 3were assigned to an unburned oil treatment, and 3 wereassignedtoaburnedoil treatment,whichwasamixtureofunburned and burned oil in a 50:50 by volume ratio (the

The Journal of Biological Sciences

R u t g e r s C a m d e n - J o u r n a l o f B i o l o g i c a l S c i e n c e s | V O L 5 | M a y 2 0 1 9 | 3

burnedoilwasdilutedduetoalackofresultsinburnedoilreplicates in unreported pilot studies). There were tworeplicateswithineachindividualcontainer,foratotalof36replicates,6withineachtreatment.

FilterPaperTreatmentFor each container, two 7.62 cm x 12.65 cm filter papers(mediumrectanglesdepictedinFig.1)weresoakedwith1.5mL of the assigned treatment. Amicropipettewas used topipet100µLina5x3gridpattern.Each100µLincrementwasseparatedby2.54cm(1.27cmfromedgeoffilterpaper),withthreeincrementsacrossthewidth(7.62cm)ofthefilterpaperandfivedownitslength(12.65cm).Twofilterpaperswereplacedineachcontainersothatthefilterpapersonlyslightlyoverlapinthemiddle.Eachedgeofthecontaineristhenleftwith7.62cmx2.54cmuncoveredVogelAgar(smallrectanglesdepictedinFig.1),whichservedastheinoculationsites.Thisgriddingpatternensuresthatthefilterpapersaresaturated and not oversaturated and that the amount oftreatmentisnormalized.

UnburnedoilwasPennzoil550035091MotorOilLubricant.Burnedoilwastakenfromacarafteranoilchange.

InoculationToinoculate,100μLofsuspension(2.50x105conidia)waspipetteddirectlyontothemiddleoftheedgewherethe7.62cmx2.54cmuncoveredVogelAgarandsideofthecontainermeet.Suspensionswerevortexedbetweeneachinoculation.

ConditionsandMeasurementsThe containers were stored in incubators set to 25 ºC,covered with parafilm. Parafilm was sterilized with 91%isopropyl alcohol. Growth was recorded and documentedevery 24 hours by measuring, with a ruler, the lateralappearanceofmacroscopicmycelialgrowth(incm),relativeto the edge of the filter paper nearest the inoculation site.Pictureswerealsotakenofeachindividualreplicate.

ResultsThe lateral growthwasmeasuredat24hour intervals,butonly the data from the 72 hour interval was analyzed.Pictureswerealsotakenevery24hours.

72hoursafterinoculation,therewasnostatisticaldifferencebetween lateral growth across both strains of N. crassa(mutant (without hydrophobins) and wild type (withhydrophobins)) (two-way ANOVA, p > 0.05) (Fig. 2).Additionally,thereisadifferencebetweenlateralappearanceof mycelial growth across oil treatments(burned/unburned/control) (two-way ANOVA, p < 0.05)(Fig.2).Therewasnointeractionbetweenoiltreatmentandfungalstrain(two-wayANOVA,p>0.05)(Fig.2).

Figure 2. Lateral growth ofN. crassa 72 hours after inoculation.Significantdifferencebetweendistilledwater (control)andeitheroil-basedtreatment.n=6foreachtreatment.Wild-typeFGSC2489representedbyblackbarsandmutantFGSC13319representedbywhitebars.

There is also no significant difference between MutantBurnedgrowthandWildTypeBurnedgrowth(TukeyHSDp>0.05),andMutantUnburnedgrowthwasnotsignificantlydifferentfromWildTypeUnburnedgrowth(TukeyHSDp>0.05)(Fig.2).

Average Mutant Unburned was not significantly differentfromMutantBurned growth (TukeyHSDp>0.05) (Fig. 2).Average Wild Type Unburned growth was also notsignificantly different from Wild Type Burned growth(TukeyHSDp>0.05)(Fig2.).

Compared to the control replicates, which were grown indistilledwater, bothMutantBurned andMutantUnburnedhad significantly less growth (TukeyHSDp<0.05) (Fig.2).The same is true for Wild Type Burned and Wild TypeUnburned compared to distilled water, each havingsignificantlylessgrowth(TukeyHSDp<0.05)(Fig.2).Therewas no significant difference betweenMutant Control andWildTypeControl(p>0.05)(Fig.2).

Inunburnedtreatmentsacrossbothstrains,theunburnedoilspilled from the filter papers into the inoculation site. Inburned oil treatments, there appeared to be localizedconcentrationsofburnedoilatthemarginofhyphalgrowth(bestseeninFigure3,middleimage).Burnedoiltreatmentsappeared to present with more fuzzy white macroscopicgrowththantheunburnedoiltreatments(Fig.3).

02468

101214

Distilled Water Unburned Burned

Late

ral G

row

th (c

m)

Treatment

A A

B B B B

The Journal of Biological Sciences

R u t g e r s C a m d e n - J o u r n a l o f B i o l o g i c a l S c i e n c e s | V O L 5 | M a y 2 0 1 9 | 4

Figure 3. Growth phenotypes. Images of wild-type FGSC 2489 indistilledwater(A),burnedoil(B),andunburnedoil(C),aswellasimagesofhydrophobinmutantFGSC13319indistilledwater(D),burnedoil(E),andunburnedoil(F).

Discussion Basedon the results of this trial, burnedandunburnedoilsignificantly decreased the lateral growth of themyceliumandhydrophobinpresencedidnothaveaneffect inanyoiltreatment. There was no significant difference in lateralgrowthbetweenburnedandunburnedoiltreatments.

Mutant growth was greater than wild type growth inunburned oil, but not significantly so. However, this isunlikely to be due to an actual effect of the lack ofhydrophobins, as this difference mostly appears to be theresultofasinglehigh-growthoutlierinthemutantunburnedtreatment.Thisoutlier resulted in the relativelyhigherrorbarsforthemutantunburnedtreatment.Morereplicationisnecessary to determine the validity of this outlier. Highermutant growth in any oil treatment relative to wild typegrowthwouldalsonotbeexpectedbecausethepresenceofhydrophobinsservestodecreasesurfacetensionatoil-waterinterfaces(Linderetal.,2005).Soincreasedlateralgrowthintheabsenceofhydrophobinswouldcontradictthecurrentunderstandingofhydrophobins.

Theinteractionbetweenthehydrophobicunburnedoilandthewater-basedagaralsoledtoa“flooding”effectthatcouldhaveconfoundedtheresultsintheunburnedoiltreatments.Thiscouldhaveaffectedtheresultsastheinoculationswouldhaveimmediatelyencounteredtheoiltreatment,whichhasbeenshowninthisstudytoimpairgrowth.Theburnedoiltreatments,whichdidnotexperiencethis“flooding,”didnotencountertheirtreatmentuntiltheyreachedthefilterpaper.

The lack of a significant effect of hydrophobins on lateralgrowthwassurprising,andsothestrainswerere-testedfor“wettability”aftertheconclusionoftheexperimenttoensurethattherehadbeennocross-contamination.Thereisnoclearexplanationforthislackofeffect,andfuturestudiesshouldattempt to further characterize the role and impact ofhydrophobinsathyphaltipsexposedtooil.

Althoughthereisnosignificantdifferencebetweenunburnedandburnedoil, it isdifficult todrawdefinitive conclusionsabout the effect of toxic compounds in the burned oil andtheireffectonthelateralgrowth.Firstly,theburnedoilwasdiluted with unburned oil. The “flooding” effect in theunburnedoiltreatmentsmustalsobeconsidered.Qualitativeobservations support this explanation, as the inoculationsites at 24 hours appeared to be covered with a film ofunburned oil, but this “flooding” was not observed in theburnedoiltreatments.Therealsoappearedtobea“pooling”of the dark pigment characteristic of the burned oil at themarginofhyphalgrowthintheburnedoilreplicates.Itmaybepossible that thehyphaearesomehowtranslocatingthetoxins concentrated in the burned oil that they are notcapableofimmediatelydegrading.

The significantly impaired lateral growth in the oiltreatments, compared to the distilled water controltreatments,couldalsobeduetonutrientrichnessratherthanonly environmental impairment. The presence of massivehydrocarbons in these treatments could impede lateralgrowth as the fungimight prefer to grow amore heavily-branched mycelium when it encounters these largehydrocarbonsinadditiontotheVogelAgar.Thiscouldbeafeasible explanation due to N. crassa having a relativelylimitedcapacityfordegradingcrudeoil(Ekundayo,F.O.etal.,2012).AlthoughN. crassa candegrade crudeoil, itmaybethat it cannot degrade crude oil to a degree thatwould beoverallbeneficialforitsgrowth.

There is also an obvious effect on the composition of themycelium due to burned oil exposure as observed by thedominance of the fuzzy white mycelial structures in thesetreatments. This change in appearance could possibly berelated to the reproductive structures produced along themycelium, and consequently this difference could have aneffectonmycelialgrowth.

A B C

D E F

The Journal of Biological Sciences

R u t g e r s C a m d e n - J o u r n a l o f B i o l o g i c a l S c i e n c e s | V O L 5 | M a y 2 0 1 9 | 5

Future studies should attempt to observe andquantify thebranchingpatternswhenN.crassaisexposedtoburnedandunburned oil. Quantifying the relative amounts of thedifferentreproductivestructuresofN.crassawhenexposedtooiltreatmentscouldalsobeofinterest.Higherreplicationandbettercontrolofthelocalizationoftheoiltreatmentsinfuture trialswould lead tomoredefinitive results. Enzymeassays could be used to characterize the biochemicalmechanismbywhichN.crassabreaksdownthecompoundsintheoiltreatments.Afuturestudycouldthenalsoattempttoincreasetheexpressionofrelevantenzymesusinggeneticmodification.

Anyreplicatedtrialsofthisexperimentaldesignshouldalsoseektouseamoreconcentratedburnedoilsolution.Heretheburned oil was diluted to a 50:50 by volume ratio withunburnedoil.Thiswasdonebecausepilotstudieshadfoundthat the concentrated burned oil solution led to almost nogrowthofN.crassa.Futuretrialsshouldattemptan80:20or70:30byvolumeratioofburnedoiltounburnedoil.

FutureresearchshouldalsoaimtofurtherinformtheroleN.crassa couldplay inbioremediation.Theremustbe a clearunderstanding of what byproducts are formed whenintroducingabioremediativemicroorganismintoapollutedenvironment,andsoidentifyingthecompoundsproducedasthe result of N. crassa degradation of oil is important.Consideration must also be given to the other effects,independent of pollution, that a specific microorganismwould have on the other populations and the abioticenvironment composing the ecosystem into which it isintroduced. Experiments growing N. crassa in an oil-contaminated environment that is also inhabited by otherfungal species would be an example of a useful study forinformingthisconcern.

Acknowledgements WethankDr.KwangwonLeeforgivingustheopportunitytocarryoutthisprojectthroughhisPrinciplesandPracticesofBiologicalResearch (PPBR) course, aswell ashis generous

giftsofVogelSalts,N.crassastrains,culturetubes,andcell-countingcassettes.

WethankDr.NathanFriedforhisroleinteachingthePPBRcourse,aswellashisassistance ingenerating ideasforourproject, analyzing data, and providing feedback for ourwritingprocess.

Weespecially thankMs.SarahJohnsonforansweringallofour technical inquiries and for providing us with thematerialsnecessarytomakethisprojecthappen.

WethankDr.KatieMalcolmandDr.JenniferOberlefortheirhelpfulinputbasedontheirexpertisewithfungi.

References Akintunde, W.O., Olugbenga, O.A., and Olufemi, O.O. (2015). Some Adverse Effects of Used Engine Oil (Common Waste Pollutant) On Reproduction of Male Sprague Dawley Rats. Open Access Maced J MedSci3,46–51.

Bayry, J., Aimanianda, V., Guijarro, J.I., Sunde, M., and Latgé, J.-P. (2012). Hydrophobins—Unique FungalProteins.PLOSPathogens8,e1002700.

Ekundayo, F. O., Olukunle, O. F., and Ekundayo, E. A. (2012). Biodegradation of Bonnylight crude oil by locally isolated fungi from oil contaminated soils in Akure, Ondo state. Malaysian Journal of Microbiology.

Linder, M.B., Szilvay, G.R., Nakari-Setälä, T., and Penttilä, M.E. (2005). Hydrophobins: the protein amphiphiles of filamentous fungi. FEMS Microbiol Rev29,877–896.

Luke, A.K., andBurton, S.G. (2001). A novel application for Neurospora crassa: Progress from batch culture to a membrane bioreactor for the bioremediation of phenols. Enzyme and Microbial Technology 29, 348–356.