Characterization of Salt Pond, Falmouth, MA: Its residents and their habitat

Grace Chong

University of Southern California

Final Report Microbial Diversity 2018

21 August 2018 1. ABSTRACTThegoalofthisprojectwastocharacterizetheseasonallystratifiedcoastalSaltPondinFalmouth,MA.Environmentalwatersampleswereprobedwithionchromatographyandvariouschemicalanalysestodeterminewhatnonlivingthingswerepresentintheenvironment,andtechniquesfromlightmicroscopy,16Sribosomalsequencing,andspectroscopywereusedtoprobethevastarrayof lifesupportedby thephysicalandchemicalgradientspresent in thisenvironment.Adramaticshiftinthephototrophiccommunitywasobservedevenintheshort2-weektimespanofthisproject,andsulfatereducingbacteriaweresuccessfullyenrichedandisolatedfromthisenvironment. Overall, the information revealed by this mini-project will aid the direction offuture studies that wish to “dig deeper” into the inhabitants of Salt Pond and similarenvironments.2. INTRODUCTIONSalt Pond is a seasonally stratified coastal environment in Falmouth, near Cape Cod,Massachusetts (Figure 1). Its water column contains strong gradients of dissolved oxygen,salinity,density,andchemicalcomposition,asseeninFigure2,asnapshotofenvironmentaldatafromearlyAugust2018,providedbyresearchers fromWoodsHoleOceanographic Institution(WHOI)intheirrecenteffortstotrackchangesincertainenvironmentalparametersasthepondbecomesmore or less stratified at different times of the year. These physical and chemicalgradientsmakeSaltPondan interestingenvironmenttostudy,perhapsasananalogofotherstratifiedmarineenvironments,suchastheBlackSea.However,this localenvironment isnotvery well characterized, beyond a couple specific topics such as dimethylsulfide (sulfurcompound)cycling[1–3]andmagnetotacticmicrobes[4–7].

Figure1.ImagesfromGoogleMapsforthesamplinglocation:SaltPond,Falmouth,(CapeCod),MA. Approximate location of sampling site is indicated by the red pin, with coordinates:41°32'37.8"N70°37'38.1"W.

Figure2. SnapshotofYSIprobedataobtainedatvariousdepthsofSaltPond,Falmouth,MA,courtesy of Taylor Sehein and Emily Maness from the Edgecomb Lab at WHOI. This graph,estimated for 8/3/18, is the average of their data from timepoints 7/30/18 and 8/6/18.ByusingenvironmentalwatersamplestakenfromvariousdepthsofSaltPondfromAugust3rdto August 15th, 2018, and using a variety of techniques from lightmicroscopy, 16S ampliconsequencing, bioinformatics, absorption spectroscopy, ion chromatography, colorimetricchemicalassays,mediadesign,andattemptedenrichmentofphototrophic,magnetotactic,andsulfatereducingbacteria,wewilladdressfourresearchquestionsthatcharacterizeSaltPondanditsinhabitants:1)Whoisthere?2)Whatistheirenvironmentlike?3)Howdoesthephototrophiccommunitychangeovertime?Lastly,4)Canweenrich/isolatebacteriafromthisenvironment?

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3. MATERIALSANDMETHODS3A.SamplingSamplingoccurredatSaltPondinFalmouth,MA.Arowboat(fromtheEdgecomblab,WHOI)wasused to obtain samples approximately from the middle of the pond near the followingcoordinates:41°32'37.8"N70°37'38.1"W(Figure1).There,theboatwasanchored.Aropewasusedtoestimatethedepthofthepond(i.e.,depthofthesediment-water interface)foreachsamplingday.Aperistalticpumpandrubbertubing(fixedtoaropewithcementweight)wereusedtocollectbulkwatersamples.SeeFigure3forasummaryofsamplingdepthsanddates.Allsamplingtripsoccurredinthemorningaround9aminordertoavoidhittingpeakwindtimes,whichmakesrowingtheboatinthedesireddirectionchallenging.

Figure3.DiagramillustratingsamplesobtainedfromSaltPondacrossdifferentsamplingdates,type of sample, and water depths. A) Water samples collected on 8/3/2018. B) Plexiglassmembranestickusedtocollectmembrane-permeablewatersamplesafterequilibratingfor11days.C)Watersamplescollectedon8/15/2018.OnAugust3,2018,>1Lofwatersampleswerecollectedateachofsixdepths(Figure3A).Fromtoptobottom:30cm,3m,3.75m,4m,4.25m,4.5m(i.e.4.2m,1.5m,0.75m,0.5m,0.25m,and0mfrombottom).Thepondwas4.5mdeepasofthissamplingdate.AppropriatefilteredorunfilteredsampleswerefilteredimmediatelyforlaterDNAextractionandchemicalassays,tobestoredat-20°C(DNA)or4°C(chemicals)untilfurtherprocessing.

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FromJuly26toAugust6,2018,a~1mlongcustommadeplexiglass“membranestick”apparatus(seeFigure3BandFigure4)wasembeddedintothesedimentatthebottomofthepond,withthehelpofdiversfromWHOI.3columnsofcylindricalholes(~27holespercolumn)havebeendrilledintothisapparatus.Each“hole”holdsabout1.5mLeachandarespaced3cmcenter-to-centerfromotherholesaboveorbelow.Theholes were filled with water, and both sides of this apparatus were covered withdialysis tubing (Fisher brand, regenerated cellulose membrane, nominal molecularweightexclusionis3500daltons)sothatonlythingssmallerthan3500daltons(~35aminoacids),suchasionsorotherchemicalsfromtheenvironment,couldpermeatethemembrane.About25cmofthismembranestickwasabovesediment,and50cmbelow.After~11days,thissamplewasretrieved,themembranewasremoved,andwatersampleswereextractedforchemicalanalyses(storedat4°C).

Figure4.Photoofplexiglass“membranestick”describedinFigure3B.OnAugust15,2018,athirdsamplingtripwasperformed(SeeFigure3C).SimilartothesamplingtriponAugust3,>1Lofwatersampleswereobtainedfromvariousdepths(approx.3.5,3.75,and4mfromsurface,or0.5,0.25,0mfrombottomasofthissamplingdate).Theywerestoredat4°Cuntilspectralanalysis.3B.LightMicroscopyLightmicroscopywasperformedonwetmountsofeachsampleobtainedon8/3/18(Figure3A)using a Zeiss Axio Imager A2microscopewith the 100x objective using the transmitted light(brightfield)method.3C.16SsmallsubunitribosomalDNAampliconsequencingSamplepreservation:WhileatthesamplingsiteonAugust3rd,asmuchaspossiblewassyringepumpedthroughSterivex0.22µmfilterstobepreservedfor16Sampliconsequencing.Atotalof360mL,240mL,150mL,90mL,90mL,and120mLwerefilteredintoindividualfiltersforeachof sixdepths (4.25m,1.5m,0.75m,0.5m,0.25m, and0m frombottom - SeeFigure3A)respectively.Filterswereplacedimmediately insterile50-mLfalcontubesfortransport.Thentheywerestoredat-20°Cuntilprocessingfor16SrRNAampliconsequencing.Oncebackatthelab,thefiltersfromeachofthese6watersampleswereextractedbycuttingopentheSterivexfilter’splasticcasingusingarazorbladeheatedinthehottestpartofaBunsenburnerflame(approx.3inchesabovetheburner).EachfilterwascutinhalfforDNAextraction.DNAExtraction&Quantification:DNAforeachofthese6samples(6samplesx2halvesofeachfilter=12DNAextractions)wasextractedusingtheQIAampPowerFecalDNAKitfollowingtheinstructionmanualfromAugust2016(exceptthatinstep6weused13200xgcentrifugation,andinStep19weused50uLelutionbufferonly).TheextractedDNAfromeachofhalfofeachfilterwascombinedwithitsotherhalf(12halffiltersextracted=>6finalDNAsamples).ThisDNAwas

quantified usingQuantusDNAquantificationmachinewith theQuantiFLUORONE dsDNA kit(bothfromPromega).TheextractedDNAwasstoredin-20°Cduringtheremaining~2weeksofthecourse,tobeusedasPCRtemplateforthenextstep.AnyDNAleftoverattheendofthecoursewasplacedinthe-80°Cfreezer.PCR Amplification, DNA Gel Verification, & Troubleshooting: Then, PCR amplification wasperformed. Aliquots of the 6 DNA extraction samples were diluted so that the amount oftemplateineachPCRrunwasaboutthesame.SeveralrunsofPCRwereperformed,followedbyrespectiveDNAgelelectrophoresis(1%agarosegels in1XTAEbuffer,with1uLofPromega’sDiamondnucleicaciddyeper100mLofgel,runat~100Vfor~1houreach).SeeAppendixIforPCRreactionmixesandthermocyclerprotocolsforeachrun.PCRamplificationRound1protocolswerefollowedusingspecificprimersforbacteriaorarchaea,foratotalof12PCRreactions(6bacterialruns,6archaealruns).ForeachsubsequentstepofPCR,we triedsomethingdifferent to troubleshootanyof thePCRamplifications thatdidnotwork.Forexample,inPCRRound2,weadjustedtheconcentrationofanythingthatdidnotworkinRound1to½,1x,or2xthetemplateconcentrationthatwehadpreviouslyused.ForPCRRound3,wetried increasingthenumberofcycles,andalsousing2uLoftheoriginalDNAtemplate(undiluted).Lastly,forPCRRound4,wetriedaconcentrationgradient(10,25,or50ng/uLoftemplateperPCRreaction),andtemperaturegradientspreadacross4tubesfrom45to65°C.Finally, all PCRproducts thatmadea clearly visiblebandona gelwere storedat -20°CuntilprocessedbycourseTA&Facultytoaddbarcodes,makelibraries,andsendoutforsequencingviatheHiSeqmethod.Data analysis: Sequence data were processed using Qiime 2 using the DADA2 pipeline andcomparingsequencestothe13_8releaseoftheGreengenesreferenceOTUs.Attheend,taxabarplotsweregenerated.3D.ChemicalAnalysesImmediatepreservation:Atthesamplingsiteon8/3/18(Figure3A),50mLofeachsamplewasimmediately filtered througha0.22µMfilter, tobesaved for ionchromatographyandotherchemical assays. For 1.2mLof each filtered samplewas added immediately toHPLC vials (2replicatesper sample) containing0.3mLof91mMzincacetate, topreserve sulfide for lateranalysis.Similarly,4.5mLofeachunfilteredsamplewasaddedto0.5mLof1MHCltofixtheiron (prevent oxidation) and save it for later determination of total Fe(II) and Fe(III)concentration.Allmaterials(exceptfiltratesforDNAprocessing)werestoredat4°Cuntillateranalysis.Similarly, for the samples in the “membrane stick” retrieved on 8/6/18 (Figure 3B), themembranewas removedand samples fromeachdepth (every3 cm)werealiquoted for zinc

acetate(1.2mLofsamplein0.3mMzincacetateinHPLCvials),ironassay(0.8mLsamplein0.1mL 1MHCl), and phosphate/ammonium assays (remaining ~1.5mL). A few samples for zincacetatewerelostduetomembranebreakage(causedduringtransportormembraneremoval,mostlywherethemembranewasstapledtotheplexiglass).Refractometermeasurements:Approx.1mLofeachdepthsamplecollectedon8/3/18(Figure3A)wasusedtomeasuredensity(g/mL)andsalinity(ppt)usingarefractometer.IonChromatography:Filteredsamplesfrom8/3/18and8/6/18(Figures3A-B)werediluted1:100indistilledwaterfor ionchromatography inaDionex ICS-2000machinewithan IonPacAS-22anion exchange column and IonPac AG-22 cation exchange column. This was to determineconcentrationsof any lithium, sodium,ammonium,potassium,magnesium, calcium, fluoride,chloride,nitrite,nitrate,sulfate,bromide,phosphate,andacetateionsthatwerewithinrangeofdetection.Ammonium assay: Filtered samples from 8/3/18 and 8/6/18 (Figures 3A-B) were tested forammoniumusingacolorimetricphenol-lessassayadaptedbyT.JewellfromKathleenTreseder’s“MicroplateNutrientAnalysis”,originallyfrom[8].Standards0,0.05,0.10,0.20,0.50,and1ppmammonium sulfate solution were prepared. A sodium salicylate solution (Solution 1) wasprepared by adding 3.4 g of sodium salicylate, 2.5 g of sodium citrate, and 2.5 g of sodiumtartrate,and0.0125gofsodiumnitroprussidedissolvedinatotalof50mLofwater.Asolutionof1.5Msodiumhydroxidein2%bleachwasalsoprepared(Solution2).40µLofeachstandardorsamplewasaddedtoawellina96wellplate(x3wellstotalfor3technialreplicates).Then80µLofSolution1and80µLofSolution2wereaddedtoeachwell.Thesolutionswereincubatedforabout60minutesatroomtemperature,thenreadat650nmonaSpectraMaxM2(MolecularDevices)platereader.200µLofwater(x3wellsperplate)wereusedasaplateblank.Ironassay:TheFerrozineassay [9]wasused toevaluateunfilteredsamples from8/3/18andmembrane-filtered (exclusion size 3500 Daltons) samples from 8/6/18 (Figures 3A-B). TodeterminetotalFe(II)andFe(III),0.1mLofeachpreservedsample(initiallystoredina9:1ratioofsampleto1MHCl)wasaddedto0.9mLof1MHCl,mixed,andallowedtoincubateat4°Cforabout20hours.Standardsof0,50mM,100mM,150mM,and200mMferroussulfatein1MHClwerealsoprepared.Then,10µLofeachsampleorstandardwasaddedtoeachof3wells(technicalreplicates)ina96wellplate.90µLof10%(w/v)hydroxylaminehydrochloridesolution(in 1 M HCl) and 90 uL of 0.1% (w/v) Ferrozine in 50% (w/v) ammonium acetate solution(Ferrozinesolutionstoredindarkat4C)wereaddedtoeachofthewellsandmixed.Thesolutionswereincubatedforabout10minutesatroomtemperature,thenreadat562nmonaSpectraMaxM2(MolecularDevices)platereader.200µLofwater(x3wellsperplate)wereusedasaplateblank.Phosphate assay: Filtered samples from 8/3/18 and 8/6/18 (Figures 3A-B) were tested forphosphate concentrations using themicroplate phosphate assay describedby [10], followingtheir protocol exactly as written, except that standards with higher range of phosphate

concentrations(0,0.1,1,10,100µMpotassiumphosphate)wereused.Thesolutionswerereadat620nmonaSpectraMaxM2(MolecularDevices)platereader.200µLofwater(x3wellsperplate)wereusedasaplateblank.Sulfideassay:Filteredsamplesfrom8/3/18and8/6/18(Figures3A-B)weretestedforsulfideconcentrationsusingtheClineassay[11].HPLCvialscontained0.3mLof91mMzincacetateand1.2mLofsamplesthathadbeenfilteredon-site.Standardsolutionscontaining0,10,or100µMofsodiumsulfidewereprepared.Whenreadytoassay,asolutionof0.2%DPPDS(N,N-dimethyl-p-phenylenediaminesulfate)wasadded1:1toasolutionof1%Ferricammoniumsulfate,and0.5mLofthismixturewasaddedtoallofthesamplevialsandstandards.Theywereincubatedin the dark for 30minutes. Then 200uL of each vialwas transferred to a 96well plate (x 3replicateseach).Thesolutionswerereadat665nmonaSpectraMaxM2(MolecularDevices)platereader.200µLofwater(x3wellsperplate)wereusedasaplateblank.Thenextday,allsamplesandstandardswerediluted1:4inwaterandre-readonnew96wellplates,sincesomeweretoodarkblue(toohighOD)forthesensitivityoftheinstrument(?)–however,immediatedilutionandplatemeasurementispreferred.3E.AbsorbancespectroscopyAbsorbancespectroscopywasperformedonwatersamplesfrom8/3/18and8/15/18(Figure3A,3C) using a custom built optical setup and a Spectral Evolution PSR-1100f instrument (Serialnumber1794478)withDarwinSPsoftware.Aftertryingseveralothermethods,e.g.absorbanceoflighttransmittedthrough50-mLPfennigbottles(~3cmpathlength)or~10cmpathlengththroughlarge(200-mL)rectangulartissueculturebottles(toextendpathlengthforbettersignalofsampleswithlowpigmentconcentrations),thefinalmethodusedwasabsorbanceofalightsourceshinedontoa25mmfilter(0.22µMporesizeMetricelmembranefilterfromGelmanSciences, Inc). Samples were poured into a 10-mL glass column atop a vacuum filtrationapparatusallowedforefficientcollectionofcellsonfilters.Filterswereplacedontopofablackagarpetridish(slightlywet, flat,darkenvironment–thewetnesshelpsthefilterstostayflatinsteadofdryingupandcurling),alightsourcewasshinedonit,andafiberopticcablegoingtothelightdetectorwaspointedatit(SeeFigure5)andtheabsorbancewasmeasured.Anemptymembranefilterwasusedasablank.

Figure5.Photoofabsorbancespectroscopysetuptomeasureabsorbanceoflightbypigmented

cellscollectedonafilter.

Forsamplesfrom8/3/18(Figure3A),intheorderoftoptobottomdepths,70,110,35,35,25,and23mLoforiginalwatersampleswerefiltered.Allsubsequentsamplesfrom8/15/18(Figure2C)anditsenrichmentswerecollectedwith5mLfiltrateonly.Forcomparisons,allspectrawerenormalizedtothesamevolumeoffilteredsample(i.e.5mL).3F.Phototrophicenrichments50-mLPfennigbottleswerefilledwithfreshSaltpondwatercollectedfrom3differentdepthson8/15/18(Figure3C)andincubatedatvariouswavelengthsoflight(750,850,and960nm)inordertoenrichforlowerlightphototrophs.After4days,5mLofeachsamplewasvacuumfilteredontoa0.22µMmembranefilter,inordertomeasureitsabsorbancespectrumasdescribedaboveinSection3E.3G. Enrichment and isolation of sulfate-reducing bacteria (attempted enrichments ofmagnetotacticbacteria)MagneticEnrichmentPart1:On8/3/18,water from6depthsofSaltPondwere immediatelyplacedinto125-mLglassbottleswithamagnettapedperpendicularlytothesideofthebottle,behindtherectangularwhitelabel,inordertoenrichformagnetotacticbacteriafromtheSaltPondenvironment.Themagnetwasplacedwithitssouthpolefacingthebottle,sincethesouthpoleofcommercialmagnetscorrespondstotheearth’sNorthpole,andwillattractNorth-seekingmagnetotacticmicrobes in theNorthern hemisphere. These bottleswere incubated at roomtemperaturecoveredinfoiluntilablackpelletofcellsaccumulatedinsidethebottleatthespotclosesttothemagnet.Enrichment Media:Media to enrich for magnetotactic bacteria (sulfate-reducing or oxygen-reducing) was prepared with 1X Salt Water Base (342.2 mM sodium chloride, 14.8 mMmagnesiumchloridehexahydrate,15,1mMcalciumchloridedehydrate,and6.71mMpotassiumchloride),20mMMOPSbuffer,10mMammoniumchloride(Nsource),5.3mMsodiumsulfate(S source and electron donor for anaerobic cultures), and 1.4 mM potassium phosphate (Psource),0.0001%resazurin(oxygenindicator)and0.5g/Lofdithiothreithol(DTT,areductant),brought to pH 7, and autoclaved. After autoclaving, the still-hot medium was immediatelytransportedtoahotstirplateanddegassedwithnitrogengasuntiltheresazurinturnedclear(i.e.,themediumisanoxic).Undernitrogengas,theremainingfiltersterilizedingredientswereaddedtotheautoclavedmedium:10mMsodiumsuccinate(carbonsourceandelectrondonor),0.035 mM of iron(ii) chloride tetrahydrate (to facilitate magnetosome formation bymagnetotacticbacteria),1Xtraceelementssolution,and1Xvitaminssolution.SeeAppendixIIforthefinalconcentrationsofthevarioustraceelementsandvitaminsthatwereadded.MagneticEnrichmentPart2–Agarshaketubes:Themagneticpelletfrommagneticenrichmentbottles3,4,and5(correspondingtodepths0.25,0.5,0.75mabovesediment-waterinterface,respectively)wereharvestedafter1weekbycarefullypouringoutmostoftheliquidinthebottleandresuspendingthevisiblemagneticpelletintheremaining<1mLofliquidinthebottle.Thiswasbroughttoatotalof1mLbyaddingappropriateamountsoffilteredwaterfromthesame

depth.Eachresuspensionwasusedtostartanoxicandoxygen-gradientagarshaketubes(SeeFigure6).Thesetubeswerepreparedinanoxicconditions(undernitrogengas)byadding4.5mLofanoxicmediawasplacedintoeachof24sterile10-mLtesttubes(3inoculax2conditionsx4dilution tubes = 24 tubes). 0.5mL of inoculumwas added to the first tube. Then, 1mLwaspipettedfromthistubetothe2ndtube,andsoontothe4thtube.0.5mLfromthe4thtubewasaddedtothe1sttubetomakethetotalvolumeineach5mL.Then1.5mLof3%agar(sterile)wasaddedtomakethefinalagarconcentration~0.75%,andtubeswereimmediatelystopperedandinvertedtomix.Aftertheagarhadcooled,about1mLofmeltedcandlewaxwasdrippedontothetopsof3ofthedilutionseries(correspondingtothe3inocula)tosealthetubesfromoxygen.Theother3dilutionserieswereun-stoppered,inordertoallowanO2gradienttoform.(Note:Thisisnotsterile,sonothingwasdonewiththeO2tubesbesidesfurtherobservation.Infuture,leavestoppersonorsomethingequivalenttopreservesterility.Thestoppersdonotstopoxygenfromentering.)Oxygengradientscouldbeobservedthroughapinkgradientformingatthetopsoftheoxygentubes.Tubeswereincubatedatroomtemperature,coveredinfoil,andobserveddailyforgrowth.

Figure6.Diagramofagarshaketubes–anoxicorO2-gradientdilutionseries

Extractionofanaerobicsinglecoloniesandfreezerstockpreservation:Anaerobicglycerolstockswerepreparedinananaerobicchamberbyresuspendingeachsinglecolonyin5mLoffilteredsaltpondwaterfromtheappropriatedepth.Thiswascombined1:1with20%glycerol(finalconc10%),andaliquotedinto5-mLglassflat-bottomedbalchtubesandsealedwithrubberstopperandaluminumcap.Thesewerefrozenat-80.Atotalof5coloniesweresavedinfrozenstocks.

4. RESULTSANDDISCUSSION4A.Whoisthere?Transmitted lightmicroscopyrevealedthevastand fascinatingmorphologicaldiversity inSaltPond,someofthoseobservedon8/3/18arerepresentedbelowinFigure7(largeobjectsnottoscale).Perhapsmostobviouswerethelargepigmentedalgae,aswellastheprevalentsmallrodsthatweremostlikelycyanobacterialikeSynechococcus–andoddly,eventhelargerpigmentedorganismslikealgaewerefoundevennearthebottomofSaltPond,depthswheresuitablelightqualitymightnotbeexpectedtopenetrate.Anothernotablefeaturewasthepresenceofbrightobjectswithincells,perhapsendosporesorsulfurglobules.Fascinatingwasthelargeflagella-likefilament,about100micronsinlength,thatwasseenonsomeofthecellssurroundingalargeparticleofwhatispotentiallyorganicmatter.Perhapsmostmysteriouswerethelongandangularrod-shapedmicrobeswithbrightspotsthatmadethemappearlikemagicalswords,foundonlyatthebottomofthepond(thoughtheymayalsoexistwheretheywerenotseen).

Figure7.SnapshotofmorphologicaldiversityinSaltPond.Next,16Sampliconsequencingandbioinformaticsfromsamplescollectedon8/3/18revealsthetop~10orsotaxonomicgroups(andmuchmoredetail,ifdesired)(Figure8andAppendixIII).Themostcommongroupoforganismswere thecyanobacteriaorderSynechococcales,whichincludessomeofthemostabundantprimaryproducersontheplanet;unsurprisingly,theywerefoundespeciallyclosertothesurfaceofthepond.AlsonotablewasagroupofarchaeafromtheParvarchaealorderWCHD3-30,whichwasseenin2017inanoxygenminimumzonenearCosta

Rica [12],and is seennow in theoxygenminimumzoneofSaltPond.OthercommongroupspresentincludeBacteriodetes(variousheterotrophs),Flavobacteriales(somearefishpathogens,longskinnyrodslikelyalsorepresentedinmicroscopyimages),Rhodobacterales(includespurplenon-sulfurbacteria),Desulfobacterales(includessulfurreducingbacteria,foundespeciallyinthelower depths), Chlorobi & Chloroflexi (includes green sulfur and non-sulfur bacteria), andActinobacteria(includessporeformers).Manyothergroupswereunknown(perhapspreviouslyuncharacterized),orcomposedsmallerpercentagesofthepopulation.

Figure8.Snapshotof16Staxonomicdata,brokendownintokingdom,phylum,class,andorder-leveltaxonomy.These4setsofgraphseachcontain6columnswhich,fromlefttoright,indicatethedepthsofSaltPondsampledfromtoptobottom.Majorgroupsineachgrapharelabeled.Meanwhile,absorbancespectroscopyonwatersamplesobtainedon8/3/18reflectthepresenceand dominance of certain groups of phototrophs (Figure 9). From this we can see peakscharacteristicforchlorophyllafromcyanobacteriaandeukaryoticalgae(680nm),andpeaksforbacteriochlorophylls foundmost strongly in green bacteria such as the green sulfur bacteriaChlorobi and the green nonsulfur bacteria Chloroflexi (720, 780 nm), and peaks forbacteriochlorophyll a, which is the dominant peak for purple bacteria such as the purplenonsulfurbacteriaRhodobacter,allofwhichwerecommontaxonomicgroupsalsoseenin16Sdata.Thesedataindicatethatasyougodownindepthsofthepond,groupsthatharvestlightatshorterwavelengths(e.g.680nm)becomelessdominant,whiledeeperlevelshavemoregreenandpurplebacteriathatthriveonlongerwavelengthsoflightcapableofreachingdeeperwaters.

Figure9.(Left)SpectraoffilteredwaterfromvariousdepthsofSaltPondon8/3/18,normalizedtothevolumeofsamplefiltered.Acomparisonofthedifferentdepthsindicatesthatgroupsofpigmentedmicrobeswhocapturelightatlowerwavelengthsdecreaseinrelativeabundanceasdepthincreases.(Right)Photoofthefilters.4B.Whatistheirenvironmentlike?Refractometermeasurementsofsamplesfrom8/3/18confirmourexpectationsthatdensityandsalinityincreaseacrossdepths(Figure10).

Figure10.Graphsofdensityandsalinitymeasurementsfrom8/3/18indicatethattheyincreasecorrespondingtoincreasingdepth.

Meanwhile,ionchromatography(Figure11)indicatesthatthereareverylowlevelsoflithiumandfluoridedetected,lowtomoderatelevelsofnitrate,bromide,calcium,potassium,sulfate,andmagnesium,andveryhighconcentrationsofsodiumandchloride. Itwas interestingthatmany of these ion concentrations had a peak increase or decrease close to the oxic-anoxicinterfacewhichisperhapsreflectiveofthemicrobeslivingthere.Forexample,thedropinsulfateconcentrationaroundthe4meterdepthmaycorrespondtopeak insulfatereducingactivity,whichprovidestheverynoticeablesmellofsulfideinthedeeperlevelsofSaltPond.

Figure11.Graphsofionchromatographdatafrom8/3/18samples,ionconcentrationvs.waterdepth. Data has been organized into three graphs depending on the scale of concentration(<1mM,1-60mM,and200-800mM).Theammoniumassaydidnotwork,possiblyduetohumanerror(standardcurvealsodidnotwork). However, it was also likely that we would not have detected the ammoniumconcentrations, as preliminary data from WHOI (Taylor Sehein, unpublished) has foundammonium concentrations 10-100x smaller than the smallest nonzero concentration in ourstandardcurve.Theironassaystandardsworked,buttheassaydidnotdetectanynonzeroconcentrationsofironinoursamples.ThisisperhapsbecauseironintheenvironmentwouldprecipitateoutduetothehighconcentrationsofsulfideinSaltPond.Thephosphateandsulfideassaysworked,indicatingthatinthewatercolumn(8/3/18)therearelow levels until the very bottom, where phosphate and sulfide concentrations dramaticallyincrease(Figure12).

Figure12.Graphsofphosphateandsulfideconcentrationsvs.depth.Ionchromatographyandchemicalassayswerealsoperformedonwatersamplescollectedbythe“membranestick”(Figure3B)whichallowedusto“zoomin”ontheareajustaboveandbelowthesediment-water interface.Here,wealsoreport its ionchromatographyresults(Figure13)andphosphateandsulfideresults(Figure14),whichgavedetectablemeasurements.

Figure13.Graphsofionchromatographdatafrom8/6/18samples,ionconcentrationvs.depthabove or below the sediment-water interface. Data has been organized into two graphsdependingonthescaleofconcentration.

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Figure14.Graphsofphosphateandsulfideconcentrationsfromthe“membranestick”vs.depthaboveorbelowthesediment-waterinterface.4C.Howdoesthephototrophiccommunitychangeovertime?By comparing absorbance spectra (Figure 15) from filters of water samples obtained at thebottom0m,0.25m,and0.5mdepthson8/15/18withthosetakenfrom8/3/18, itbecomesclearthatthephototrophiccommunityhaschangedovertimeofthis2-weekperiod.Previously,thedominantphototrophicpigmentbelongedtochlaofcyanobacteria&algae(680nm),butthispeakisalmostdisappearedaftertwoweeksandinsteadthemajorpeakforgreenbacteria(720nm)becomesdominantinstead.Otherpeaks,includingtheothergreenbacterialfeatureat780nm,andthepurplebacteriaat~880nm,alsobecomemorepronounced.A largedrop incarotenoids(400-550nm)isalsoobserved.Overall,thephototrophiccommunitybecamered-shiftedduringthistwo-weekperiod.Enrichmentoflower-lightphototrophs(peaks>750nm)wasattemptedbyincubating8/15/18samplesat750,850,and960nm.Spectroscopyresults(Figure16)suggestthattheenrichmentperiod (3-4days)was tooshort toseenoticeablechanges inpigmentsdetected. Instead, thedominantphototrophevenaftertheseenrichmentswasthepeakat720nm(greenbacteriaex.Chlorobi),whichwasslightlyunexpectedduetothenon-optimallightquality.Perhapsmoretimewouldhaveenrichedforothertypesofphototrophs.

Figure15.(Left)Photoofthefiltersfrom8/3/18and8/15/18usedforthiscomparison.(Right)Spectraofthosefilters.Arrowsindicatehowmajorpigmentschangedoverthistwo-weekperiod.

Figure16.(Left)Photoofthefiltersfrom8/15/18,andafterenrichmentsfor3-4daysatvariouslightqualities,filteredon8/19/18.(Right)Absorbancespectraofthosefilters.

4D.Canweenrich/isolatesomebacteriafromhere?Sulfatereductionproducessulfideandreactswithironinthemedium,producingavisibleblackironsulfideprecipitate.After5days,ablackbandofthisprecipitatewasobservedinanoxicagartube1(mostconcentrated),whichhadbeeninoculatedwithmagneticenrichmentfromdepth3(0.75mabovesedimentasof8/3/18).AftertransferringtheagarcontainingthisbandtoaBalchtube filledwithanoxicmediaand incubatingat roomtemperature for3days, the insideandedgesoftheagarchunksbecamecoatedwithathinlayerofblackprecipitate, indicatingthatmore sulfate reduction had occurred. Thus, successful enrichment of a sulfide reducingcommunityhadbeensuccessful(Figure17).

Figure 17.Photos documenting the enrichment of sulfate reducers, as seenby the black ironsulfideprecipitatethatoccursasaresultofsulfidereactingwithironinthemedium.Asimilarbandofblackprecipitatewasalsoobservedintheoxygen-gradienttubecorrespondingto thesamesampledescribedabove.Since itappeared justbelowthebottomof theoxygengradient, where the resazurin becomes clear, the enrichment was possibly microaerophilic.However,thiswasnotobserveduntilitwastoolatetostartanewenrichment.Also,thoughthemediumwasdesignedinhopesofcapturingmagnetotacticbacteriausingmagneticenrichmentsasinocula,duetotimelimitations,wedonotknowifthesearemagnetotacticornot.After~8daysofagartube incubations,7singlecolonieswereobserved inanoxicagartube1(mostconcentrated)ofthedilutionseriesinoculatedbythemagneticenrichmentfromdepth5(0.25mabovesediment).Anaerobicglycerolstockswerepreparedafterextractingthebottom5coloniesinanoxicconditions(theother2coloniesgotlostwhentryingtoremovethewaxplug).Insummary,microbesfromSaltPondweresuccessfullyenrichedandisolated(?),thoughfurtherwork needs to bedone to verify their complete isolation and their sequence identity beforesubsequentcharacterizationcancontinue.

5. CONCLUSIONSFromthismini-project,wewereexposedtothevastmorphologicaldiversityinSaltPond,andsaw interesting correlations inhowcertain specieswere reflected inmicroscopy,prokaryoticcommunitycomposition,andspectroscopy.Usinganassortmentofchemicaltechniques,wesawwherecertainenvironmentalparameters changedramatically.Wesaw that thephototrophiccommunity shifted within the two-week timescale of this project, and we were capable ofenrichingandperhapsisolatingsomeofthesulfate-reducingbacteriafromthisenvironment.Overall,thechemical,physical,andbiologicalgradientsfoundinSaltPondmakeitanexcitingenvironment for discovery. Hopefully, information learned from thismini-projectwill inspireothersandhelptoinformfuturestudieson“wheretolook”ifinterestedinspecificprocessesororganisms.6. AcknowledgementsSpecial thanks toKurtHanselmann,whohasgiven themosthelpandadvice throughout thiswholeproject.Also,thankstoeveryoneelsewhocontributedtosamplingtrips,includingKurt,GabrielaKovacikova,AndrianGajigan,DeeSanders,JamieHall,SarahGuest,andJaredLivingston.Meanwhile,SarahG.,ScottDawson,andRachelWhitakerhelpedtomakeallDNAprocessingandanalysispossible.GeorgeO’TooleandMadelineLópezMuñozhelpedmethroughthechallengesof trying to capture and grow (possibly) strict anaerobes. Dee Sanders and RebeccaWipflerhelpedwiththechemicalassaysandionchromatography.EliseCowleyandJosedelaTorrealsohelped from afar with advice on various chemical assays. David Hershey and Yang Hai gavepointersandsharedenthusiasminthedevelopmentandtroubleshootingofexperimentswithmagnetos.PatrickDegnan,WhitneyEnglandandJoseVargas-Muñizhelpedwithbioinformaticsstuff.TaylorSehein,EmilyManess,andGinnyEdgecombfromWHOIsharedtheirresearchaboutotherthingsgoingoninSaltPond,andalsolentustheirboatforsamplingtrips.Thanksagaintothecourseorganizers,faculty,TAs,CAs,andotherstudentswhomadethiscourseamemorableexperience. Lastly, thanks to the various funding sources who support this course and thestudentswhoattendit.

7. References1. WakehamSG,HowesBL,DaceyWH:Dimethylsulfide inastratifiedcoastalsaltpond.

Nature1984,310:770–772.2. Wakeham SG, Howes BL, Dacey JWH, Schwarzenbach RP, Zeyer J:Biogeochemistry of

dimethylsulfide in a seasonally stratified coastal salt pond.GeochimCosmochimActa1987,51:1675–1684.

3. Zemmelink HJ, Houghton L, Frew NM, Dacey JWH: Dimethylsulfide and major sulfurcompoundsinastratifiedcoastalsaltpond.LimnolOceanogr2006,51:271–279.

4. Bazylinski DA, Schlezinger DR, Howes BH, Frankel RB, Epstein SS: Occurrence anddistributionofdiversepopulationsofmagneticprotistsinachemicallystratifiedcoastalsaltpond.ChemGeol2000,169:319–328.

5. Simmons SL, Sievert SM, Frankel RB, Bazylinski DA, Edwards KJ: SpatiotemporalDistribution ofMarineMagnetotactic Bacteria in a Seasonally Stratified Coastal SaltPond.ApplEnvironMicrobiol2004,70:6230–6239.

6. Simmons SL, Bazylinski DA, Edwards KJ: South-SeekingMagnetotactic Bacteria in theNorthernHemisphere.Science(80-)2006,311:371–375.

7. Moskowitz BM, Bazylinski DA, Egli R, Frankel RB, Edwards KJ:Magnetic properties ofmarinemagnetotacticbacteria inaseasonallystratifiedcoastalpond(SaltPond,MA,USA).GeophysJInt2008,174:75–92.

8. WeatherburnMW:Phenol-hypochlorite reaction for determinationof ammonia.AnalChem1967,39:971–974.

9. Stookey LL: Ferrozine---a new spectrophotometric reagent for iron.Anal Chem 1970,42:779–781.

10. Hoenig M, Lee RJ, Ferguson DC: A microtiter plate assay for inorganic phosphate. JBiochemBiophysMethods1989,19:249–251.

11. Cline JD: Spectrophotometric determination of hydrogen sulfide in natural waters.LimnolOceanogr1969,14.

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AppendixI–PCRprotocolsPCRmix1and2

Volume Units Ingredient

25 uL GoTaqGreen2xMasterMix

22 uL PCRwater

1 uL Fprimer10uMstock(515F-ENP)

1 uLRprimer10uMstock(926R-ENPforBacteriaor958R-ENPforArchaea)

1 uL TemplateDNA

PCRThermocyclerprotocol1

Step Temp Time

1 95C 5min

2 95C 1min

3 55C 1min

4 72C 1min30sec

5 GotoStep225moretimes

6 72C 10min

7 4C Forever

PCRThermocyclerprotocol2

Step Temp Time

1 95C 2min

2 95C 30sec

3 55C 30sec

4 72C 1min30sec

5 GotoStep224moretimes

6 72C 10min

7 12C Forever

PCRmix3

Volume Units Ingredient

25 uL GoTaqGreen2xMasterMix

21 uL PCRwater

1 uL Fprimer10uMstock(515F-ENP)

1 uLRprimer10uMstock(926R-ENPforBacteriaor958R-ENPforArchaea)

2 uL TemplateDNA

Cont.AppendixI–PCRProtocolsPCRThermocyclerprotocol3

Step Temp Time

1 95C 2min

2 95C 30sec

3 55C 30sec

4 72C 1min30sec

5 GotoStep229moretimes

6 72C 10min

7 12C Forever

PCRmix4

Volume Units Ingredient

25 uL GoTaqGreen2xMasterMix

x uLPCRwater(AdjustedtoTemplateDNAforfinalPCRreactionvolumeof50uL)

1 uL Fprimer10uMstock(515F-ENP)

1 uLRprimer10uMstock(926R-ENPforBacteriaor958R-ENPforArchaea)

x uLTemplateDNA-GRADIENT10ng/rxn,25ng/rxn,or50ng/rxn

PCRThermocyclerprotocol3

Step Temp Time

1 95C 5min

2 95C 1min

3 GRADIENT,4temperatures45-65C 1min

4 72C 2min

5 GotoStep229moretimes

6 72C 10min

7 10C Forever

AppendixII.TraceElementsFinalConcentrations20uM HCl7.5uM FeCl3⋅6H2O0.48uM H3BO30.5uM MnCl2⋅4H2O6.8uM CoCl2⋅6H2O1.0uM NiCl2⋅6H2O12nm CuCl2⋅6H2O0.5uM ZnCl223nM Na2SeO3150nM Na2MoO4

VitaminsFinalConcentrations10uM 10mMMOPS0.1ug/mL pH7.2Riboflavin0.03ug/mL Biotin0.1ug/mL ThiamineHCl0.1ug/mL L-Ascorbicacid0.1ug/mL d-Ca-pantothenate0.1ug/mL Folicacid0.1ug/mL Nicotinicacid0.1ug/mL 4-aminobenzoicacid0.1ug/mL pyridoxineHCl0.1ug/mL Lipoicacid0.1ug/mL NAD0.1ug/mL Thiaminepyrophosphate0.01ug/mL Cyanocobalamin

AppendixIII.16Staxonomicdatarepresentedatthe“order”level,withamoredetailedlegend.ThebargraphdepictsdatafromvariousdepthsofSaltPondfromtoptobottom(bars lefttoright).Thelegendproceedsfrommostabundanttolessabundant(toptobottom)andisbrokenintotwopieces(lefttoright)sincetherewasnotenoughspaceonthepage.