Quantification of ammonia oxidation rates and the distribution of ammonia-oxidizing Archaea and ...

12 Pages·2012·0.72 MB·English

by  J. M. Beman

ORIGINALRESEARCHARTICLE
published:24July2012
doi:10.3389/fmicb.2012.00263
Quantification of ammonia oxidation rates and the
distribution of ammonia-oxidizing Archaea and
Bacteria in marine sediment depth profiles from
Catalina Island, California
J.M.Beman1*,VictoriaJ.Bertics2,3,ThomasBraunschweiler2-|- andJesseM.Wilson1
1SchoolofNaturalSciences,UniversityofCalifornia,Merced,Merced,CA,USA
2DepartmentofBiologicalSciencesandWrigleyInstituteforEnvironmentalStudies,UniversityofSouthernCalifornia,LosAngeles,CA,USA
3DepartmentofMarineBiogeochemistry,HelmholtzCentreforOceanResearchKiel,Kiel,Germany
Editedby: Microbial communities present in marine sediments play a central role in nitrogen bio-
BessB.Ward,PrincetonUniversity, geochemistryatlocaltoglobalscales.Alongtheoxidation–reductiongradientspresentin
USA
sedimentprofiles,multiplenitrogencyclingprocesses(suchasnitrification,denitrification,
Reviewedby:
nitrogen fixation, and anaerobic ammonium oxidation) are active and actively coupled to
Zhe-XueQuan,FudanUniversity,
oneanother–yetthemicrobialcommunitiesresponsibleforthesetransformationsandthe
China
AndreasSchramm,AarhusUniversity, rates at which they occur are still poorly understood.We report pore water geochemical
Denmark (O , NH+, andNO−)profiles, quantitativeprofilesofarchaealandbacterialamoA genes,
2 4 3
*Correspondence: andammoniaoxidationratemeasurements,frombioturbatedmarinesedimentsofCatalina
J.M.Beman,SchoolofNatural
Island,California.AcrosstriplicatesedimentcorescollectedoffshoreatBirdRock(BR)and
SciencesandSierraNevadaResearch
withinCatalinaHarbor(CH),oxygenpenetration(0.24–0.5cmdepth)andtheabundanceof
Institute,UniversityofCalifornia,
Merced,5200NorthLakeRoad, amoAgenes(upto9.30×107genesg−1)variedwithdepthandbetweencores.Bacterial
Merced,CA95343,USA. amoAgeneswereconsistentlypresentatdepthsofupto10cm,andarchaealamoAwas
e-mail:[email protected]
readilydetectedinBRcores,andCHcoresfrom2008,butnot2007.Althoughdetectionof
-l-Presentaddress: DNAisnotnecessarilyindicativeofactivegrowthandmetabolism,ammoniaoxidationrate
ThomasBraunschweiler,Institute
measurements made in 2008 (using isotope tracer) demonstrated the production of oxi-
ofMicrobiology,SwissFederal
InstituteofTechnologyZürich, dizednitrogenatdepthswhereamoAwaspresent.Ratesvariedwithdepthandbetween
Zürich,Switzerland. cores,butindicatethatactiveammoniaoxidationoccursatupto10cmdepthinbioturbated
CH sediments, where it may be carried out by either or both ammonia-oxidizing archaea
andbacteria.
Keywords:nitrification,amoA,sediments,bioturbation,archaea
INTRODUCTION denitrification–whichisthoughttodominateNlossinsediments
MarinesedimentsareEarth’slargestmicrobialhabitat,harboring atwaterdepths<100m(Kuypersetal.,2006;Francisetal.,2007)–
an estimated 1031 microbial cells with a total biomass rivaling N must be present in oxidized forms such as nitrite (NO−) or
− 2
that of all plants (Whitman etal., 1998). Sedimentary micro- nitrate(NO ).ThisisalsothecaseforNlossviaanaerobicammo-
3 −
bialcommunitiesplayasubstantialroleinglobalbiogeochemical niumoxidation(anammox),asanammoxusesNO asanelectron
2 +
cycles of carbon (C),nitrogen (N),and sulfur (S) – nearly 50% acceptor(Strousetal.,2006).Dissolvedammonium(NH )must
4
of N removal from the ocean, for instance, occurs in sediments therefore first be oxidized, or reduced N present within organic
(Codispoti etal., 2001; Deutsch etal., 2011). Coastal sediments materialmustberegeneratedandsubsequentlyoxidized,beforeN
areparticularlysignificantsitesforNcyclingduetohumaninflu- canberemovedanaerobically.
ence on the global N cycle: agricultural fertilizer use and fossil The oxidation of reduced N occurs via the two-step process
fuelcombustionhavemorethandoubledtheamountofNflow- of nitrification: ammonia-oxidizingarchaea(AOA)andbacteria
ing through terrestrial ecosystems, yet over 50% of this N is (AOB)oxidizereducedNH3/NH+4 toNO−−2,andnitrite-oxidizing
removed in aquatic and coastal ecosystems before it reaches the bacteria (NOB) oxidize nitrite to NO (Francis etal., 2007;
3
sea(Seitzingeretal.,2006;GruberandGalloway,2008).Theover- Erguderetal.,2009).Giventheimportanceofnitrificationtosed-
all size of the N sink in sediments (where N is converted by imentaryandglobalNcycling,AOAandAOBhavebeenstudied
anaerobic microbial processes into gaseous forms that may flux extensivelyinestuarineandcoastalsediments(FreitagandProsser,
out of the system) is nonetheless poorly constrained, leading to 2003; Mortimer etal.,2004; Bernhard etal.,2005; Francis etal.,
debate about whether the oceanic N cycle is currently in bal- 2005;BemanandFrancis,2006;Bernhardetal.,2007;Mosierand
ance (e.g., Codispoti etal., 2001; Gruber and Galloway, 2008; Francis, 2008; Abell etal., 2010; Wankel etal., 2011) using 16S
Deutsch etal., 2011). In order for these outputs to occur via rRNA or the ammonia monooxygenase subunitA gene (amoA)
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Bemanetal. Ammoniaoxidationinsedimentprofiles
asmolecularmarkers.Mostofthesestudieshavetargetedsurface sulfidicsediments(Caffreyetal.,2007).Inanundergroundcoastal
sediments, and few have examined variability in nitrifier distri- aquifer, Santoro etal. (2008) found that AOA and AOB appear
butionsandactivitywithdepth.Surprisingly,FreitagandProsser toshiftinrelativedominancebasedonsalinityandammonium
(2003) and Mortimer etal. (2004) detected AOB 16S rRNA at concentrations (Santoro etal., 2008). Based on pyrosequencing
depths of up to 40 cm in sediments from Loch Duich in Scot- of 16S rRNA,AOA comprised 35% of archaeal sequences in an
land;basedonthisobservationanddetectableratesofnitrification oxiccoralreefsedimentsample,butformedasmallerproportion
downto8cmdepth,Mortimeretal.(2004)arguethatthisisevi- (<10%)ofthearchaealcommunityinananoxicsample(Gaidos
dence of “anoxic nitrification,” possibly coupled to manganese etal.,2011).Fewotherdataareavailablefromsediments.Quan-
reduction.Dollhopfetal.(2005)alsoshowedthatsedimentbio- tifying the distribution of AOA relative to AOB and in relation
turbation supplies oxygen toAOB present at 6 cm depth in salt tonitrificationratesmaythereforeenhanceourunderstandingof
marshsediments. sedimentary N biogeochemistry, as no study has collected sedi-
In contrast to AOB, however, the depth distribution of the mentdepthprofilesofAOA,AOB,andammoniaoxidationrates
recentlydiscoveredAOAinsedimentsislargelyunknown.Sulfide inparallel.
inhibits sedimentary nitrification (Joye and Hollibaugh, 1995), ThepurposeofthisstudywasconsequentlytoquantifyAOA,
butErguderetal.(2009)arguethatAOAtoleratehigherconcen- AOB, and ammonia oxidation rates in sediment cores from
trations of sulfide than AOB based in part on their presence in Catalina Island, California, USA (Figure 1). In a previous
FIGURE1|LocationofCatalinaIslandalongthecoastofCalifornia(B), lowerleft,andcollectionofBirdRocksedimentsisshownin(D).
andofCatalinaHarborandBirdRocksamplinglocations(A).Catalina BurrowdensityatCatalinaHarborin2008was∼120burrowopening
Harborsedimentsamplinglocationisshownin(C)withscalebarat persquaremeter.
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Bemanetal. Ammoniaoxidationinsedimentprofiles
study of Catalina Island sediments, Bertics and Ziebis (2009) sedimentwasrequiredtoallowforcoring,whilesamplesfromBR
detected increases in pore water nitrate where decreases in pore werecollectedon21NovemberbelowtheseasurfaceviaSCUBA
water ammonium concentrations were also observed; canonical in an area near a large rock formation. At both sites, sediment
correspondence analysis revealed that changes in the microbial sampleswerecollectedusing5cmdiameter,39cmlengthacrylic
community with sediment depth were correlated to changes in cores;threeintactsedimentcoresof5–25cmsedimentdepthwere
ammoniumconcentrations–indicatingthatammoniumisakey collectedateachsite,andcoreswereplacedinanicechestatambi-
factorinfluencingmicrobialcommunitiesinCatalinaIslandsed- enttemperaturefortransportbacktothelaboratory.In2008,six
iments. In the present study, AOA and AOB amoA genes were sedimentcoreswerecollectedinapproximatelythesamelocation
quantified in sectioned, triplicate cores collected at two loca- inCHaswassampledin2007,withthreeparallelcorescollected
tions, and cores were collected during two sampling periods for15Nmeasurementson14April, andthreeparallelcorescol-
at one of these locations. Coupled biogeochemical measure- lectedfornutrientmeasurement,oxygenmeasurements,andDNA
ments included microsensor oxygen profiles, measurements of samplingon15April.
dissolved nitrogen in pore waters, and nitrification rate mea- Followingoxygenanalyses(seebelow),eachoftheninecores
surementsusing15Nisotopicallylabeledammonium.Measurable wassub-sampledforammoniumandnitrateconcentrationanal-
rates of nitrification were found throughout two cores, and ysesandDNAextraction.One-centimeterslicesweretakenfrom
both AOA and AOB amoA genes were present at depths of each core starting at the surface down to 10 cm for the CH
upto10cm. cores (CH1–CH6) and 5 cm for the BR cores (BR1–BR3). BR
cores extended to a depth of only 5 cm owing to the difficulty
MATERIALSANDMETHODS in obtaining longer cores from porous sediments via SCUBA.
SITEDESCRIPTION Pore water was collected from each 1-cm slice by centrifuga-
Samples were collected from two locations on or near Catalina tion (10 min at 5000 × g) using 50 ml Macrosep® Centrifugal
Island,California,USA.Thefirstsite,“CatalinaHarbor”(CH;33◦ Devices (Pall Corporation, Life Sciences) flushed with nitro-
27.080(cid:4)N,118◦29.293(cid:4)W),wasashallow(<2m)intertidallagoon gen gas. The recovered pore water (∼3 ml) was immediately
in CH on the western side of the island (Figure1). The lagoon frozen at −20◦C for later determination of dissolved nitrogen
was a low energy, highly bioturbated area consisting of muddy compounds.
sand with the majority of grains being <500 μm (Bertics etal.,
2010). Thetwomostabundantburrowingmacrofaunawerethe POREWATERAMMONIUMANDNITRATEANALYSES
bayghostshrimpNeotrypaeacaliforniensis,Dana,1854(Crustacea: ANDMICROSENSOROXYGENPROFILES
Decapoda:Thalassinidea)andtheMexicanfiddlercrabUcacrenu- Porewaterammoniumconcentrationsweredeterminedbyflow
lata,Lockington,1877(Crustacea: Decapoda: Ocypodoidea). N. injection analysis modified for small sample volumes (Hall and
californiensis inhabits intertidal areas stretching from Alaska to Aller,1992); 50μlof porewaterwasinjectedforeachsediment
Baja California, and is known to build complex branching bur- slice in triplicate. The sum of nitrate and nitrite was deter-
rowsthatextendto∼76cmdepthandhaveseveralopeningstothe mined spectrophotometrically after reduction of samples with
surface(MacGinitie,1934;Brenchley,1981;SwinbanksandMur- spongy cadmium (Jones, 1984). One milliliter of pore water
ray,1981). U.crenulata isfoundfromSantaBarbara,California from the respective core slices was used for the colorimetric
toCentralMexicoandtypicallymaintainssimpleJ-shapedbur- analysis of nitrite concentrations, and nitrite + nitrate concen-
rowswithasingleentranceandthatextendtoadepthof∼20cm; trations(afterreduction)onaspectrophotometer(Stricklandand
U. crenulata frequently leaves these burrows during low tide to Parsons,1972).
forage on algae, bacteria, and detritus on the sediment surface Each of the nine intact cores was analyzed for oxygen con-
(Zeiletal.,2006). tentontheverticalaxisusingaUnisenseoxygenmicrosensor–a
The second site, “Bird Rock” (BR; 33◦ 25.788(cid:4)N, 118◦ miniaturizedamperometricsensorwithaguardelectrode(Revs-
30.314(cid:4)W), was located 1.5 km off the eastern shore the island bechandJørgensen,1986;Unisense© 2007).Foreachcore,three
in ca. 20 m of water. This site consisted of regions with boul- high-resolution microprofiles of oxygen were measured in ver-
derslyingontopof morepermeablesandyandgravelsediment tical intervals of 200–250 μm using Clark-type amperometric
(Nelson andVance, 1979), and regions of rocky outcrops – the oxygen sensors (Revsbech and Jørgensen,1986; Revsbech,1989;
largestofwhichextendsoutofthewaterandformsasmallisland Unisense©, Aarhus, Denmark) following a two-point calibra-
namedBR.Thesandyregionwheresamplingoccurredsupported tion. Sensors were attached to computer-controlled motorized
dense patches of the giant kelp Macrocystis pyrifera and other micromanipulators (Märzhäuser,Wetzlar, Germany) and driven
brownalgae,alongwithassociatedmeio-andmacrofaunalcom- vertically into the sediment in micrometer steps. Signals were
munities. Typical water velocities in the area range from 1 to amplified and transformed to millivolt (mV) by a two-channel
7 m s−1 and the swell surrounding BR ranges from 1 to 3 m picoammeter (PA 2000; Unisense©) and directly recorded on a
inheight(MorrowandCarpenter,2008),makingthissiteanarea computerusingthesoftwareProfix®(Unisense©).
ofhightidalactivityincontrasttoCH.
DNAEXTRACTIONANDQUANTIFICATIONANDQUANTITATIVE
SAMPLECOLLECTION PCRANALYSES
In2007,sedimentsamplesfromCHwerecollectedon19Novem- ForDNAextraction,ca.500mgofsedimentfromeach1cmdepth
berduringhightide,asaminimumof10cmofwaterabovethe intervalwasstoredat−80◦C,andDNAwasextractedfrom200to
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Bemanetal. Ammoniaoxidationinsedimentprofiles
700mgof sedimentusingtheZRSoilMicrobeDNAKit(Zymo oxidation (15R ) were calculated using an equation modified
ox
Research,Irvine,CA,USA;2007samples)ortheMPBiomedicals fromWardetal.(1989):
FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH, USA;
2an00d8thseammpalneus)f.acDtuNrAer’swparsoqtoucaonlti(fiLeifdeuTesicnhgnothloegPieiscoCGorrepeonraatsiosany, 15Rox = (nt −(nnoNO+−x−)×n[NO+−3)×+tNO−2], (1)
Carlsbad,CA,USA). NH4 oNH4
QuantitativePCR(qPCR)analyseswereidenticaltothoseused
lBboyiwoBtienecmghnarenoalecottgiaoieln.s(,2cMh01eam2d)ii.ssoAtrnryc,:hW1ae2Ia,.5lUaμSmALo)AS,Y2qBPmRCMRPraMesmsgaCiyxsl2Fu,s0(eE.d4ptμihceMenfotorle-f wtniomhNeeHret+,nnisotNtihOse−xth,iniesitatihta%elma1t%5eNaseuinnrreidtchhaemt%NenO15t−3Nof+oNfNuHnO+4la−2batepltoehdoelNbmOege−3ians+nuirNnedOg−2oaft,
eachprimer,1.25unitsAmpliTaqpolymerase(LifeTechnologies the ex4periment, n + is at% 15N of NH+, and [NO− +NO−]
Corporation, Carlsbad, CA,USA),40 ng μL−1 BSA (Life Tech- NH4 − 4 3 2
istheconcentrationoftheNO pool.Allstatisticalanalyseswere
nologiesCorporation,Carlsbad,CA,USA),and1ngDNAinafinal x
conductedinMATLAB.
volumeof25μL.β-AOBwerequantifiedusingthesamereaction
chemistry but without additional MgCl . Primers (and relevant
2 RESULTS
referencesforprimersequences),cyclingconditions,qPCRstan-
MICROSENSOROXYGENPROFILESANDPOREWATER
dards,standardcurvecorrelationcoefficients,andPCRefficiencies
DISSOLVEDNITROGENCONCENTRATIONS
are listed in Table 1. All qPCR assays were performed on a
Oxygen concentrations in overlying water were similar in both
StratageneMX3005PqPCRsystem(AgilentTechnologies,LaJolla, CHandBRsedimentsin2007(typically150–210μM),butoxy-
CA,USA). gen concentrations declined to 0 μM at a depth of 2400 μm in
CHcores(Figure2),whereasmorepermeableBRsedimentscon-
15NH+4 OXIDATIONRATEMEASUREMENTS tained>114μMO2at2400μm,andoxygenwasdetectabledown
Ammonia oxidation rates were measured by injecting 99 atom to a depth of 5000 μm (0.5 cm; Figures 2A–C). In CH cores
percent(at%)15NH+4 solutiontoaconcentrationof33μmolL−1 collected in 2008, oxygen penetrated up to 3000μm, consistent
through small silicone-sealed holes drilled into the acrylic core
withwhatwasobservedin2007.Therewassubstantialvariation
cylinder. Theaccumulationof 15NlabelintheoxidizedNO− +
NO− pool was measured after incubation for ∼24 h. The δ215N among measurements made in individual cores, however, and
valu3e of N O produced from NO− +NO− using the“denitri- amongmanyof thecores.Forexample,triplicatemeasurements
2 2 3 inBRcore1(Figure2A),CHcore1(Figure2D),andCHcore
fiermethod”(Sigmanetal.,2001)wasmeasuredusingmethods
6 (Figure2I) exhibit high variation, and measured oxygen pro-
describedinPoppetal.(1995)andDoreetal.(1998):N2Opro- filesdifferedacrosscorescollectedatthesametimeinthesame
duced from NO−2 + NO−3 was transferred from the reaction samplinglocation.
vial, cryofocused, separated from other gases using a 0.32 mm Dissolved nitrogen in pore water also differed between the
i.d. × 25 m PoraPLOT-Q capillary column at room tempera- twosamplinglocations,butdisplayedconsistentpatternsbetween
ture,andintroducedintoionsourceMAT252massspectrometer sampling periods in CH (Figure 3). In BR pore water, ammo-
throughamodifiedGC-CIinterface. Isotopicreferencemateri- nium(NH +)wasmaximalat1cmanddeclinedfrom28to9.9
4
als (USGS-32, NIST-3, and UH NaNO3) bracketed every 12–16 μM moving into the sediments. Combined nitrate and nitrite
samples and δ15N values measured on-line were linearly corre- (NO−+NO−)concentrationsexhibitedmoderatevariationwith
lated (r2 = 0.996–0.999) with accepted reference material δ15N depth3 inBRc2ores,rangingfrom23to33μM.CHsedimentsdif-
values.
feredfromBRinabsolutevaluesandobservedtrendsofdissolved
Initial at% enrichment of the substrate at the beginning of +
nitrogenwithdepth:in2007,NH increasedwithdepth,from23
bthaelaenxcpeerbiamseednto(nnNNHH+4,+seceoEnqc.en1)trwataiosncsalcausslautmedinbgytihsoattotpheem15aNss taond>r1e0a0chμedMa;mNaOx−3im+umNOva−2luweoasf41ty4pμicMallaytl1owcmin,pClaHteapuoerdeawta1t0e–r
activityofunlabeledNH4+was0.3663at%15N.Ratesofammonia 12μMfrom4to6cm,andwasbelow3.5μMfrom2to3and7
4
Table1|Primers(andrelevantreferencesforprimersequences)cyclingconditionsusedforqPCR,qPCRstandardsandstandardcurve
correlationcoefficients,andqPCRefficiencies.
Assay Primers(reference) Cyclingconditions qPCRstandard r2 Efficiency(%)
ArchaealamoA Arch-amoAFandArch-amoAR 95◦C(4min);30×of95◦C(30s), CloneGOC-G-60-9 0.989–0.994 83.1–101
(Francisetal.,2005) 53◦C(45s),72◦C(60swithdetection (GenBankaccessionno.
step);dissociationcurve EU340472)dilutionseries
Betaproteobact amoAFandamoA2R 95◦C(5min);40×of94◦C(45s), CloneHB_A_0206_G01 0.973–0.998 85.7–109
erialamoA (Rotthauweetal.,1997) 56◦C(30s),72◦C(60s),detection (GenBankaccessionno.
stepat81◦C(7s);dissociationcurve EU155190)dilutionseries
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Bemanetal. Ammoniaoxidationinsedimentprofiles
–2000
A B C
–1000
m)
μ
h ( 0
pt 40 80 120 160 240 40 80 120 200240 40 80 120 240
e
D
1000
2000
3000
4000
5000
–2000
D E F
–1000
m)
μ
h ( 0
pt 120 160 200 240 40 80 120 160 200 240 120 160 200 240
e
D 1000 Oxygen (μM) Oxygen (μM) Oxygen (μM)
2000
3000
4000
–2000
G H I
–1000
m)
μ
h ( 0
pt 40 80 160 200 240 40 80 200 240 40 80 200 240
e
D
1000
2000
3000
4000
FIGURE2|Microsensorprofilesofoxygeninsedimentcores. andnegativevaluesrepresentoverlyingwater)andthehorizontal
DatafromBirdRockcoresfrom(A–C),andCatalinaHarborcores axisdisplaysoxygenconcentrationsinmicromolar.Errorbarsdenote
from2007(D–F)and2008(G–I)areshown;verticalaxisdepicts onestandarddeviationoftriplicatemicrosensorprofilestakenfor
depthinsediment(0μmdepthrepresentsthesedimentsurface eachcore.
to10cm.ThesameoverallpatternwasobservedinCHsediments inboth2007and2008:NO−+NO− variedfrom3.3to33μM
3 2
in2008:NH + increasedfrom6.6to76μMwithdepthwhereas at 6 cm depth in 2007, and from 2.6 to 15 μM at 5 cm depth
4
NO− +NO− concentrations were always less than 10 μM,and in2008.
3 2
exceeded5μMonlyat2,5,and6cmdepthinthecores.Onaver-
age, concentrationsof bothNH+ andNO− +NO− werelower QUANTIFICATIONOFAOAANDAOB
4 3 2
in2008comparedwith2007,butthesedifferenceswerenotsig- Toexaminewhetherammoniaoxidizerswerepresentinthesesed-
nificant owing to variability between replicate cores. Inter-core iments,weextractedDNAandquantifiedtheabundanceofAOA
variabilitywasgenerallymuchhigherforNO−+NO−thanNH+ and AOB based on amoA genes. AOA amoA genes, AOB amoA
3 2 4
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Bemanetal. Ammoniaoxidationinsedimentprofiles
NH4+ NO3- + NO2- Archaeal amoA Bacterial amoA
(μM) (μM) (genes g-1) (genes g-1)
0 25 50 75 100 125 0 10 20 30 40 50 104 105 106 107 108 104 105 106 107 108
1 1 1 1
Bird Rock
A B C D
2007
2 2 2 2
m)
c NH4+ (μM)
h (
pt NO3- + NO2- (μM)
de 3 3 3 3
nt Archaeal amoA
e (genes g-1)
m
di Bacterial amoA
e 4 4 4 4 (genes g-1)
S
5 5 5 5
0 25 50 75 100 125 0 10 20 30 40 50 104 105 106 107 108 104 105 106 107 108
1 1 1 1
Catalina Harbor
2 E 2 F 2 G 2 H 2007
3 3 3 3
m)
h (c 4 4 4 4 NH4+ (μM)
pt 5 5 5 5
e NO3- + NO2- (μM)
d
nt 6 6 6 6
e Archaeal amoA
m 7 7 7 7 (genes g-1)
di
e Bacterial amoA
S 8 8 8 8 (genes g-1)
9 9 9 9
10 10 10 10
0 25 50 75 100 125 0 10 20 30 40 50 104 105 106 107 108 104 105 106 107 108
1 1 1 1
Catalina Harbor
I J K L
2 2 2 2 2008
3 3 3 3
m)
c 4 4 4 4
h ( NH4+ (μM)
pt 5 5 5 5
e
d NO3- + NO2- (μM)
nt 6 6 6 6
me Archaeal amoA
di 7 7 7 7 (genes g-1)
Se 8 8 8 8 B(gaecnteersi agl- 1a)moA
9 9 9 9
10 10 10 10
FIGURE3|Sedimentprofilesofdissolvedinorganicnitrogenand areshownforindividualcorescollectedatBirdRock(D),CatalinaHarborin
+
ammoniaoxidizers.Averageporewater[NH ]isshownforBirdRockin 2007(H),andCatalinaHarborin2008(L).In(C–L),dashedlinesdenote
4
2007(A),CatalinaHarborin2007(E),andCatalinaHarborin2008(I),and depthsweredatavaluesareomittedduetoqPCRinhibitionofsamples,and
porewater[NO−+NO−]isshownforBirdRock(B),CatalinaHarborin2007 colorshadingdenotesdifferentcores.Lightgreen/lightbluedenotesBR1
3 2
(F),andCatalinaHarborin2008(J).ArchaealamoAgenes(g-sediment−1)are (C,D),CH1(G,H),andCH4(K,L);“mid”green/“mid”bluedenotesBR2(C,D),
shownforindividualcorescollectedatBirdRock(C),CatalinaHarborin2007 CH2(G,H),andCH5(K,L);darkgreen/darkbluedenotesBR3(C,D),CH3
(G),andCatalinaHarborin2008(K).BacterialamoAgenes(g-sediment−1) (G,H),andCH6(K,L).In(G),archaeal amoAwasonlydetectableinonecore.
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Bemanetal. Ammoniaoxidationinsedimentprofiles
genes,orboth,werepresentinallsamplesfromalldepths,sam- NO−+NO−poolin15N.Becausethevaluesweobservedareinthe
3 2
pling locations, and time points (Figure 3). AOB amoA genes rangeexpectedforsedimentarydenitrification,thissuggeststhat
werequantifiedineverysamplecollectedin2007atCHandBR, littleornoammoniaoxidationoccurredinthiscore(weenriched
whereasAOAwereundetectableintwoofthreeCHcorescollected the15NH+poolto76.7at%).Instead,themeasuredvalueseffec-
in2007,andwerepresentatlowerabundanceintwoofthreeBR tivelyrepr4esentinsituδ15NofNO−+NO−,andthesevalueswere
cores.BothAOBandAOAamoAgenesvariedwithdepthinBRand usedtocalculate15NH+ oxidation3 ratesi2ntheothercores.(Two
4
CHcores:AOAamoAgenesrangedfrom4.01×106to1.22×107 exceptions were the lower δ15N values measured at 7 and 9 cm
genesg−1 inBRcore3and2.03×104 to1.73×105 genesg−1 depth,whereweinsteadlinearlyinterpolatedtheinsituδ15Nval-
incores1and2(Figure3),whileAOBamoAgenesrangedfrom ues.)Incontrasttotheδ15NvaluesobservedinCHcore5,δ15N
6.55×104to3.26×107genesg−1intheBRcores.AOAandAOB ofNO−+NO−inporewaterexceeded330‰inCHcores4and
3 2
amoAgeneswerehighlyvariableacrossthereplicatecores,how- 6(Figures4A,C).Porewaterδ15Nwashighlyvariablethroughout
ever,andthispatternheldforCHcoresfromboth2007and2008: each core, and between both cores, and spiked at several depth
formostsedimentdepths,thecoefficientofvariationamongrepli- intervals–indicatingthatlabeled15NH+wasbeingoxidizedrela-
catecoreswas>1.Thisisclearlyindicativeofheterogeneityand tivelydeepwithintheCH4andCH6cor4es(Figures4D,E).15NH+
4
patchinessinamoAgenesinthesesediments,andmoststrikingis oxidationrateprofilesshowedmaximaat6cminCH4,andat3cm
thatfactthatAOAamoAgeneswereundetectedintwosediment inCH6,whererateswerealsoelevatedat5and7cm(Figure4).In
corescollectedatCHin2007,butweredetectedinthethirdrepli- bothcores,15NH+oxidationrateswerereadilydetectableat9cm
4
cate separated by <50 cm. Another possibility is that the amoA depth.Ratesrangedfrom0to7.15nmolL−1day−1inCH4and0
primersdidnotsuccessfullyamplifythearchaealamoAsequence to18.3nmolL−1day−1inCH6.
types present in these samples; if so, this indicates that entirely
different AOA communities inhabit these cores, and is consis- DISCUSSION
tentwithheterogeneityandpatchinessofamoAgenesinCatalina GEOCHEMISTRYOFCATALINASEDIMENTS
sediments. Oxygen typically penetrates only a few millimeters into coastal
WhenAOAamoAgeneswerequantifiedintheCH3corecol- sediments owing to rapid consumption during organic matter
lectedin2007,theywerecorrelatedwithamoAgenesfromAOB degradation, or chemical re-oxidation of reduced compounds
(r2=0.936,P<0.001)withanAOB:AOAslopeof7.78(Figure3). (Revsbechetal.,1980;GundersenandJørgensen,1990).However,
Itisunlikelythatthiscorrelationisanartifactof differentDNA the depth of oxygen penetration can be increased via bioturba-
extractionefficienciesfordifferentdepths,asDNAwasextracted tion/bioirrigation (Aller, 1982; Ziebis etal., 1996a; Bertics and
from 0.15 to 0.25 g of sediment at each core depth and yielded Ziebis,2009),sedimentpermeabilityandincreasedbottomwater
316–741ngofDNA,whilebothAOAandAOBamoAgenesvaried flow velocity, and/or increased wave action (Booij etal., 1991;
bymorethananorderofmagnitude.In2008,AOAandAOBamoA Precht etal.,2004). Sediment topography features that generate
genesweremoreweaklyrelated(r2=0.49–0.55,P<0.05)intwo pressure differences can also lead to advective transport of oxy-
ofthecores,anduncorrelatedinthethird(r2=0.03,P>0.05).As genated water into the sediment (Ziebis etal., 1996b). At BR,
theserelationshipsindicate,weobservedrelativelylittlevariability sedimentscontained>114μMO at2400μmandoxygenwas
2
inAOBamoA:AOAamoAratioswithcoredepthinBRandCH detectabledowntoadepthof5000μm(0.5cm;Figures2A–C).
sediments,yettherewereobviousdifferencesbetweencores,sam- This is consistent with oxygen transport via advective processes
plinglocations,andsamplingperiodsintherelativedominanceof severalcentimetersintothesediment,especiallygiventheporous
AOBandAOAamoAgenes.Withaloneexception,AOBamoAwas natureofthesecoarseBRsediments.Incontrast,inCHsediments,
1.9–46timesmoreabundantthanAOAamoAinallBRsamples(at oxygenwasnotdetectedbelow2400μmin2007(Figures2D–F)
1cmdepthinBRcore3AOAamoAwasmorenumerous),while and 3000 μm in 2008 (Figures 2G–I) – suggesting that oxygen
theratioofAOBtoAOAamoArangedfrom0.24(5cmdepth)to diffuses to a consistent depth at CH. An important caveat to
8.6(4cmdepth)intheCH3corecollectedin2007.AOAamoA thisisthefactthatmacrofaunacantransportoxygenmorethan
wasnotamplifiableinCHcores1and2from2007andAOBamoA 50cmdeep(Ziebisetal.,1996a)andbioturbationhasbeenshown
wasthereforepresentinsubstantialgreateramounts.Incontrast, totransferoxygenmultiplecentimetersdeepintoCHsediments
AOAamoAgenesweremoreabundantthanAOBinthe2008CH (BerticsandZiebis,2009).Thepresenceofbioturbationisthere-
cores, with AOA amoA:AOB amoA ratios ranging from 0.86 to forealikelyexplanationforthevariationwithinandamongmany
2.9 in CH core 4, 0.77 to 2.9 in CH core 5, and 0.5 to 5.1 in of the CH cores – e.g., CH core 1 (Figure 2D) and CH core 6
CHcore6. (Figure2I).
NO− + NO− profiles also differed between BR and CH
3 2
δ15NANDNITRIFICATIONRATEPROFILES sediments, in that high concentrations (>20 μM) were seen
δ15NofNO−+NO−inporewaterwasmeasuredfollowinga24h throughout BR cores while concentrations reached a maximum
incubation3of intac2t cores collected in 2008 to calculate 15NH+ valueof14μMat1cminCHcoresfrom2007andwerealways
oxidationrates.δ15NofNO−+NO−inCHcore5exhibitedonl4y lessthan10μMincoresfrom2008.However,severalsubsurface
modestenrichment,ranging3from132.8‰atthesurfaceto54.0‰ peaks of NO− +NO− occurred in CH in both 2007 and 2008,
3 2
at10cmdepth(Figure4B).Thispatternistypicalforsediments andmayreflecteither(1)transportofoxidizedcompoundsinto
(e.g., Lehmann etal.,2007) where denitrification at depth pref- thesedimentviabioturbation,or(2)productionofNO−+NO−
3 2
erentially removes isotopically light N, enriching the remaining inthesedimentviatheactivityofnitrifyingbacteriaandarchaea
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Bemanetal. Ammoniaoxidationinsedimentprofiles
δ15N of NO3- + NO2- (‰)
0 100 200 300 400 0 10 20 3 0 40 50 60 0 100 200 300 400
1 1 1
A B C
2 2 2
m) 3 3 3
c 4 4 4
h (
pt 5 5 5
e
d
t 6 6 6
n
e
m 7 7 7
di
Se 8 8 8
9 9 9
10 10 10
15NH4+ oxidation rate nmol L-1 d-1
0 5 10 15 20 0 5 10 15 20
1 1
D E
2 2
m) 3 3
c 4 4
h (
pt 5 5
e
d
t 6 6
n
e
m 7 7
di
Se 8 8
9 9
10 10
Catalina Harbor 4 Catalina Harbor 5 Catalina Harbor 6
FIGURE4|Measuredδ15NofporewaterNO−+NO−followingincubationwithadded15NH+label(A–C),and15NH+oxidationrates(D,E)inCatalinaHarbor
3 2 4 4
in2008.Notedifferencesinscalesin(A–C);oxidationrateswerenotcalculatedinCatalinaHarborcore5owingtothelackofclearisotopicenrichment.
(i.e.,insitunitrification).CHcoresdisplayedthetypicalincrease DNAextractedfromsedimentsmaynotbederivedfromactiveor
+
in NH that is expected with increasing sediment depth due to viablemicroorganisms–indeed,itispossibletorecoverancient
4
microbial remineralization of organic material. Concentrations DNA from sediment cores (Coolen and Overmann,1998) – yet
of both NH+ and NO− +NO− were on average lower in 2008 the presence of, and variability in, oxidized nitrogen at 4–6 cm
4 3 2
whencomparedwith2007–althoughthesedifferenceswerenot depthinCHcoresisindicativeofactiveproduction.Weassessed
significantowingtovariabilitybetweenreplicatecores.Adecrease thisusingdirectbiogeochemicalmeasurements(seebelow)rather
in recruitment of shrimp and a decrease in microbial mat for- than extraction of RNA, yielding quantitative rates rather than
mation was previously observed in these sediments from 2007 relative levels of gene expression. Our DNA data are neverthe-
to 2008 (Bertics etal.,2010) and may explain this shift in sedi- lessconsistentwithotherstudiesprofilingAOBinsediments:AOB
mentgeochemistry.Henceinterannualvariabilityingeochemical DNAhasbeendetectedat40cmdepthinLochDuichsediments
conditionsandmicrobialactivitycanoccurinCH,butitoccurs (FreitagandProsser,2003;Mortimeretal.,2004),6cmdepthin
againstabackdropofsubstantialspatialvariability. salt marsh sediments (Dollhopf etal., 2005), and at least 2 cm
depthinestuarinesedimentsfromPlumIslandSound(Bernhard
ABUNDANCEOFAOAANDAOBINCATALINASEDIMENTS etal.,2007),wherepotentialnitrificationwasmeasuredatupto
Ammonia-oxidizingarchaeaandAOBwerealsohighlyvariablein 4cm.Inthesestudies,AOBtypicallyrangedfrom104to107amoA
CatalinaIslandsedimentsbasedontheabundanceofamoAgenes. genesg−1,andourdataaresimilar(3.6×104to9.3×107amoA
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Bemanetal. Ammoniaoxidationinsedimentprofiles
genesg−1).However,inadditiontoAOB,wereportamoAgenes with NH+ – and positively correlated with NO− – in 2008
4 2
fromAOAatupto5cmdepthinBRsedimentcores,and10cm (Table2).
depthinCHsedimentcores,wheretheyrangedfrom7.2×104to
1.3×107genesg−1. NITRIFICATIONINCATALINASEDIMENTS
Previous studies have shown that although AOA and AOB AmmoniaoxidationratemeasurementsindicatedthatAOAand
arepresumablyfunctionallyequivalent,theirrelativedominance AOBwereactivelynitrifyingthroughouttwoofthethreecollected
varies across gradients of salinity present in sediments (Caffrey coresin2008.ModestenrichmentintheCH5coresuggeststhat
etal.,2007;MosierandFrancis,2008;Santoroetal.,2008).Studies although we recovered amoA genes, either this DNA was not
+
in soils suggest that pH (Nicol etal., 2008) and NH4 concen- derived from living organisms, or these organisms were inac-
trations (reviewed by Erguder etal.,2009) also alter the relative tive during our incubation. Evidence for the later includes the
abundanceofAOAandAOB–morespecifically,anexceptionally relatively low δ15N values measured at 7 and 9 cm depth, as
+
highaffinityforammoniabenefitsAOAwhenNH4 concentra- in a previous study conducted in the same location in 2008,
tionsarelow(Martens-Habbenaetal.,2009).Whileweobserved Bertics etal. (2010) found the highest rates of nitrogen fixation
relatively little variability in AOB amoA:AOAamoA ratios with at depth of 7 and 9 cm in the most bioturbated location they
depth in BR and CH sediments, AOB amoA genes were more sampled. Hence one possible explanation for the “light” δ15N
abundant in BR sediments and CH sediments from 2007, while of NO− + NO− at these depths is the oxidation of recently
3 2
AOA amoA genes were more abundant than AOB in the 2008 fixed nitrogen, i.e., while ammonia oxidation appeared inactive
CHcores. DifferentDNAextractionkitswereusedforCHsed- at the time of our sampling, it may have been previously active
iments collected in 2007 and 2008, and it is possible that the within or near these sediment layers. Another explanation for
MP Biomedicals kit (used in 2008) is less effective in extracting these local minima in the pore water profile is that this rep-
bacterialDNAandsoexplainsthedifferencesobservedbetween resents NO− and/or NO− of differing δ15N that is present in
3 2
the two sampling periods. When comparing measured values, groundwater.
however, 2008 values lie within the range of AOB and AOA IntheCH4andCH6cores,15NH+oxidationrateswerereadily
4
amoA gene abundances observed across both sites in 2007; this detectableatmostdepthsup9cminbothcores,andupto10cm
argues against extraction bias, as one would expect much lower depth in the CH6 core. Relatively few 15N-based rate measure-
orhighernumbersforoneorbothofthegenes.Inanycase,the mentshavebeenconductedwithinsediments(Ward,2008),but
evidence for interannual variability in ammonia oxidizer popu- ourexperimentalapproachwassimilartothatusedbyMortimer
lations is mixed, given that: (1) measured NH+ values are still etal.(2004)andourmeasuredrates(0–18.3nmolL−1day−1)were
4
farinexcessofKm value(123nM)fortheloneculturedmarine similartovaluesof 4.86–89.6nmolL−1 day−1 measuredat2–6
AOA,Nitrosopumilusmaritimus (Martens-Habbenaetal.,2009), and10–12cmdepthinLochDuich(Mortimeretal.,2004).How-
while Km values for some AOB are as low as 10 μM (Casciotti ever,ourmeasurementsweremuchlowerthanthemaximumrates
etal., 2003 and references therein), and (2) high spatial varia- measuredat0–2cminLochDuich(1.6×106nmolL−1day−1)
tionwithinthesesedimentsmightobscuretemporaltrends. Put and most other measurements in the literature (Ward, 2008).
anotherway,ourdatadonotconclusivelyindicatewhetherAOA Theseresultsthereforecaptureactive15NH+oxidationatdepths
4
orAOBaremoredominantinthesesediments,butareindicative of up to 10 cm in Catalina Island sediments, but also indicate
of substantial spatial variation and possibly temporal variation thatratesaregenerallylowandvariablewithdepthandbetween
as well. This parallels our geochemical results, but there was replicatecores.
little correspondence between AOA and AOB and nutrient and One possible explanation for measurable ammonia oxida-
rate data: no significant correlations were observed in the 2007 tion at depth is the periodic supply of oxygen to aerobic
data (all P > 0.05), whereas AOA were negatively correlated nitrifiers:previousworkhasshownthatalterationofsedimentby
Table2|Correlationcoefficients(r2)forcomparisonsbetweenqPCRdata,nutrientconcentrations,and15NH+oxidationratesaveragedacross
4
triplicatecorescollectedinCatalinaHarborin2008.
LogAOAamoA AOAamoA LogAOBamoA AOBamoA [NH+] [NO−] [NO−] 15NH+oxidationrate
4 2 3 4
LogAOAamoA 0.19 0.07 0.55* 0.44* 0.22 0.28
AOAamoA 0.09 0.02 0.48* 0.54* 0.36 0.26
LogAOBamoA 0.08 0.03 0.01 0.06
AOBamoA 0.02 0.02 0.01 0.02
NH+ 0.30 0.03 0.02
4
NO− 0.41* 0.01
2
NO− 0.10
3
*P<0.05.
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Bemanetal. Ammoniaoxidationinsedimentprofiles
macrofaunacanalterredoxchemistryandmicrobialcommunities of 790nmolSO2− cm−3 day−1 separatedbyonly3–5cmfrom
4
in CH sediments (Bertics and Ziebis, 2009, 2010; Bertics etal., areas displaying rates of <5 nmol SO2− cm−3 day−1 (Bertics
4
2010),andburrowswerepresentinthemajorityof thecoreswe and Ziebis, 2010). It is therefore possible that in some patches
collected.PreviousworkbyDollhopfetal.(2005)infactshowed of CH sediment, high sulfate reduction rates inhibit nitrifica-
thatnitrificationratesandAOBabundancewererelatedtoburrow tion, while in other areas, low sulfate reduction rates allow for
abundance.Abiotic“anoxicnitrification”(Mortimeretal.,2004) thepresenceofnitrification–therebyexplainingthehighlevelsof
may also explain oxidation of ammonia at up to 9 cm depth – variationinnitrificationratesseenbetweenreplicatecoresinCH.
however,AOBhavebeendetectedatgreaterdepthsinothersedi- ThishypothesisissupportedbyGilbertetal.(1998),inwhichthe
mentaryenvironments,andamoAgenesfrombothAOBandAOA authorsfoundthatbioturbationledtotheclosepresenceofoxic
werereadilyquantifiedwhereactiveammoniaoxidationwasalso and anoxic microenvironments, which in turn strengthened the
measured. Asaresult, ourfindingsareconsistentwithprevious proximityandexchangesbetweennitrificationanddenitrification
work indicating that bioturbation sustains nitrification by pro- insediments.
vidingperiodicintrusionsofoxygen(Dollhopfetal.,2005;Ward, Our results are consistent with ammonia oxidation being
2008,andreferencestherein). broadly but patchily distributed in marine sediments, where
Hydrogen sulfide is a confounding issue for nitrification in this key process may be coupled to anaerobic N cycling and
sediments because it can completely inhibit nitrification (e.g., loss. The high degree of heterogeneity observed for substrates,
Joye and Hollibaugh, 1995); yet in spite of relatively high sul- products, genes, and biogeochemical activity – laterally, with
fate reduction rates occurring in CH sediments (Bertics and depth, and through time – demonstrates that sedimentary N
Ziebis, 2010), pore water hydrogen sulfide was not previously cyclingisextraordinarilycomplex. Understandingthiscomplex-
detected (Bertics and Ziebis, 2009), possibly because dissolved ity and variability will be critical for balancing the N cycle
sulfide reacts with the high levels of iron (Bertics and Ziebis, in an era of global change (Gruber and Galloway,2008; Beman
2009), leading to the precipitation of iron sulfides (Berner, etal.,2011).
1970). Hydrogen sulfide may also be oxidized by sulfide oxidiz-
ers present in nearby sediments (Meyers etal., 1987) – in fact, ACKNOWLEDGMENTS
hydrogensulfideisoxidizedbyorganismsusingnitrateasanelec- ThisworkwassupportedbyNSFgrantsOCE08-24997(toJ.M.
tronacceptorinoceanicoxygenminimumzones(Canfieldetal., Beman and Brian Popp) and 10-34943 (to J. M. Beman), and
2010). Some combination of these processes likely explains the theEugeneCotaRoblesFellowship(awardedtoJesseM.Wilson).
lack of sulfide inhibition of ammonia oxidation in cores CH4 WethankSusanAlford,C.J.Bradley,JackieMueller,NatalieWals-
andCH6. grove,andElizabethGierforassistanceinpreparingandanalyzing
However, the variation in 15NH+ oxidation rates that we samples,andBrianPoppforaccesstotheStableIsotopeBiogeo-
4
observed (e.g., between cores and with depth) may stem from chemistrylabattheUniversityofHawaii.Wealsowishtothank
productionofhydrogensulfide:similartotheratemeasurements JedFuhrmanandWiebkeZiebisforhelpfuldiscussions,andthe
reported here, sulfate reduction rates are heterogeneous in CH WrigleyMarineScienceCenteronCatalinaIslandforsupporting
bioturbatedsediments, withareashavingsulfatereductionrates fieldwork.
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