Ecophysiological Characterization of Ammonia-Oxidizing Archaea and Bacteria from Freshwater

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Published Ahead of Print 8 June 2012.
10.1128/AEM.00432-12.
2012, 78(16):5773. DOI:
Appl. Environ. Microbiol.
Mukherjee, George Bullerjahn and Annette Bollmann
Elizabeth French, Jessica A. Kozlowski, Maitreyee
from Freshwater
Ammonia-Oxidizing Archaea and Bacteria
Ecophysiological Characterization of
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Ecophysiological Characterization of Ammonia-Oxidizing Archaea and
Bacteria from Freshwater
Elizabeth French,aJessica A. Kozlowski,a* Maitreyee Mukherjee,bGeorge Bullerjahn,band Annette Bollmanna
Miami University, Department of Microbiology, Oxford, Ohio, USA,aand Bowling Green State University, Department of Biological Sciences, Bowling Green, Ohio, USAb
Aerobicbiologicalammoniaoxidationiscarriedoutbytwogroupsofmicroorganisms,ammonia-oxidizingbacteria(AOB)and
therecentlydiscoveredammonia-oxidizingarchaea(AOA).Herewepresentastudyusingcultivation-basedmethodstoinvesti-
gatethedifferencesingrowthofthreeAOAculturesandoneAOBcultureenrichedfromfreshwaterenvironments.Thestrainin
theenrichedAOAculturebelongtothaumarchaealgroupI.1a,withthestraininoneenrichmentculturehavingthehighest
identitywith“Candidatus Nitrosoarchaeumkoreensis”andthestrainsintheothertworepresentinganewgenusofAOA.The
AOBstrainintheenrichmentculturewasalsoobtainedfromfreshwaterandhadthehighestidentitytoAOBfromthe Nitro-
somonas oligotropha group (Nitrosomonas cluster 6a). We investigated the influence of ammonium, oxygen, pH, and light on the
growthofAOAandAOB.ThegrowthratesoftheAOBincreasedwithincreasingammoniumconcentrations,whilethegrowth
ratesoftheAOAdecreasedslightly.IncreasingoxygenconcentrationsledtoanincreaseinthegrowthrateoftheAOB,whilethe
growthratesofAOAwerealmostoxygeninsensitive.Lightexposure(whiteandbluewavelengths)inhibitedthegrowthofAOA
completely,andtheAOAdidnotrecoverwhentransferredtothedark.AOBwerealsoinhibitedbybluelight;however,growth
recoveredimmediatelyaftertransfertothedark.OurresultsshowthatthetestedAOBhaveacompetitiveadvantageoverthe
testedAOAundermostconditionsinvestigated.FurtherexperimentswillelucidatethenichesofAOAandAOBinmoredetail.
N
cycle. The first and rate-limiting step of nitrification is the oxidation
ofNH3toNO2
wasattributedtoonlyasmallsubsetoftheProteobacteria;mostfresh-
waterandterrestrialammonia-oxidizingbacteria(AOB)belongtoa
distinct group in the Betaproteobacteria, while a few marine AOB
species belong to the Gammaproteobacteria (29, 32, 33). The AOB
haveachemolithoautotrophicmetabolism,oxidizingNH3toNO2
via the intermediate NH2OH (hydroxylamine) and fixing carbon
fromCO2(carbondioxide)viatheCalvincycle(1).
Recently, genes encoding ammonia monooxygenase (amoA),
the first enzyme in the process of ammonia oxidation, were dis-
coveredtogetherwitharchaeal16SrRNAgenesinametagenomic
study (60) and a soil fosmid library (57). At the same time, Nitro-
sopumilusmaritimus,thefirstarchaealammoniaoxidizer,wasiso-
lated in pure culture from a saltwater aquarium (30). Ammonia-
oxidizing archaea (AOA) in pure and enrichment cultures have
essentiallythesamemetabolismasAOB;theyoxidizeNH3stoichio-
metrically to NO2
(15,20,30,36,43,56).However,thegenomesofN.maritimusand
“Candidatus Nitrosoarchaeum limnia” revealed differences be-
tweenAOAandAOB,suchastheuseofthe3-hydroxypropionate/
4-hydroxybutyrate pathway for HCO3
hydroxylamineoxidoreductase,andthepresenceofmanycopper-
containing enzymes (5, 63).
AOA and AOB often co-occur in the same environment, but
the contributions of AOA and AOB to the total ammonia oxida-
tion still need to be elucidated. Many previous studies focused on
the influence of environmental factors on niche differentiation
betweenAOAandAOBusingcultivation-independentmolecular
methods. From those studies, it can be concluded that AOA are
frequently found in environments with lower substrate (NH4
and O2) availability and AOB are frequently found in environ-
ments with higher substrate availability (see references 4, 13, 17,
itrification, the microbial oxidation of NH3(ammonia) to
NO3
?(nitrate),isoneofthekeyprocessesoftheglobalnitrogen
?(nitrite).Untilrecently,aerobicammoniaoxidation
?
?and fix carbon from bicarbonate (HCO3
?)
?fixation, the absence of
?
24, 42, and 55, among others). However, most of these studies
were conducted using methods that target the abundance and/or
expression of the archaeal and bacterial amoA genes. Unfortu-
nately, it is not possible to draw direct conclusions about the ac-
tivity of the AOA and AOB on the basis of the abundance and
expression of the amoA gene, because amoA mRNA has been de-
tected in AOB for weeks and 16S rRNA (ribosomes) has been
detected for up to a year after the onset of starvation (8, 25, 26).
The response of AOA toward starvation and resuscitation has not
yet been investigated. In addition, it has been shown that not all
amoA-encoding Thaumarchaeota are autotrophic ammonia oxi-
dizers (39, 64). While studies focusing on the analysis of abun-
danceandactivityofmicrobesusingmolecularmethodsgivevery
valuable insights, it is also necessary to investigate the response of
microbes to environmental factors using cultivation-based ap-
proaches, because these experiments will demonstrate changes in
physiological activity more conclusively.
Here we present a study that used a cultivation-dependent ap-
proach to investigate the responses of AOA and AOB to environ-
mental factors. Cultures of three phylogenetically distinct AOA
fromfreshwatersedimentsinOhiowereenriched,andthegrowth
Received 21 February 2012 Accepted 30 May 2012
Published ahead of print 8 June 2012
Address correspondence to Annette Bollmann, bollmaa@muohio.edu.
*Present address: Jessica A. Kozlowski, University of Alberta, Department of
Biological Sciences, Edmonton, Alberta, Canada.
This paper is dedicated to the memory of John W. Hawes (Center for
Bioinformatics and Functional Genomics and Department of Chemistry and
Biochemistry, Miami University).
Supplemental material for this article may be found at http://aem.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.00432-12
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of the AOA was characterized under different conditions and
compared with that of AOB from freshwater in an enrichment
culture. Factors of interest include the NH4
O2concentration, and light wavelength and intensity. These fac-
tors have strong effects on the physiology and niche differentia-
tion of AOB (7, 23, 44, 54) and are, therefore, also very likely to
influence the physiology and niche differentiation between AOA
and AOB.
?concentration, pH,
MATERIALS AND METHODS
Sampling. Near-shore sediment samples were taken from Lakes Acton
(AC; 39°57=N, 84°74=W) and Delaware (DW; 40°39=N, 83°05=W) in fall
2008. Additional sediment core samples were collected from Lake Acton
in summer 2009.
Medium.Themineralsalts(MS)mediumusedtoenrichandcultivate
AOA and AOB contained 10 mM NaCl, 1 mM KCl, 1 mM CaCl2· 2H2O,
0.2 mM MgSO4· 7H2O, and 1 ml liter?1trace elements solution (9, 61).
HEPES buffer was added in a 4-fold molar ratio to the NH4
tion,andthepHwasadjustedto7.5beforeautoclaving.Afterautoclaving,
sterile KH2PO4solution was added to obtain a final concentration of 0.4
mM (9, 61).
Enrichment of AOA (AOA-AC2, AOA-AC5, and AOA-DW enrich-
ment cultures). Sediment samples (1 g) were inoculated into 50 ml MS
mediumwith0.25mMNH4
The enrichments were incubated at 27°C in the dark. NH4
monitored weekly using a colorimetric assay (9, 28). When the cultures
reached late logarithmic growth phase (depletion of about 80% of the
initialNH4
a 10% (vol/vol) inoculum. The cultures were passed through 0.45-?m-
pore-sizefiltersforthefirstfivetosixtransferstoexcludeAOB(9;Annika
Mosier, personal communication). In addition to filtration, the enrich-
mentculturesfromDWwerealsotreatedwith100?gml?1streptomycin
to eliminate AOB. After several transfers, when the cultures depleted
NH4
cellulose filters for molecular characterization. The filters were stored at
?20°C.
AOB culture. We used the previously described AOB strain from a
freshwater enrichment culture G5-7 (AOB-G5-7) to compare the growth
of AOA to that of AOB (6, 7). The strain belongs to the Nitrosomonas
oligotropha cluster and is adapted to low NH4
Members of this AOB cluster have been found in many freshwater envi-
ronments around the world (11, 12, 14, 22, 53; E. French and A. Boll-
mann, unpublished data).
Growth experiments. All growth experiments were conducted in MS
medium with 0.5 mM NH4
cotton stoppers unless otherwise noted. We tested the influence of differ-
entfactors(NH4
rate of NO2
(AOA-AC2,AOA-AC5,andAOA-DW)andtheAOBenrichmentculture
(AOB-G5-7). All cultures were inoculated with 10% (vol/vol) condi-
tioned late-log-phase cells and incubated in the dark at 27°C. Samples (1
ml) were taken at regular intervals and centrifuged at 16,000 rpm for 20
min. The supernatant was stored at ?20°C for further chemical analysis.
To investigate the influence of different NH4
with15?Mto5mMNH4
concentrations. The influence of pH was investigated by adjusting the
initial pH in the medium to values between 6 and 9. The influence of the
O2concentration was investigated by equilibrating the medium in serum
bottles under anaerobic conditions overnight. After equilibration, the
bottles were sealed with rubber stoppers. Different calculated O2concen-
trationsintheheadspacewereachievedbyexchangingthecorresponding
volumeoftheheadspacewithsterilefilteredair.Theinfluenceoflightwas
investigated by incubating the cultures 18 cm above light-emitting diode
panels emitting 30 ?mol photons m?2s?1at the wavelengths 5,000 to
7,000K(whitelight),623?3nm(redlight),and470?5nm(bluelight)
?concentra-
?immediatelyuponarrivalinthelaboratory.
?levels were
?concentration),theyweretransferredtofreshmediumusing
?atregularintervals,20mlwascollectedon0.1-?m-pore-sizenitro-
?concentrations (6, 7).
?at pH 7.5 in 125-ml Erlenmeyer flasks with
?concentration,O2concentration,pH,andlight)onthe
?/NO3
?production of the three AOA enrichment cultures
?concentrations, medium
?waspreparedwiththecorrespondingHEPES
and 3 ?mol photons m?2s?1at the wavelength 470 ? 5 nm (blue light).
The light intensity inside the glass bottles was 25 ?mol photons m?2s?1
(high light conditions) and 2.5 ?mol photons m?2s?1(low light condi-
tions),asmeasuredwithaLI-250Alightmeter(LI-CORBiosciences,Lin-
coln,NE),indicatingthattheglassfilteredapproximately15%ofthelight.
To investigate the influence of light-to-dark and dark-to-light transitions
onthegrowthofAOAandAOB,cultureswereincubatedinthedarkuntil
50% of the NH4
sametime,culturesthatwereincubatedinthelightweretransferredfrom
thelighttothedark.Controlswereincubatedforthecompletecycleinthe
dark.
Evaluationofgrowthexperiments.NO2
were determined in the supernatants using colorimetric assays (9, 49).
NO2
time (see Fig. S1 in the supplemental material). Growth rates were calcu-
latedfromthelinearincrease(slope)ofthelog-transformedNO2
concentrations over time, assuming that NO2
cultures is correlated with the growth of AOA and AOB (3, 9, 30). The
increaseinNO2
and the correlation coefficients were always?0.97 but in most cases were
even ?0.99.
Molecular analysis. (i) DNA isolation from AOA enrichment cul-
tures. DNA was isolated from the nitrocellulose filters using a Qiagen
DNeasy blood and tissue kit (Valencia, CA) with the following modifica-
tions.Acid-washedzirconiumbeads(1g)and500?lhigh-saltbuffer(1M
NaCl, 5 mM MgCl2· 2H2O, 10 mM Tris, pH 8) (2) were added to the
nitrocellulose filters. The filters were homogenized using a bead beater
(Biospec Products, Bartlesville, OK) at 4,800 rpm for 30 s. This was re-
peated three times, and the samples were stored in between cycles on ice
for 10 min. After bead beating, 500 ?l Qiagen buffer AL and 50 ?l protei-
naseKwereaddedandthemixturewasincubatedat56°Cfor30min.The
reactionmixturewasspundownat8,000rpmfor1minandtransferredto
spin columns supplied by the manufacturer. The spin columns were
treated according to the manufacturer’s recommendations, and the DNA
was eluted with 100 ?l elution buffer AE (Qiagen).
(ii) PCR. GoTaq green master mix (Promega, Madison WI) was used
forallstandardPCRs,accordingtothemanufacturer’srecommendations,
using the primers and protocols summarized in Table S1 in the supple-
mental material.
(iii) Cloning and sequencing. PCR products were cleaned using a
WizardSVgelandPCRproductcleanupsystem(Promega,Madison,WI)
and cloned into the pGEM-T Easy vector system (Promega, Madison,
WI).TransformantswerescreenedforinsertsusingPCRwithM13prim-
ers,andthePCRproductswerecleanedupandsequencedusingaBigDye
Terminator(version3.1)cyclesequencingkit(LifeTechnologyCorpora-
tion, Carlsbad, CA) on an Applied Biosystems 3730xl DNA analyzer (Life
Technology Corporation).
(iv) DNA sequence analysis. All sequences were edited with the
4Peaksprogram(A.GriekspoorandT.Groothuis,TheNetherlandsCan-
cer Institute). The sequences were aligned using ARB software (35). Phy-
logenetic trees were constructed using the neighbor-joining algorithm in
ARB, and parsimony and maximum likelihood methods were performed
usingthePHYLIPprogram(16).Treesconstructedwithallthreemethods
showed the same overall grouping; therefore, only the tree constructed
with the neighbor-joining method has been presented.
(v) FISH. The catalyzed reporter deposition (CARD)-fluorescence in
situ hybridization (FISH) protocol (45, 48) was used with the following
modifications:thehybridizationtemperaturewas46°C,andthefirstwash
was performed at 48°C, followed by an amplification step at 46°C. All
probes(seeTableS2inthesupplementalmaterial)werelabeledattheir5=
ends with horseradish peroxidase and used at a final concentration of 50
ng ?l?1. All filters were counterstained with DAPI (4=,6-diamidino-2-
phenylindole) for total cell counts. Direct microscopic counts were per-
formed by fluorescence microscopy (Zeiss Axiophot HB0100; Carl Zeiss
Inc., North America) at ?1,000 magnification.
?was consumed and then transferred to the light. At the
?andNO3
?concentrations
?/NO3
?concentrations were log transformed and plotted against
?/NO3
?
?/NO3
?production in the
?/NO3
?productionwaslinearforseveraldaysto1week,
French et al.
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Nucleotide sequence accession numbers. All sequences were depos-
ited in GenBank under the numbers JQ669389 to JQ669394.
RESULTS
Enrichment of AOA. AOA were enriched from the sediment of
Lakes Acton (AOA-AC2 and AOA-AC5 cultures) and Delaware
(AOA-DW culture) under autotrophic conditions with NH4
the sole electron donor in the medium. On the basis of the AOA
amoAsequences,allenrichmentculturesbelongtowatercolumn/
sediment group I.1a of the Thaumarchaeota (Fig. 1). The strain in
the AOA-AC2 culture was 81 to 81.7% (amoA) and 92.8 to 93.1%
(16S rRNA gene) identical to the strains in the other two enrich-
ment cultures, while the strains in AOA-AC5 and AOA-DW were
87.1% (amoA) and 97.9% (16S rRNA gene) identical to each
other.TheamoAsequencesofthestraininAOA-DWwere98.2to
98.5% identical to those of clones from the sediment of Lakes
Acton, Delaware, and Pleasant Hill (C. Li and A. Bollmann, un-
published),98.5%identicaltothoseofclonesfromthefreshwater
sediment in the San Francisco Bay (38), and 98.1% identical to
those of clones from a drinking water distribution system in The
Netherlands (59). The amoA sequences of the strain in AOA-AC5
were 99% identical to the amoA sequence of a clone from a paddy
soil in Japan (18). The strain in the third enrichment culture,
AOA-AC2, is closely related to “Ca. Nitrosoarchaeum koreensis”
(99.8% identity for amoA and 99.6% identity for 16S rRNA gene)
?as
and“Ca.Nitrosoarchaeumlimnia”(94.3%identityforamoAand
98.5% identity for the 16S rRNA gene) (5, 27). In contrast to the
strain in AOA-AC2, the strains in AOA-AC5 and AOA-DW were
not closely related to described AOA isolates or strains in enrich-
ment cultures, such as N. maritimus and Nitrososphaera viennen-
sis, among others (70 to 82% identity for amoA and 81 to 93%
identity for the 16S rRNA gene) (Table 1).
CARD-FISH was used to determine the proportion of AOA in
the enrichment cultures at the end of the logarithmic growth
phase.AOA-DWcontained85%AOA,AOA-AC2contained91%
AOA, and AOA-AC5 contained 81% AOA (Table 2). AOB and
nitrite-oxidizingbacteria(NOB)werenotdetected,astestedbyPCR
amplification with AOB-specific 16S rRNA and amoA primers (see
TableS1inthesupplementalmaterial)(resultsnotshown)andFISH
withAOB-andNOB-specific16SrRNAprobes(Table2;seeTableS2
inthesupplementalmaterial).
Influence of NH4
andAOB.Duringstratificationinthesummer,theNH4
tration in Lake Acton increases to up to 400 ?M (41), which falls
within the tested range of NH4
mM NH4
doubled the growth rate of the AOB in AOB-G5-7, while the
growth rates of the AOA in the enrichment cultures decreased or
remained constant (Fig. 2). The growth rate of the AOA in
?concentration on growth rates of AOA
?concen-
?concentrations of 15 ?M and 5
?concentrations up to 1 mM NH4
?. Increasing NH4
?
FIG1 Neighbor-joiningphylogenetictreeoftheAOAinenrichmentculturesbasedonamoAgenesequences(595bp).Bootstrapvaluesof?50of100replicates
are shown at the nodes. SF, San Francisco.
Enrichment and Characterization of Three AOA
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AOA-DW at the lowest NH4
icantlyhigherthanthegrowthrateathigherNH4
(see Table S3 in the supplemental material). The same tendency
was observed for the other two cultures, although the statistical
support was less strong (Fig. 2; see Table S3 in the supplemental
material). The AOA strains in the enrichment cultures exhibited
different tolerances to high NH4
AOA-DWgrewatNH4
AOA-AC2 grew at NH4
strain in AOA-AC5 grew at NH4
(Fig. 2). The lag phase of AOA and AOB differed; the strain in
AOB-G5-7becameactive1to3daysafterinoculationatalltested
NH4
with increasing initial NH4
weeks before logarithmic growth could be detected at NH4
centrationsbetween1mMand5mMNH4
S4 in the supplemental material).
Influence of O2concentration on growth of AOA and AOB.
Lake Acton stratifies during the summer and has an anaerobic
zone as well as a zone with low oxygen availability (1 mg liter?1
O2) (41). We therefore investigated the response of the strains in
our enrichment cultures to 0.5 to 2% O2(calculated) in the head-
space, which corresponded to 0.2 to 0.8 mg liter?1O2in the me-
dium. The growth rate of the AOB in AOB-G5-7 decreased with
decreasing O2concentration, and the growth rates at all different
O2concentrations were significantly different from each other
(Fig. 3; see Table S5 in the supplemental material). The strains in
the AOA enrichment cultures grew at all O2concentrations in the
headspace, with the exception of that in the AOA-AC2 enrich-
ment culture at 0.5% O2. The decrease of the growth rates with
decreasing O2concentration in the AOA cultures was less steep
than the decrease of the growth rates in the AOB enrichment cul-
ture. However, at low O2concentrations the growth rates in the
AOA-AC2 and AOA-AC5 enrichment cultures were significantly
lower than the growth rates at 21% O2(Fig. 3; see Table S5 in the
supplemental material).
InfluenceofpHongrowthofAOAandAOB.Weinvestigated
the growth of the strains in all cultures at pH 6 to 9, the range at
which nonacidophilic ammonia oxidizers grow (31, 32, 33). The
growth rates of the strains in all cultures showed bell-shaped
curves in relation to the pH, with maximum growth rates at pH 7
?concentration (15 ?M) was signif-
?concentrations
?concentrations; the strain in
?concentrationsupto1mM,thestrainin
?concentrations up to 2 mM, and the
?concentrations up to 5 mM
?concentrations,whereasthelagphaseoftheAOAincreased
?concentrations up to more than 2
?con-
?(seeFig.S2andTable
to7.5(Fig.4).ThestrainintheAOA-AC2culturedidnotgrowat
pH 6, while the other AOA strains and the AOB strain did. The
growthratesofthestrainsinAOA-AC5andAOA-DWculturesat
pH 9 were similar to the growth rates at pH 7.5, while the growth
rates of the strains in AOA-AC2 and AOB-G5-7 cultures differed
significantlyfromtheirrespectiveratesatpH7.5(Fig.4;seeTable
S6 in the supplemental material).
Influence of light on growth of AOA and AOB. The investi-
gated intensities represent a range of light, but below light satura-
tion, at which phytoplankton in freshwater systems are able to
grow (51). White light (30 ?mol photons m?2s?1) strongly in-
hibitedthegrowthoftheAOAintheAOA-DWculturebuthadno
effectontheAOBintheAOB-G5-7culture(Fig.5).TheAOAdid
not grow in white light and did not begin to grow after being
transferredfromthelighttothedark.However,growthcontinued
whentheAOAculturesweretransferredfromthedarktothelight.
To get a better insight into which wavelength of light had the
strongest influence on the growth of AOA and AOB, we con-
ducted similar experiments with red (623 ? 3 nm) and blue
(470 ? 5 nm) light. Strains in both cultures grew in the red light,
butwhilethegrowthoftheAOBintheAOB-G5-7culturewasnot
influenced by the red light, the growth rate of the AOA in the
AOA-DWculturewassignificantlylowerintheredlightandafter
transfer from the light to the dark (Fig. 5; see Tables S7 and S8 in
the supplemental material). Blue light at 30 ?mol photons m?2
s?1had the strongest effect on the growth of strains in both cul-
tures (Fig. 5). In the blue light, strains did not grow in any of the
cultures, and growth of the AOA in the AOA-DW culture did not
recover after transfer from the light to the dark. In contrast, the
AOBintheAOB-G5-7culturerecoveredimmediatelyaftertrans-
ferfromthelighttothedark,butthegrowthratewassignificantly
lowerthanthegrowthrateinthecontinuousdark(seeTableS7in
thesupplementalmaterial).Transferoftheculturesfromthedark
into blue light stopped growth immediately. Strains in both cul-
turesgrewinthelessintensebluelight(3?molphotonsm?2s?1),
but the growth rate of strain in the AOA-DW culture was signifi-
cantlylowerinthelowbluelightthaninthedark(Fig.5;seeTable
S8 in the supplemental material).
DISCUSSION
Enrichment of AOA cultures AOA-DW, AOA-AC2, and AOA-
AC5.Weenrichedandcharacterizedthegrowthofthreedifferent
freshwater AOA belonging to thaumarchaeal group I.1a within
the newly described phylum Thaumarchaeota (10, 52). The AOA
in one of the cultures, AOA-AC2, is closely related to “Ca. Nitro-
soarchaeumkoreensis,”whilethestrainsintheothertwocultures,
AOA-AC5 and AOA-DW, are only 70 to 82% (amoA) and 81 to
93% (16S rRNA gene) identical to other cultivated isolates and
TABLE 2 Quantitative analysis of compositions of AOA-AC2, AOA-
AC5, and AOA-DW enrichment cultures
Organism
Composition (%)a
AOA-AC2AOA-AC5AOA-DW
Crenarchaeota
Bacteria
Nitrospira (NOB)
AOB
91.0
9.5
ND
ND
81.2
3.3
ND
ND
85.4
9.2
ND
ND
aThe cell numbers were determined using CARD-FISH (percentage of DAPI counts)
(n ? 1). Samples were taken at the end of the logarithmic phase. ND, not detected.
TABLE 1 Identities of AOA in the AOA-AC2, AOA-AC5, and AOA-
DW enrichment cultures in comparison with previously cultivated AOA
Species (reference)
% identitya
AOA-AC2AOA-AC5AOA-DW
amoA
16S
rRNAamoA
16S
rRNAamoA
16S
rRNA
Nitrosopumilus maritimus (30)
Nitrososphaera viennensis (56)
“Ca. Nitrososphaera gargensis”
(20)
“Ca. Nitrosocaldus yellowstonii”
(15)
“Ca. Nitrosoarchaeum limnia” (5)
“Ca. Nitrosoarchaeum koreensis”
(27)
“Ca. Nitrosotalea devanaterra”
(34)
88.6
69.6
70.9
96.2
82.7
81.9
79.8
70.4
72.7
92.9
83.7
82.7
78.8
71.1
72.1
92.9
83.7
82.2
71.180.471.181.870.081.0
94.3
99.8
98.4
99.6
81.9
81.6
92.6
92.9
81.5
81.2
92.9
92.8
77.9 88.576.7 89.776.189.3
aComparisons are based on 16S rRNA genes (794 bp; corresponding to 109 to 915 in
Escherichia coli numbering) and amoA genes (595 bp).
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enrichmentculturestrains,suchasN.maritimusandNitrosospha-
era viennensis (Table 1). This finding indicates that the AOA in
these two enriched AOA cultures belong to a new genus of the
ammonia-oxidizing Thaumarchaeota, assuming that the identity
between the two genera is, on average, 96.4% on the basis of the
16S rRNA gene sequence (66). This new genus/group includes
many ribotypes from non-salt water systems, such as freshwater
systems (38; Li and Bollmann, unpublished) and drinking water
systems (59), as well as soil and hot spring environments (67), as
indicated by highly identical clones (Fig. 1).
Purecultures.Inthisstudy,nopureculturesoftheAOAwere
obtained. It is safe to assume that the heterotrophic satellite com-
munity is providing some compound that enabled the AOA to
grow in the enrichment culture. Similar observations have been
made with other AOA as well as with AOB. Potential compounds
that positively influence the growth of AOA could be small or-
ganic compounds such as pyruvate, which improved growth and
enabled isolation of N. viennensis (56). However, the addition of
pyruvate during serial dilution has not led to isolation of any of
these strains to date, indicating that different compounds might
beimportantfordifferentAOA.Furtherresearchwillbenecessary
to elucidate the interactions between AOA (and AOB) and the
heterotrophicsatellitebacteriainammonia-oxidizingenrichment
cultures.
Growth of AOA and AOB. Overall, the growth experiments
showedthatthegrowthratesoftheAOAwerealmostalwayslower
than the growth rates of the AOB. All our experiments have been
conducted under strict chemolithoautotrophic conditions. The
resultsindicatethatthestraininAOB-G5-7hadanadvantageover
the strains in the three tested AOA cultures under the conditions
investigated.Innature,however,conditionsareoftenlessdefined
withrespecttoenergy-generatingprocesses.Ithasbeensuggested
thatnotallThaumarchaeotaarechemolithoautotrophicammonia
oxidizers;somecarrytheamoAgenebutarenotactivelyoxidizing
NH4
inpureandenrichmentcultures(39,56,65).Onthebasisofthese
observations and our data, one could speculate that AOA in nat-
ural samples utilize a mixotrophic and/or heterotrophic lifestyle
ratherthanacompletelyautotrophiclifestyle,whichcouldexplain
their success in nature compared to that in the laboratory.
Increasing NH4
the growth rates and lag phases of AOA and AOB, with AOB
growingfasterandhavingshorterlagphasesthanAOA(Fig.2;see
Fig. S2 and Tables S3 and S4 in the supplemental material). After
?, and others utilize mixotrophic or heterotrophic lifestyles
?concentrations had different influences on
FIG 2 Influence of NH4
SD; n ? 3). NH4
?concentration on the growth rates of the strains in enrichment cultures AOA-AC2, AOA-AC5, AOA-DW, and AOB-G5-7 (mean ?
?concentrations are shown on a linear scale (A) and on a logarithmic scale (B).
FIG 3 Influence of the calculated O2concentration in the headspace of the
bottle on the growth rates of the strains in enrichment cultures AOA-AC2,
AOA-AC5, AOA-DW, and AOB-G5-7 (mean ? SD; n ? 3).
FIG4 InfluenceofthepHofthemediumonthegrowthratesofthestrainsin
enrichment cultures AOA-AC2, AOA-AC5, AOA-DW, and AOB-G5-7
(mean ? SD; n ? 3).
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comparing these results with data provided by other studies that
determined the Kmof AOA for NH3/NH4
1,000timeslowerthantheKmofAOB(27,36,43),wesuggestthat
AOBhaveanadvantageoverAOAathigherNH4
(?10?M).Thisassumptionissupportedbythedetectionofhigh
abundancesofAOBinenvironmentswithhigherNH4
tofertilizationandotherprocesses,whileAOAaremoreabundant
in low-NH4
The AOA in enrichment cultures AOA-DW and AOA-AC5
showed lower tolerance to high NH4
strain in the AOA-AC2 culture, with the highest concentrations
supporting growth at 1 mM NH4
(AOA-AC5).Theseconcentrationsarelowerthanthehighesttol-
erances toward NH4
Nitrosoarchaeumkoreensis”(10mM),andthestrainintheAOA-
AC2 enrichment culture (5 mM), which is closely related to “Ca.
Nitrosoarchaeum koreensis” (27, 56). These results indicate that
the strains in the AOA-DW and AOA-AC5 cultures are less toler-
ant to high NH4
isolates in other enrichment cultures. Similar observations have
been made for AOB; members of the Nitrosomonas oligotropha
cluster, which are also commonly found in freshwater environ-
ments, are less tolerant to high NH4
adapted to low NH4
?to be approximately
?concentrations
?inputdue
?and unfertilized environments (17, 21, 24, 62, 64).
?concentrations than the
?(AOA-DW) and 2 mM NH4
?
?observed for N. viennensis (15 mM), “Ca.
?concentrations than other AOA isolates and
?concentrations and better
?concentrations, while members of the Ni-
trosomonas europaea/N. eutropha cluster are primarily found in
environments with high NH4
AOA and AOB responded differently when cultured over a
range of O2concentrations. The strains in the AOA-AC5 and
AOA-DWculturesgrewatalltestedO2concentrationsatthesame
rate,whilethestrainintheAOA-AC2culturedidnotgrowat0.5%
O2, and the growth rate of the strain in the AOB-G5-7 culture
decreased with decreasing O2concentrations (Fig. 3; see Table S5
in the supplemental material). Environmental surveys often de-
tected AOA at the oxic-anoxic interface (4, 13, 17, 47), indicating
anadaptationtolowoxygenconditions.ThelowKmforO2found
for N. maritimus as well as other AOA (27, 36, 43) and the envi-
ronmental data support the hypothesis that AOA are very likely
better adapted to low O2than AOB and may therefore have a
competitiveadvantageattheoxic-anoxicinterface,whileAOBare
active under more aerobic conditions.
AOAandAOBgrewatmostofthetestedpHvalues,withAOA
growing at almost the same rate over a wide pH range and AOB
showing a more bell-shaped curve, with the highest growth rate
occurring at pH 7 to 7.5 (Fig. 4; see Table S6 in the supplemental
material). AOA are found over a wide pH range in different envi-
ronments, such as soils and hot springs (15, 19, 21, 40, 46), but
most cultivated AOA, such as N. maritimus, N. viennensis, “Ca.
Nitrosoarchaeumkoreensis,”and“Ca.Nitrosotaleadevanaterra,”
?concentrations (6, 7, 31, 32, 33).
FIG 5 Influence of white, red, and blue light with an intensity of 30 ?mol photons m?2s?1and blue light with an intensity of 3 ?mol photons m?2s?1on the
growth rates of the strains in enrichment cultures AOA-DW and AOB-G5-7 (mean ? SD; n ? 3).
French et al.
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have rather narrow pH ranges for growth and activity compared
with the AOA strains tested in enrichment cultures (27, 30,
56, 58).
The strain in AOB-G5-7 was more tolerant to light than the
straininAOA-DWandalsorecoveredfasterafterexposure,while
the strain in AOA-DW did not fully recover from light exposure
(Fig. 5; see Tables S7 and S8 in the supplemental material). In the
environment, maximum numbers of thaumarchaeal amoA and
16S rRNA copies have been detected at levels below where photo-
syntheticallyactiveradiation(PAR)inthewatercolumndropped
to 0, indicating that no light was penetrating to this depth (4, 47).
Inthesamestudy,AOBandAOAweredetectedinlowabundance
in more shallow waters of the Pacific, indicating that AOB as well
as some AOA strains could be more tolerant to light than those
thatarethemostabundantinthelowerpartsofthewatercolumn
(47). The light response of AOA and AOB could be due to differ-
ences in the reaction of the copper-containing enzymes to light.
AOBareverysensitivetobluenear-UVlight(23,50).Theauthors
discussedthatthisinhibitioncouldbeattributedtotheabsorption
of light by the oxygenated state of the copper-containing ammo-
nia monooxygenase, which leads to inactivation of the enzyme
(50). Genome studies of AOA showed a large number of copper-
containing enzymes such as multicopper oxidases and blue cop-
per proteins (5, 63), suggesting that some of the copper-contain-
ing enzymes in AOA could be sensitive to light as well, leading to
inhibition of overall metabolism in AOA by light. During the
preparation of the manuscript, Merbt et al. (2012) published a
study investigating the response of two AOB (Nitrosomonas euro-
paea and Nitrosospira multiformis) and two AOA (N. maritimus
and“Ca.Nitrosotaleadevanaterra”)towhitelight(37).Thestudy
confirmed our findings.
Conclusion.TheresultsofthisstudyshowthatAOBareableto
outcompete AOA under almost all conditions tested. These find-
ings are in accordance with those of other cultivation-based stud-
ies,aswellasobservationsmadeintheenvironmentusingmolec-
ular approaches. Further investigation must be done using other
cultivation-based experiments, such as continuous cultures,
which enable us to cultivate AOA and AOB under more strin-
gently controlled conditions, and in situ incubations, which en-
able us to investigate the response of AOA and AOB to environ-
mental changes under conditions which allow AOA and AOB to
utilizemetabolicfunctionsastheywouldnaturallyintheenviron-
ment.
ACKNOWLEDGMENTS
We thank Annika Mosier (University of California, Berkeley) for helpful
discussionsatthebeginningoftheproject,MichaelVanniandBethMette
(Department of Zoology, Miami University) for support with sampling,
Lynn Johnson (Instrumentation Laboratory, Miami University) for con-
struction of the light installations, Anne Bernhard (Connecticut College,
New London, CT) for providing her AOA amoA ARB alignment file, and
Anne Morris Hooke (Department of Microbiology, Miami University)
for critical reading of the manuscript.
ThisworkwassupportedbystartupfundsofMiamiUniversityandby
National Science Foundation grants DEB-1120443 to A.B. and OCE-
0927277 to G.B.
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