urakawa, hidetoshi, willm martens-habbena, carme huguet, jose r. de la...

Ammonia availability shapes the seasonal distribution and activity of archaeal and

bacterial ammonia oxidizers in the Puget Sound Estuary

Hidetoshi Urakawa,1,a,* Willm Martens-Habbena,1 Carme Huguet,2,b Jose R. de la Torre,1,c

Anitra E. Ingalls,2 Allan H. Devol,2 and David A. Stahl 1

1 Department of Civil and Environmental Engineering, University of Washington, Seattle, Washington2 School of Oceanography, University of Washington, Seattle, Washington

Abstract

A combination of molecular and activity measures was used to explore seasonal patterns of distribution,activity, and diversity of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) inrelationship to ammonia availability in Hood Canal, a fjord within the Puget Sound, Washington State estuarysystem. A greater contribution of AOA to nitrification, relative to AOB, was supported by complementaryquantification of transcripts of a gene (amoA) coding for one subunit of the ammonia monooxygenase,quantification of intact glycerol dialkyl glycerol tetraether lipids, and kinetic modeling. High AOA : AOB ratiosand nitrification rates measured by 15NHz4 dilution technique were associated with low concentrations ofammonia, whereas transient increases in AOB were most closely associated with regionally and seasonallyelevated concentrations of ammonia in the water column. Additionally, single cell nitrification rates andtranscript numbers were calculated for AOA and AOB populations. Together these observations offer additionalecological support for the idea that niche separation of AOA and AOB is in part determined by ammoniaconcentrations in estuarine ecosystems.

The oxidation of ammonia to nitrite by autotrophicmicroorganisms was long thought to be mediated by a fewrestricted groups of bacteria belonging to the Beta- andGammaproteobacteria. The discovery and high abundanceof ammonia-oxidizing archaea (AOA) in pelagic (Mincer etal. 2007; Beman et al. 2008), estuarine sediment (Mosierand Francis 2008), freshwater (Herrmann et al. 2009), soil(Leininger et al. 2006), hot springs (de la Torre et al. 2008),and engineered systems (Urakawa et al. 2008) haveprompted a reevaluation of microbial controls on thenitrogen cycle that will require a deeper understanding ofthe ecophysiology of AOA and their relationship to thebetter characterized ammonia-oxidizing bacteria (AOB;Prosser and Nicol 2008; Urakawa et al. 2011). The isolationand description of the first AOA, Nitrosopumilus maritimusstrain SCM1 (Könneke et al. 2005), and the demonstrationthat its genome sequence shares highly similar gene contentand gene organization with marine planktonic archaeaoffered a framework for combined physiological environ-mental studies (Walker et al. 2010; Urakawa et al. 2011).Physiological characterization of SCM1 revealed anextremely high affinity for ammonia. This organism growsexponentially until total ammonia is depleted to below10 nmol L21 (Martens-Habbena et al. 2009). In turn, theextremely high-ammonia affinity demonstrated in culture

was consistent with the observed high abundance of marinegroup I archaea in nutrient-poor pelagic waters (Karneret al. 2001). However, there have been only limited studiesdirectly associating AOA with nitrification in the marineenvironment.

We previously reported exceptionally high numbers ofAOA in Hood Canal, a fjord in the Puget Sound estuarysystem (Urakawa et al. 2010; Horak et al. 2013). Theirnumbers, as inferred from amoA gene copy number,exceeded 105 cells mL21 in water column samples takenin May (Horak et al. 2013) and July (Urakawa et al. 2010).This fjord has a shallow sill near its mouth (55 m) anddepths exceeding 150 m throughout much of its length. It isa highly dynamic system as a result of seasonal shifts infreshwater input and illumination as well as the replace-ment of bottom waters near the end of summer with densenitrate-rich, acidic, waters upwelled along the coast andentering the Puget Sound estuary through the Strait of Juande Fuca and Admiralty Inlet (Feely et al. 2010). The fjordexperiences pronounced seasonal stratification, hypoxia,and acidification as a result of poor ventilation and theseasonal intrusion of water from coastal upwelling thatcontributes to high primary production (3000 mg C m22 d21;Newton et al. 2002). Thus, Hood Canal waters arecharacteristically of much lower pH than are most openmarine systems, ranging from a pH of 7.3 to 7.9 in 2008(Feely et al. 2010). Because increasing stratification andacidification are predicted consequences of climate change(Feely et al. 2008), this economically important marineecosystem is part of a long-term monitoring system(associated with the Northwest Association of NetworkedOcean Observing Systems, http://www.nanoos.org). Weanticipate that this fjord will provide a valuable model toresolve the relative contribution of AOA and AOB to

* Corresponding author: [email protected]

Present addresses:a Florida Gulf Coast University, Department of Marine and

Ecological Sciences, Fort Myers, Floridab Institute of Environmental Science and Technology, Auton-

omous University of Barcelona, Cerdanyola, Spainc San Francisco State University, Department of Biology, San

Francisco, California

Limnol. Oceanogr., 59(4), 2014, 1321–1335

E 2014, by the Association for the Sciences of Limnology and Oceanography, Inc.doi:10.4319/lo.2014.59.4.1321

1321

nitrification and the environmental variables controllingtheir activities (Bouskill et al. 2012). It also serves as amodel for the study of ocean acidification and the capacityof natural systems to respond to gradual acidificationthrough adaptive evolution (Lohbeck et al. 2012).

Archaea may often be more significant contributors tonitrification than the better-described bacterial ammoniaoxidizers in pelagic waters (Beman et al. 2008; Martens-Habbena et al. 2009; Santoro et al. 2010). However, theircontribution to nitrification in estuaries has been poorlycharacterized where significant AOB populations are oftenco-resident (Urakawa et al. 2010; Bouskill et al. 2012;Horak et al. 2013). Thus, the relative contribution of AOAand AOB to ammonia oxidation in these and similarcoastal settings remains unclear. In order to betterconstrain niche separation of AOA and AOB, we herepresent complementary molecular and membrane lipidanalyses along with kinetic modeling that together indicatethat the greater part of nitrification in this estuary iscontrolled by AOA, whereas AOB were most closelyassociated with regional and seasonally elevated concen-trations of ammonia in the water column. Together theseobservations offer additional ecological support for theidea that niche separation of AOA and AOB is in partdetermined by ammonia concentration.

Methods

Site description—Hood Canal is a long and narrow fjord(96 km in length) located in the Puget Sound estuary system,which consists of three interconnected basins separated bysills (Fig. 1). This fjord reaches a depth of 180 m and isseparated from the main basin of Puget Sound and the deepwaters of the Pacific Ocean by a broad shallow sill (55 mdepth). The water circulation is mainly controlled by asalinity-driven density gradient created by two layerexchange flows. The outgoing surface water mainly consistsof riverine inflow, and the deep incoming water consists ofsalty nitrate-rich water from Admiralty Inlet. Similar toother fjords, the shallow moraine sill partially closes offdeep-water ventilation. Poor ventilation coupled with highproductivity contributes to seasonal oxygen depletion(hypoxia) of the bottom waters, often adversely affectingthe animal communities (Newton et al. 2002; Keister andTuttle 2013). Hood Canal has been the site of ongoing stateand federal studies in which four buoys (ocean remotechemical–optical analyzer) record vertical water columnprofiles daily for long-term observations. We selected threehydrologically distinct stations containing two of the threePuget Sound Regional Synthesis Model (PRISM) stations:(1) PRISM 10 (P10; 47.91 N, 122.62 W) of approximately55 m depth near the sill, (2) PRISM 12 (P12; 47.42 N,123.11 W) in the fjord interior of approximately 150 mdepth, and (3) Lynch Cove (LC; 47.37 N, 123.01 W), a highlyproductive shallow (15 m) embayment at the terminus of thefjord receiving direct riverine input and influenced bysediment resuspension through tidal disturbance.

Water and sediment sampling and determination ofwater chemistry—A Seabird conductivity–temperature–

depth rosette equipped with 10 liter Niskin samplingbottles, a fluorometer, and an oxygen sensor deployedfrom the R/V Clifford A. Barnes (University of Washing-ton, School of Oceanography) was used to obtain verticalprofiles of pressure, salinity, temperature, chlorophyll a(Chl a), oxygen, and water samples (six depths) duringAugust, October, and December 2008 and May 2009. Theconcentrations of nitrogen species (ammonia, nitrite, andnitrate) were determined colorimetrically as describedpreviously (Grasshoff et al. 1999; Holmes et al. 1999;Zhang and Fischer 2006). Cells for deoxyribonucleic acid(DNA) extraction were collected by filtering 1 to 3 liters ofseawater through 0.22 mm pore size Sterivex cartridge filters(Millipore). Glycerol dialkyl glycerol tetraether (GDGT)membrane lipid content was determined for differentparticle size fractions by filtering 40 liters of seawaterthrough three filters in a series, as follows: a 60 mm poresize nylon mesh filter, a 0.7 mm glass-fiber filter, and a0.22 mm pore size Sterivex cartridge filter, as describedpreviously (Ingalls et al. 2012). All samples were stored inshipboard liquid nitrogen and at 280uC in our laborato-ries. Prior to this series of cruises, water column samplesand surface sediment samples (0–3 cm) were collected fromthree sites in Hood Canal (P10, P12, and PRISM 402[P402; 47.35 N, 123.02 W]) in August 2007 (Fig. 1) andused for construction of amoA gene sequence clone libraries(Table 1).

Fig 1. Map of Hood Canal and field stations (P10, P12, andLC) used in this study. P402 was only used for clone libraryconstructions prior to the seasonal study.

1322 Urakawa et al.

Quantification of AOA and AOB—DNA was extractedby a modified phenol–chloroform extraction method, asdescribed previously (Urakawa et al. 2010). Copy numbersof the bacterial and archaeal amoA gene were determinedusing the LightCycler FastStart DNA Master SYBR GreenI kit (Roche) and capillary system (LightCycler, Roche),as described previously (Martens-Habbena et al. 2009;Urakawa et al. 2010). Briefly, the amoA-1F and amoA-2Rprimer set was used to amplify a region of the bacterialamoA genes (Rotthauwe et al. 1997). The standard curveswere generated using genomic DNA of Nitrosomonaseuropaea ATCC19718 in a dilution series of 102–107 genecopies. The archaeal amoA was quantified using theCrenAmoAQ-F and CrenAmoAModR primer set (Minceret al. 2007). The standard curve was generated usingNitrosopumilus maritimus SCM1 genomic DNA in adilution series of 101–106 copies of the amoA gene. Datawere analyzed with the second derivative maximum methodusing the Light Cycler Software (version 3.5.3; Roche). Theoverall average quantitative polymerase chain reaction(qPCR) efficiency was 84.1% (R2 5 0.99).

Phylogenetic analysis of AOA and AOB—DNA wasextracted using a FastDNA SPIN Kit for Soil (MPBiomedicals) according to the manufacturer’s instructions.Archaeal and bacterial amoA genes were amplified asdescribed previously (Rotthauwe et al. 1997; Francis et al.2005). PCR products (594 base pairs [bp] for AOA, 450 bpfor AOB) were cloned into the TOPO TA Cloning kitvector (Invitrogen) and sequenced at the University ofWashington sequencing facility. Environmental clonesequences were aligned with amoA gene sequences retrievedfrom GenBank, and neighbor-joining phylogenetic treeswere constructed with the Jukes–Cantor correction usingthe ARB software package. Operational taxonomic unitsand diversity indices (Shannon and Simpson) were deter-mined using the furthest neighbor algorithm in DOTUR

(Schloss and Handelsman 2005). Good’s coverage estimateof libraries was calculated from the equation C 5 1 2 (n1 3N21), where n1 is the number of clones that occurred onlyonce, and N is the total number of environmental clonesexamined. DNA sequences were deposited in GenBankunder accession numbers AB828780 to AB828967 forarchaeal amoA genes and numbers AB828968 toAB829111 for bacterial amoA genes.

Transcript analysis of the amoA gene—Six samples fromour data set were selected for transcript analysis. We choseone deep-water sample from each time point sampled atP12 and two samples from Lynch Cove. These sampleswere selected based on the model prediction of nitrificationcontribution rates at which high archaeal and bacterialactivities were observed. Sterivex filters were cut in half andthe individual halves inserted singly into bead-beating tubes(lysing matrix E tube; MP Biomedicals), as describedpreviously (Urakawa et al. 2010), followed by addition of0.3 mL of 1 3 ribonucleic acid (RNA) extraction buffer(50 m mol L21 sodium acetate, 10 m mol L21 ethylenedi-aminetetraacetic acid, pH to 5.2 with acetic acid), 0.34 mLof low-pH phenol saturated with 0.1 mol L21 citrate buffer(pH 4.3), and 40 mL of 10% sodium dodecyl sulfate. Cellswere disrupted at speed 6.0 in the bead beater (FastPrepFP120 instrument; MP Biomedicals) for 40 s. After beadbeating, the tube was centrifuged at 16,000 3 g for 5 min;the aqueous phase was then transferred to a 2.0 mL PhaseLock Gel tube (Eppendorf), and an equal amount ofphenol–chloroform–isoamyl alcohol 25 : 24 : 1 was added.The tube was mixed thoroughly by repeated gentleinversion and centrifuged at 16,000 3 g for 5 min. Theaqueous phase was again transferred to a 2.0 mL PhaseLock Gel tube and an equal amount of chloroform wasadded. The contents were again mixed thoroughly byrepeated inversion and the tube was centrifuged at 16,000 3g for 5 min. The supernatant was transferred to a new 2.0 mLmicrocentrifuge tube and 4 mol L21 lithium chloride wasadded to arrive at a final concentration of 0.8 mol L21; thisstep was followed by addition of an equal volume of ice-cold100% isopropanol and 3 mL of GlycoBlue. After mixing, thetubes were held at 280uC for 30 min and RNA wasrecovered by centrifugation at 16,000 3 g for 15 min at 4uC.Supernatant was carefully removed from the pelleted RNAand the pellet gently rinsed with 1 mL of ice-cold 75%ethanol to remove residual salts. After decanting the ethanoland eliminating the remaining ethanol by air-drying, 90 mLof water was added to the dried RNA sample. Theresuspended RNA was then twice digested with DNase(DNA-free-kit, Ambion), following the manufacturer’sprotocol, and complementary DNA (cDNA) was generatedusing the Omniscript Reverse Transcription (RT) Kit(Qiagen) according to the manufacturer’s specifications.The cDNA products were then treated with RNase T1(Ambion) and quantified with SYBR Green I prior to beingused for qPCR analysis, as described previously (Urakawaet al. 2010). An identical set of reactions, minus the RTreactions, were performed for each RNA extract. Thesedigestions confirmed that the carryover of genomic DNAwas negligible.

Table 1. Number of archaeal and bacterial amoA clones fromeach site and depth.

Site and depth (m)

No. of clones

AOA AOB

P10

5 16 1020 16 1250 16 11Sediment 16 14

P12

5 16 820 15 1250 16 13

123 16 10Sediment 16 15

P402

5 16 1550 16 12Sediment 13 12

Total 188 144

Estuary nitrifying microorganisms 1323

Sample preparation for GDGT analysis—The six samplesexamined for transcript abundance were also characterizedby lipid analysis. In a previous study, a variety of GDGTlipid extraction methods were carefully compared withvarious environmental samples and an active culture of N.maritimus (Huguet et al. 2010a). Optimized core and intactGDGT lipid extraction methods employed in a previousstudy (Huguet et al. 2010b) were used in the present study.Briefly, filters were extracted by sonicating three times in abath at 25uC for 5 min each with methanol (MeOH), 1 : 1(v : v) of MeOH and dichloromethane (DCM), and, lastly,with pure DCM. After each sonication, samples werecentrifuged at 1000 3 g for 5 min to remove particles. Thesupernatants were combined and the solvent evaporatedwith a TurboVap (Caliper LifeSciences). Total lipidextracts were dried over a small pipette filled withanhydrous sodium sulfate. The total lipid extract was thendivided into polar and non-polar fractions using a glasspipette column filled with activated silica gel and sequentialelution with hexane-DCM (9 : 1 v : v) and DCM-MeOH(1 : 1, v : v), respectively. One half of the polar fraction wasretained for core GDGT measurements, and the other halfof the sample was hydrolyzed by adding 5% hydrochloricacid in MeOH (v : v) for 4 h at 70uC to measure total GDGTsin the sample (core + intact GDGTs resulting fromhydrolysis). Vials were flushed with N2 and capped. DCMand MilliQ were added and the DCM fraction was collected.This was repeated four times. The DCM fraction was rinsedsix times with MilliQ water in order to remove acid. The0.2 mm Sterivex filters that had been solvent extracted werealso hydrolyzed in order to fully extract the intact lipids thatmay have remained on the filter (Huguet et al. 2010a). Thefinal fractions were dried and a C46 internal standard wasadded (Huguet et al. 2006). The samples were thenredissolved by sonication (5 min) in hexane : propanol(99% : 1% v : v) and filtered through 0.45 mm polytetraflu-oroethylene filters prior to analysis by high-performanceliquid chromatography–mass spectrometry.

Lipid analysis with atmospheric pressure chemicalionization (APCI)—Core GDGTs were analyzed accord-ing to the procedure described by Hopmans et al. (2000)and Schouten et al. (2007). Analyses were performed intriplicate with an Agilent 1100 Series Liquid Chromato-graph coupled to an Agilent ion trap (XCT) massspectrometer equipped with an auto-injector and Chem-Station chromatography manager software. Separation wasachieved on a Prevail Cyano column (2.1 3 150 mm, 3 mm;Alltech, Deerfield) that was fitted with a Prevail Cyanoguard cartridge (7.5 3 4.6 mm, 5 mm) maintained at 30uC.GDGTs were eluted isocratically, first with hexane : iso-propanol (99% : 1%; v : v) for 5 min and then using a lineargradient of up to 1.8% volume of isopropanol over 45 min.The flow rate was 0.2 mL min21. After each analysis thecolumn was cleaned by back-flushing hexane : isopropanol(90% : 10%; v : v) at 0.2 mL min21 for 8 min and thenequilibrated back to initial conditions for 10 min. Detectionwas achieved using APCI of the eluent with the followingconditions; nebulizer pressure, 60 psi; vaporizer tempera-ture, 400uC; N2 drying gas flow, 6 L min21 at 200uC;

capillary voltage, 23 kV; and corona, 5 mA (, 3.2 kV). Thescanning range was 730–1350 m/z and the target mass was1300 m/z. The C46 GDGT internal standard, as describedby Huguet and colleagues (2006), was used to determineabsolute abundances.

Nitrification rate measurement—Nitrification rates (asseparate measurements of ammonia and nitrite oxidation)were determined by the 15NHz4 isotope tracer methodfollowing the gas chromatography–mass spectrometrymethod reported previously (Horak et al. 2013). Nitrifica-tion rates were measured at three depths (near surface, mid-depth, and near bottom), with specific depths varyingamong stations. Nitrification rates were determined byfollowing accumulation of 15N label in the combined nitriteand nitrate pool. Briefly, a 10 mL water sample was mixedwith 100 mL of 1 mol L21 imidazole buffer (final pH 9.0)and 1 g of HCl-washed wet metallic cadmium powder.Samples were incubated at room temperature at 200revolutions per minute on a rotary shaker for at least 12 huntil nitrate was completely converted to nitrite. Completeconversion was confirmed by determining final nitriteconcentrations. Subsequently, subsamples containing10 nmol of nitrite were diluted with MilliQ water to a finalvolume of 10 mL, placed in septum vials, and sealed.Nitrite was converted to N2O by addition of 1 mL offreshly prepared and N2-purged solution of 0.13 g mL21

sodium azide in 10% acetic acid. 14N and 15N isotopedistributions were subsequently analyzed on a ThermoFinnigan Delta 253 Isotope ratio mass spectrometer usingnitrate and N2O standards. Nitrification rates weremodeled from the change of h15N (NO{2 + NO

{3 ) at the

beginning and end of the incubations.

Parameters for kinetic model development—Modeledspecific growth rates (m d21) for N. maritimus, Nitrosomo-nas oligotropha, N. europaea, and Nitrosococcus oceani werecalculated in the range of ammonium concentrationsbetween 1 nmol L21 and 10 mmol L21 using the equation

m~(m max|½S�)|(Kmz½S�){1

Here, m is specific growth rate (d21), mmax is maximumgrowth rate (d21), Km is half-saturation constant forammonium oxidation (mmol L21), and [S] is concentrationof ammonium (mmol L21).

Single cell nitrification activity (fmol cell21 d21) wascalculated using ammonium oxidation activity (mmol N mgprotein21 d21) and single cell protein content (fg cell21): N.maritimus (10.2 fg cell21) and N. oligotropha (126.8 fgcell21). The nitrification contribution ratio for AOA andAOB was determined for each sample using in situammonium concentrations (mmol L21), single cell nitrifi-cation rates (fmol cell21 d21) for N. maritimus and N.oligotropha, and gene copy numbers (copies L21) deter-mined in Hood Canal. The modeled nitrification rate forAOA and AOB was obtained from in situ nitrification ratesdetermined by the 15NHz4 pool dilution technique and thenitrification contribution ratio and was then used tocalculate single cell nitrification rates (fmol cell21 d21).

1324 Urakawa et al.

Results

Diversity and phylogeny of archaeal and bacterialamoA genes—A total of 188 archaeal amoA clones and144 bacterial amoA clones were analyzed in a samplepreviously collected in August 2007 (Table 1). Using anoperational taxonomic unit (OTU) level of 2%, the samenumber of OTUs (5 16) was found in archaeal andbacterial water column libraries. In contrast, the bacterialsediment libraries harbored a larger number of OTUs(5 11) than did the corresponding archaeal libraries (5 9;Table 2). The number of singletons (, 2% level) rangedfrom three (AOB sediment libraries) to nine (AOB watercolumn libraries). The library coverage (Good’s coverage)was the highest in archaeal water column libraries (5 0.89)and the lowest in bacterial sediment libraries (5 0.73). Boththe Shannon diversity index and the reciprocal Simpsonindex provided support for a greater AOA diversity in thewater column than for the AOB. In contrast, the diversity ofthe sediment AOB population was greater than that of thecorresponding AOA population. The archaeal amoA genesequences resolved into three clusters (Fig. 2A), while themajority of bacterial amoA sequences were affiliated with theNitrosospira-like (cluster 1) (Fig. 2B). The Nitrosomonas sp.Nm143 lineage was the second largest assemblage, compris-ing about 15% of the sequenced bacterial amoA clones. Fourclones (2.8% of total clones) were affiliated with the N.marina–Nitrosomonas aestuarii group.

Abundance and depth distribution of archaeal andbacterial amoA genes—The abundance and depth distri-bution of archaeal and bacterial amoA genes wereinvestigated seasonally at three hydrologically distinct fieldstations in Hood Canal (Fig. 1). Sta. P10 is on the sill andfrequently mixed, P12 is deep and highly stratified, and LCis shallow and has constantly high NHz4 below the mixedlayer (Fig. 3). The lowest and the highest sea surfacetemperature throughout the cruises were both recorded atLC and ranged from 8.7uC (December) to 20.6uC (August).The temperature and salinity below the pycnocline at P12were constant throughout the year and ranged from 8.4uCto 10.6uC (mean 6 standard deviation [SD] 5 9.3uC 60.8uC, n 5 24) and 30.2 to 30.7 (mean 6 SD 5 30.3 6 0.2,n 5 24), respectively.

Archaeal amoA gene copy numbers generally greatlyoutnumbered bacterial amoA numbers at all depths in thewater column and demonstrated a seasonal pattern

(Fig. 4A). The abundance of AOA varied over three ordersof magnitude (103–106 cells mL21), while fewer AOB variedover approximately 2 orders of magnitude (103–105 cellsmL21). Among the three stations, archaeal amoA gene copynumbers were highest at P12 (approximately 150 m depth),ranging from 104 to 106 copy mL21. The gene copynumbers corresponded to stratification patterns (Fig. 3).AOA numbers were low in the surface water, increasednear the pycnocline, and were high and constant below thepycnocline at P12 (Fig. 4A). Both archaeal and bacterialgene copy numbers reached the highest values in May andAugust and decreased in October and December (Fig. 4A).A nitrite maximum around 10 m deep was only detected inMay and August (Fig. 3). AOA numbers measured at P10(on the sill) appeared to be strongly influenced by theannual water exchange cycle. Here archaeal gene copynumbers were not high during the spring and summer butwere the highest in the winter. The occurrence of this highnumber of AOA in December can be explained by theseasonal outgoing water flow from the south of HoodCanal (i.e., P12) to Admiralty Inlet, which includes a highnumber of AOA and a high nitrate concentration but ischaracterized by low Chl a, ammonium, and nitriteconcentrations (Fig. 3). The numbers of archaeal genecopies at LC were low in August and October and high inDecember and May. Thus, there were pronounced differ-ences in the seasonal population dynamics at each of thethree stations.

AOA : AOB ratio in relationship to ammonium concen-tration—There was a low correlation between absolutenumbers of AOA or AOB and ammonium concentrationdetermined at the time of sampling ( p . 0.05). In contrast,the AOA : AOB ratio correlated with ammonium concentra-tion and varied over three orders of magnitude (Fig. 4B).The AOA : AOB ratio at Sta. P12 ranged between 0.84 and70.0 (26.8 6 4.3 [mean 6 standard error (SE)], n 5 24),where the ammonium concentration was constantly low andranged between 0 and 0.593 mmol L21 (0.065 60.127 mmol L21 [mean 6 SD], n 5 48).The AOA : AOBratio also correlated with the seasonal influx of ammonia atP10. Here, a plume of ammonium originating from the silldevelops in early spring to summer and then decreasesthrough the fall and winter. At this station we observed agood correlation between times of seasonally elevatedammonium and lower AOA : AOB ratios (Fig. 4B), suggest-ing that AOB were more competitive during periods of

Table 2. Characterization and diversity index of clone library sequences.

AOA AOB

Water Sediment Water Sediment

No. of libraries 9 3 9 3No. of clones 143 45 103 41OTU (2%) 16 9 16 11Singletons (2%) 6 5 9 3Good’s coverage 0.89 0.80 0.84 0.73Shannon index 1.79 1.58 1.51 2.08Reciprocal Simpson index 4.0 3.8 2.4 7.1


1326 Urakawa et al.

r

Fig. 2. Neighbor-joining trees showing the affiliation of (A) archaeal and (B) bacterial amoA genes. Bars indicate percent sequencedivergence. Numbers in or near triangles indicate the number of clones affiliated with each cluster. (A) A total of 2186 sequencescontaining 188 from Puget Sound for AOA and (B) 741 sequences containing 144 from Puget Sound for AOB were used for generatingthe distance matrix with Jukes–Cantor correction. Nm. indicatesNitrosomonas.

Fig. 3. Vertical profiles of sigma-T, Chl a, dissolved oxygen (DO), and dissolved nutrients. The nitrite concentration data at P12 are103 exaggerated.


measurably elevated ammonia. The highly variableAOA : AOB ratio, varying between 1.5 and 110.8 (26.7 68.4 [mean 6 SE], n 5 24), was associated with the highlyvariable ammonium concentrations measured at this site,varying between 0.01 and 7.03 mmol L21 (1.5 6 1.8 mmol L21

[mean 6 SD], n 5 48). The ammonium level at the LCstation was much higher than at P12 and presumablyresulted from a combination of variable runoff, mineraliza-tion of organic matter produced in this highly productivearea (e.g., high Chl a; Fig. 3), and tidally driven sedimentresuspension. The oxygen level in the bottom water wasconstantly below 2 mg L21 at LC (Fig. 3). Here, we observedmuch greater variation in AOA and AOB numbers, withAOB occasionally exceeding AOA. The AOA : AOB ratiowas low and varied between 0.03 and 66.6 (11.1 6 3.8 [mean6 SE], n 5 24). The ammonium concentration rangedbetween 0.03 and 6.83 mmol L21 (1.4 6 1.8 mmol L21 [mean

6 SD], n 5 24). The overall correlation between AOA : AOBratio and ammonium concentration (r 5 0.48, p , 0.001, n 554; Fig. 5A) strongly supports an association between theAOA : AOB ratio and ammonia concentration.

Kinetic model development and per-cell nitrificationrates—Our modeling effort combined three analysiselements. (1) We calculated substrate-dependent growthrates of N. maritimus, N. oligotropha, N. europaea, and N.oceani using available kinetic data (Urakawa et al. 2011;Fig. 5B). This clearly indicated that the in situ ammoniumconcentrations observed in Hood Canal would generallynot be supportive of characterized AOB. Low growth ratesfor AOB such as N. oligotropha and N. europaea could besustained at the higher range of ammonia concentrations,but these concentrations would not support N. oceani. (2)We calculated the contribution of AOA and AOB to

Fig. 4. Vertical profiles of (A) archaeal and bacterial amoA gene copies and (B) NHz4 and AOA : AOB ratio. NHz4 data are adapted

from Fig. 3. Data are mean 6 standard deviation (n 5 3).

1328 Urakawa et al.

nitrification, based on in situ ammonia concentrations,gene copy numbers, and single cell nitrification rates for N.maritimus and N. oligotropha (Fig. 5C). In most cases, themodeled contribution of AOA was greater than that ofAOB, with the exception of samples collected in Augustand October from LC. The seasonally modeled contribu-tions of AOB varied from significant (LC, October) tomoderate (LC, December) to minor (P12, August, October,December, and May; Fig. 5C). (3) Estimated nitrificationrates for AOA and AOB were calculated using in situnitrification rates determined by 15NHz4 dilution technique(Table 3). Additionally, single cell nitrification activities forAOA and AOB were estimated using rates determined forsix water samples (varying from 10.8 to 417 nmol L21 d21).The average single cell ammonia oxidation rate, estimatedby assuming one amoA gene copy per cell, varied between0.10 and 5.96 fmol cell21 d21 (mean 6 SE 5 1.15 6 0.96fmol cell21 d21) for AOA and between 0.01 and 0.22 fmolcell21 d21 (mean 6 SE 5 0.06 6 0.03 fmol cell21 d21) forAOB (Table 3).

Transcript analysis of amoA gene—The number of amoAtranscripts varied from 3.9 (6 0.1 [SE]) 3 104 to 3.8 (6 0.3)

3 105 copies mL21 for AOA and from 7.5 (6 0.1) 3 104 to22.9 (6 2.2) 3 105 copies mL21 for AOB. The concentra-tion of bacterial amoA transcripts in LC was significantlyhigher than that of P12 ( p , 0.05), suggesting that AOBactivity in LC was higher than that in P12. On the otherhand, no significant difference was found in archaeal amoAtranscripts between the two sites. The average number ofamoA transcripts per single cell was higher for AOB thanfor AOA, varying from 0.09 to 12.0 (mean 6 SE 5 2.6 61.9) for AOA and from 1.63 to 54.1 (mean 6 SE 5 14.9 68.0) for AOB (Table 4). A good correlation was foundbetween nitrification rates and abundance of archaealamoA transcripts (r 5 0.843, p 5 0.0352, n 5 6), while nocorrelation was detected between nitrification rates and thenumber of betaproteobacterial amoA transcripts (r 5 0.206,p 5 0.695, n 5 6; Fig. 6).

Quantification of GDGT lipids—We determined bothcore and intact GDGT lipids (Table 5). Core GDGT lipidsranged between 3.4 (6 0.3 [SE]) and 83.0 (6 0.9) ng L21

(mean 6 SE 5 41.7 6 12.6 ng L21). Intact GDGT lipidsvaried from 4.67 (6 0.8) to 174.1 (6 0.5) ng L21 (mean 6SE 5 50.3 6 25.3 ng L21). The ratio between core and total

Fig. 5. Ammonium concentration and niche separation of AOA and AOB. (A) Correlation between ammonium concentration andAOA : AOB ratio in Hood Canal. (B) Theoretical growth rate of ammonia oxidizers at different ammonia concentrations. The range ofammonium concentration observed in Hood Canal is shown by the double-headed arrow. Np. indicates Nitrosopumilus; Nm.,Nitrosomonas; Nc., Nitrosococcus. (C) Predicted theoretical nitrification contribution ratio for AOA and AOB.


GDGTs ranged from 0.22 to 0.75 (mean 6 SE 5 0.5 60.1), and more core lipids and fewer intact lipids weremeasured in October. Intact GDGT lipid abundance washighly correlated with nitrification rates (r 5 0.981, p 50.000556, n 5 6), while no significant relationship wasobserved between core GDGT lipids and nitrification rates(r 5 0.228, p 5 0.664, n 5 6; Fig. 6).

Discussion

We initially surveyed the phylogenetic diversity ofammonia oxidizers by selective amplification of genescoding for the amoA subunit of the archaeal and bacterialammonia monooxygenases. Although the same number ofOTUs was found in archaeal and bacterial water columnlibraries, calculated species richness indicated a greaterdiversity of AOA relative to AOB in the water column.These general patterns of diversity and presumptiveabundance are consistent with other marine pelagic studies(Mincer et al. 2007; Beman et al. 2008; Santoro et al. 2010).Most of the archaeal amoA were affiliated with WaterColumn Group A, which has been shown to be ubiquitousin marine environments (Francis et al. 2005). Only 4.3% oftotal clones affiliated with Group I.1b, a clade common interrestrial environments (Prosser and Nicole 2008). Theseresults suggested that this coastal population of AOAconsisted mainly of oceanic groups, with some possibleallochthonous input of terrestrial forms from riverine andrunoff origin or the presence of coastal adapted AOA thatare phylogenetically related to the terrestrial forms. Themajority of bacterial amoA sequences affiliated withNitrosospira cluster 1, a phylotype common in variousmarine environments, including the pelagic water columnand salt marsh sediments, but as yet having no culturedrepresentative (Bernhard et al. 2005). The second largestassemblage was the Nitrosomonas sp. Nm143 lineage,which has been observed in environmental clone librariesfrom marine sediments (Urakawa et al. 2006) and marineaquaculture systems (Foesel et al. 2008) and likely is ofprimarily marine origin (Purkhold et al. 2003).

We selected three hydrologically distinct field stations inHood Canal for a year-long observation. Analyses of theabundance and depth distribution of archaeal and bacterialamoA gene copies at these sites showed that AOA generallygreatly outnumber bacteria and that there was a seasonaloscillation (Fig. 4A). Some AOA likely derived from deepocean waters seasonally advected into the fjord (Fig. 2A).However, taken together our data indicate that the highnumbers developing over the spring and summer are almostexclusively derived from growth within the fjord. Thenitrification rates reported in the present study (10.8–417 nmol L21 d21) were similar to rates measured invarious oceanic sites (Beman et al. 2008; Santoro et al.2010) and to those of our previously published work inHood Canal (Horak et al. 2013). The single cell nitrificationrate of N. maritimus under the average ammoniumconcentration at P12 (0.065 mmol L21) was 4.24 fmolcell21 d21. These values are within the range of single cellnitrification activity reported by Ward (2011) and Santoroet al. (2010) but could be significantly underestimated if

Table

3.

Mea

sure

dn

itri

fica

tio

nra

tes,

mo

del

edn

itri

fica

tio

nra

tes,

mo

del

edsi

ng

lece

lln

itri

fica

tio

nra

tes,

an

dp

hy

sico

chem

ica

lp

rofi

les

of

the

six

wa

ter

sam

ple

su

sed

for

tran

scri

pt

an

aly

sis.

Sa

mp

leD

epth

(m)

Sig

ma

-TT

emp

era

ture

(uC

)S

ali

nit

y

Dis

solv

edo

xy

gen

(mg

L2

1)

Ch

la

(mg

L2

1)

NH

z 4

(mm

ol

L2

1)

NO

{ 2

(mm

ol

L2

1)

NO

{ 3

(mm

ol

L2

1)

Nit

rifi

cati

on

rate

(nm

ol

L2

1d

21)

tota

l

Mo

del

edn

itri

fica

tio

nra

te(n

mo

lL

21

d2

1)

Mo

del

edsi

ngl

ece

lln

itri

fica

tio

nra

te(f

mo

lce

ll2

1d

21)

AO

AA

OB

AO

AA

OB

P1

2A

ug

08

40

23

.10

9.3

29

.92

.50

.00

0.0

00

0.0

23

37

.74

17

41

5.7

81

.22

0.3

70

.02

P1

2O

ct0

85

02

3.2

99

.23

0.2

1.7

0.0

00

.00

50

.00

43

0.8

57

.65

7.5

30

.07

0.1

30

.01

P1

2D

ec0

81

20

23

.57

10

.33

0.7

2.7

0.0

00

.00

30

.04

30

.57

2.2

72

.12

0.0

80

.10

0.0

1P

12

Ma

y0

94

02

3.5

18

.43

0.2

3.5

0.0

00

.01

30

.00

37

.31

65

16

3.4

61

.54

0.1

50

.01

LC

Oct

08

62

0.6

81

1.5

27

.34

.33

.52

0.6

40

.28

18

.97

3.9

33

.83

40

.07

5.9

60

.22

LC

Dec

08

01

9.0

68

.72

4.6

5.7

0.2

40

.97

0.4

41

3.9

10

.88

.13

2.6

70

.17

0.0

8

1330 Urakawa et al.

there is more than one gene copy per cell. Although it iscommonly assumed that AOB contain two to three copiesof amoA per cell based on genome sequences of describedAOB (Santoro et al. 2010), the genome of a recentlycultured member of Nitrosospira likely has only a singleammonia–monooxygenase operon (Garcia et al. 2013).Since related Nitrosospira comprise the majority of AOBidentified in different marine systems, one amoA pergenome is probably a more appropriate conversion factor.Thus, the population sizes of AOB might have beenunderestimated in previous qPCR-based quantitative stud-ies. Available genome sequence data also indicate that theThaumarchaeota contain a single copy of amoA (Walker etal. 2010). Together, our data suggest that the in situ activityof the AOB population in Hood Canal is repressed by thelow concentration of ammonia (Figs. 4, 5). Thus, AOB(varying between 103 and105 cells mL21 in the watercolumn) may employ an opportunistic strategy to takeadvantage of transient spikes in ammonia concentration.This strategy is supported by our prior studies of theresponse of N. europaea to recovery from extendedammonia starvation (Berube et al. 2007).

The average number of amoA transcripts per cell forAOA was similar to values reported by Church andcolleagues (2010) and a laboratory study of N. maritimus(0.01 to 2.6 per cell; Nakagawa and Stahl 2013) but wasmuch higher than that reported from the Black Sea (Lam etal. 2007). In the later study Lam and colleagues (2007)reported that the number of transcripts in AOB was higherthan that in AOA. Specifically, transcripts of gammapro-teobacterial AOB were greater than those of AOA andbetaproteobacterial AOB. In the present study, althoughAOA amoA gene copy numbers outnumbered those ofAOB, higher cellular transcript numbers were observed forAOB (Table 4). This may reflect the larger cell size of AOB(Lam et al. 2007) or, as suggested by our earlier studies ofammonia-starved N. europaea, the greater stability of AOBtranscripts in the absence of cell growth (Berube et al.2007). Thus, a direct association between transcriptabundance and activity for different groups of microor-ganisms in nature must be viewed with caution (Lam et al.2007; Prosser and Nicole 2008). In addition, there isuncertainly about cellular transcript abundance simplybecause per-cell transcript numbers are calculated byrelating a pooled RNA extract to a community of unknownmetabolic status (Leininger et al. 2006; Church et al. 2010;Nakagawa and Stahl 2013). These reports reflect the many

Table 4. Total and single cell transcript numbers of AOA and AOB.

SampleArchaeal RNA 3 104

(mL21)Bacterial RNA 3 104

(mL21)Transcript ratio

AOA : AOB

Transcripts per cell

AOA AOB

P12 Aug 08 28.9(2.6) 13.8(1.9) 2.09 0.26 2.43P12 Oct 08 3.9(0.1) 7.5(0.09) 0.52 0.09 8.70P12 Dec 08 17.5(2.9) 13.4(0.34) 1.31 0.25 10.00P12 May 09 14.4(2.1) 20.6(0.78) 0.70 0.13 1.63LC Oct 08 37.8(3.3) 229.4(21.8) 0.16 11.96 12.36LC Dec 08 13.9(1.4) 191.5(4.9) 0.07 2.86 54.09

Data from archaeal and bacterial RNA are mean 6 (standard error; n 5 3), assuming a single amoA copy per AOA and AOB genome.

Fig. 6. Correlation analysis between nitrification rates and(A) gene copy numbers, (B) transcripts, and (C) GDGTs. (A) Thecorrelation coefficient between nitrification rate and archaealamoA gene copy number was 0.753 but not within the acceptancerange of significance ( p 5 0.0839).


caveats to interpreting environmental molecular data, sincethese measures and interpretation are very sensitive toextraction efficiency, primer design, assumptions of genecopy number, and the variability of species-dependenttranscriptome regulation patterns (Berube et al. 2007; Lamet al. 2007; Urakawa et al. 2010). We therefore emphasizedthe development of a kinetic model, based on the Km forammonia oxidation determined for N. maritimus andcharacterized AOB and the combination of differentmethods to infer the activities of natural ammonia-oxidizing populations.

GDGT membrane lipids are biomarkers for Archaeathat have been commonly used to track this group ofmicroorganisms in natural environments (SinningheDamsté et al. 2002). Viable cells contain intact GDGTsthat have polar head groups such as sugars or phospho-sugars attached to the glycerol moiety of the GDGT(Schouten et al. 2008; Huguet et al. 2010a,b). Soon after celldeath, the polar head groups are enzymatically cleaved,leaving only core GDGTs. Our earlier characterization ofN. maritimus indicated that actively growing cells contain6.3 fg of intact lipid per cell and only 0.001 fg cell21 of corelipids (Huguet et al. 2010b). However, more recent analyseshave prompted us to revise the value for intact lipidsdownward to 1.6 6 0.6 fg per cell (A. E. Ingalls unpubl.).This value is more in line with previously publishedtheoretical values of lipid per cell (Sinninghe Damstéet al. 2002). However, in these environmental samples, thevalues range from 0.1 to 0.7 fg intact GDGT per cell. Thelower values in the environment relative to cultures couldreflect incomplete extraction of intact GDGTs, smallercells, or cells in a variety of phases of division. Given thatnot all GDGT producing archaea contain amoA gene, it islikely that comparison of GDGTs to total archaeal 16SrRNA gene counts, including Euryarchaeota, would resultin an even lower values of lipid per cell. In any case, the lowvalues suggest that there is not a lot of intact GDGTpersisting after cell death in Hood Canal waters wherenitrification rates are high.

Core GDGTs have been widely used to estimate thebiomass of active archaeal populations (Huguet et al.2010b). However, our recent studies now strongly indicatethat intact GDGTs are more suitable to trace livingarchaeal biomass (Huguet et al. 2010a,b; Ingalls et al.2012). For example, in the present study, intact GDGTlipid abundance was highly correlated with nitrification

rates determined by 15NHz4 dilution technique (r 5 0.98,p , 0.001, n 5 6), while no significant relationship wasfound between core GDGT lipids and nitrification rates(Fig. 6). Further, the high correlation between intactGDGT and amoA transcripts (r 5 0.86, p , 0.05, n 5 6)also supports the utility of intact GDGT lipid analysis formonitoring active AOA in the marine water column. Whileit is likely that at other times and other locations this correla-tion would vary widely, the correlation within our data setsuggests that it is possible for the two parameters to reflectactivity. These correlations represent additional evidence forthe dominant role of AOA in coastal nitrification andsupport the utility of using intact GDGT lipids, as opposedto core GDGTs, to measure active thaumarchaeal biomass.Seasonal patterns of archaeal distribution were observed inthe lipid data; the archaeal biomass increased from winter tosummer and was flushed out during fall by a deep-waterrenewal event. The lipid data nicely matched this seasonalpattern of AOA gene copy numbers (Table 5; Fig. 4A). Therelative proportion of intact to total GDGT also showedseasonal patterns (Table 5) and tracked with both nitrifica-tion rates and amoA transcripts (Tables 3, 4). In the P12samples, the highest proportion of intact GDGTs (75% inAugust) corresponded with the highest nitrification rate andthe highest amoA gene counts (Fig. 4A). The lowest ratio(22% in October) corresponded to the lowest nitrificationrate measured at this site (57.6 nmol L21 d21) and low amoAgene counts (Fig. 4A).

Simple kinetic modeling was also consistent with amoAtranscript abundance, nitrification activity, and intactGDGT lipid profiles. High abundance of bacterial amoAtranscripts was observed in two LC samples, in which large(54.2% of total) and moderate (24.8% of total) contribu-tions of AOB to ammonia oxidation were predicted by themodel (Fig. 5C; Table 3). In other samples, the model pre-dicted more than a 99% contribution of AOA to nitrificationat in situ ammonium concentrations (Fig. 5C). However, itshould be noted that if the uncultivated marine Nitrosospiraspecies, dominant in our clone libraries (Fig. 2B) and ubiq-uitous in a wide range of marine environments (Bernhardet al. 2005; Mosier and Francis 2008), have substrate kineticsthat are similar to AOA, this model would substantiallyunderestimate the contribution of AOB in nitrification rates.However, the combined data sets are most consistentwith our existing model. The highest nitrification rate(417 nmol L21 d21) was observed in August at Sta. P12and was consistent with elevated archaeal amoA gene andamoA transcript numbers (Fig. 4A; Table 4). There wasgood correlation between nitrification rates and abundanceof amoA transcripts, while no correlation was detected betweennitrification rates and the number of betaproteobacterialamoA transcripts, consistent with the greater contribution ofAOA to nitrification in this estuary system (Fig. 6).

Although there is increasing evidence for AOA exertinga primary control on nitrification in both marine andterrestrial systems, this inference has been based primarilyon the abundance of gene and transcript numbers for thearchaeal amoA relative to those of the bacterial homolog(Church et al. 2010; Santoro et al. 2010). More recent, andmore direct, evidence has come from isotopic analysis of

Table 5. Core and intact GDGT lipids and intact GDGTratio.

Sample

GDGT lipidRatio*(i/total)Core (ng L21) Intact (ng L21)

P12 Aug 08 57.3(0.3) 174.1(0.5) 0.75P12 Oct 08 83.0(0.9) 22.8(0.9) 0.22P12 Dec 08 22.4(0.4) 35.0(0.4) 0.61P12 May 09 20.5(2.5) 39.9(1.8) 0.66LC Oct 08 63.8(0.8) 25.1(0.9) 0.28LC Dec 08 3.4(0.3) 4.67(0.8) 0.58

* Intact/(intact + core).

1332 Urakawa et al.

nitrous oxide, showing that the isotopic signature of theoceanic source of this nitrogen species has an isotopicsignature consistent with a primarily archaeal origin(Santoro et al. 2011). Here we have better constrained thecontribution of AOA and AOB to ammonia oxidation in amajor estuary system by using a model based on thephysiological properties of N. maritimus and cultivatedAOB to assess whether these properties are consistent withthe patterns of abundance and activities determinedthrough a seasonal cycle. The generally low ammoniaconcentrations measured at most stations throughout theyear were well correlated with AOA abundance patterns, aspredicted by the model. The robustness of the model is alsoconsistent with the similarity of Km values determined fornitrifying communities in Hood Canal and N. maritimus(Horak et al. 2013).

The AOB appeared to be of significance only at sites inwhich ammonia concentrations were more variable orderived from seasonal pulses. These results also imply thatAOB may exert significant seasonal or regional control onnitrification in coastal and estuarine ecosystems. Thus, thesefield observations are in general agreement with culture-based studies indicating that ammonium concentration is animportant factor in niche separation of marine AOA andAOB (Martens-Habbena et al. 2009). In addition toammonia concentration, there are almost certainly otherbiotic and abiotic variables that influence the distributionand abundance of the ammonia oxidizers. For example,ammonia oxidizers are known to be sensitive to light, andthis may limit their depth distribution or activity patternsthrough a diel cycle (Church et al. 2010; Luo et al. 2013).Marine nitrifiers have also been shown to be sensitive torelatively small changes in pH (Beman et al. 2011), which inthis fjord varies seasonally with mineralization of organicmaterial, and the intrusion of relatively low pH bottomwaters (pH 5 7.3–7.9) from coastal upwelling (Feely et al.2010). Interestingly, nitrification rates and other activitymeasurements, including transcript and intact GDGTanalyses, clearly indicated the major loss of AOA activityafter the deep-water renewal event in the fall. Othervariables include potentially limiting nutrients, such astrace-metal availability (Amin et al. 2013), and competitionwith other planktonic species for ammonia (Martens-Habbena et al. 2009). Since a predominant role of AOAin the nitrogen cycle is likely (Horak et al. 2013), weanticipate that this fjord will provide a useful model forbetter establishing the significance of AOA in controllingnitrogen form and availability and, in turn, environmentalvariables that control their activity and distribution inestuary ecosystems.

AcknowledgmentsWe thank the crew of the R/V Barnes for sampling in Hood

Canal. We also thank Florian Moeller, Laura Truxal, Amy Cash,and Alison Blevins for technical assistance. This work wassupported by U.S. National Science Foundation (NSF) grantsOCE-0623174 and OCE-1046017 to A. E. Ingalls, D. A. Stahl,and A. H. Devol from the Biological Oceanography andDimensions of Biodiversity and grant MIP-0604448 from theMicrobial Interactions and Processes. This work was also fundedin part by NSF grant MCB-0920741 from the Molecular and

Cellular Biosciences. We thank two anonymous reviewers forproviding valuable comments to improve this article.

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Associate editor: Wade H. Jeffrey

Received: 15 August 2013Accepted: 02 May 2014Amended: 05 April 2014

http://dx.doi.org/10.1128%2FAEM.01529-07http://dx.doi.org/10.1128%2FAEM.01529-07http://dx.doi.org/10.1128%2FAEM.00807-06http://dx.doi.org/10.1128%2FAEM.00807-06http://dx.doi.org/10.1073%2Fpnas.0913533107http://dx.doi.org/10.1016%2FB978-0-12-381294-0.00013-4http://dx.doi.org/10.1016%2FB978-0-12-381294-0.00013-4http://dx.doi.org/10.1016%2Fj.marchem.2005.09.008http://dx.doi.org/10.1016%2Fj.marchem.2005.09.008

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