hurley final report - marine biological laboratory ·...

Shifts in the relative abundance of ammonia-oxidizing bacteria and archaea in

the water column of a stratified marine environment, Salt Pond

Sarah Hurley

July 27, 2011

Department of Earth and Planetary Sciences, Harvard University

2011 Microbial Diversity Course, MBL

Abstract:

Salt Pond, a seasonally stratified kettle pond in Falmouth, Massachusetts, provides

an ideal analogue for the Black Sea and other stratified marine environments.

Nitrification in stratified water columns and oxygen minimum zones (OMZs)

connects organic matter remineralization to the anammox and denitrification, the

two known mechanisms of nitrogen loss in marine systems. We determined the

effect of physiochemical gradients through the chemocline in Salt Pond on

ammonia-‐oxidizing archaea and bacteria using amoA, the functional gene encoding

ammonia monooxygenase subunit A. Ammonia oxidizing archaea (AOA) dominated

the fresh, oxic surface water with low ammonium concentrations. Ammonia-‐

oxidizing bacteria (AOB) abundance increased through the chemocline into the

euxinic, but potentially micoraerophilic deep water. Within the AOA community,

sequences were most related to cultured and uncultured putative AOA and grouped

into surface, shallow, and deep clusters. The results of this study offer insights into

the ecology of AOA and AOB by comparing the environmental conditions in which

these groups are numerically dominant.

Introduction

Nitrogen is an essential and often limiting nutrient fundamental to the

structure and biochemistry of life. Nitrogen is readily oxidized or reduced and

serves as both an electron donor and accepter in energy metabolisms of

microorganisms. Microbial metabolisms drive the formation and consumption of

different chemical form of nitrogen and therefore drive marine nitrogen

biogeochemical cycling.

Nitrification, the stepwise oxidation of ammonium to nitrite and nitrate, is a

key process the marine nitrogen cycle responsible for the formation of the large

deep-‐sea nitrate reservoir (Figure 1). Nitrification is a microbially mediated process,

performed mostly by chemolithotrophic microbes who glean energy and electrons

from this oxidation. Ammonia released from the remineralization of particulate

organic nitrogen (PON or PN) is oxidized to substrates for denitrification and

anammox. Nitrification therefore connects the recycling of organic nitrogen to the

only two presently known nitrogen loss processes.

In productive equatorial and coastal upwelling zones, high fluxes of organic

matter and remineralization create oxygen minimum zones (OMZs), enabling

denitrification (Hogslund et al., 2008) and anammox (Hamersley et al., 2007).

Remineralization releases large amounts of ammonium, driving the high

nitrification rates often associated with OMZs. Nitrogen loss from OMZs are

estimated to account for 30-‐50% of total nitrogen loss from the oceans (Gruber &

Sarmiento, 1997).

The seasonally meromictic kettle pond, Salt Pond, in Falmouth Massachusetts

provides an ideal model system to study nitrogen cycling processes through an

oxygen gradient. The water column in Salt Pond closely resembles the chemical and

oxygen gradients typically associated with large marine anoxic basins such as the

Black Sea (Damste et al., 1993). The water column in these systems consists of an

oxic surface layer, sulfidic and anoxic deep waters, and consistent concentrations of

chemical species along isopycnals through the transition zone, or chemocline.

Studies of nitrification processes, such as ammonia oxidation, in Salt Pond can there

be translated to OMZs and larger stratified marine systems.

Ammonia oxidation, the first step in nitrification, is considered rate-‐limiting

and nitrite rarely accumulates in natural environments (Schleper & Nicol, 2010).

Traditionally, ammonia oxidation was thought to be catalyzed by Nitrosomona and

Nitrospira in the betaproteobacteria (Head et al., 1993), and Nitrosococcus in the

gammaproteobacteria (Purkhold et al., 2000; Ward & O'Mullan, 2002). Metagenomic

studies provided the first evidence for the metabolic capacity to oxidize ammonia in

marine group 1 Crenarchaeota (Venter 2004, Treusch). The first successful isolation

of an autotrophic ammonia-‐oxidizing marine archaeon, Nitrosopumilus maritiums

(Könneke et al., 2005), led to the discovery that mesophilic ammonia-‐oxidizing

archaea (AOA) are ubiquitous in the global oceans (Francis et al., 2005b). In the

open ocean and suboxic basins, AOA outnumber AOB in the water column (Coolen et

al., 2007; Lam et al., 2007).

The first step in ammonia oxidation, the oxidation of ammonia to

hydroxylamine, is catalyzed by the membrane bound ammonia monooxygenase

(AMO) enzyme (Könneke et al., 2005). AMO is encoded by the amoA, amoB, and

amoC genes. Putative crenarchaeal homologues to all three subunits have been

identified (Hallam et al., 2006; Treusch et al., 2005). Due to its functional

significance and conserved phylogeny, the amoA gene can be used as a molecular

marker to study the diversity and abundance of AOA and AOB (Coolen et al., 2007;

Lam et al., 2007).

This study attempts to address the following questions on the abundance and

diversity of AOA and AOB in the stratified, marine analog Salt Pond:

1. Who is the ammonia oxidizing community?

2. Where does this community occur in the water column?

3. How to physiochemical parameters affect the distribution of ammonia

oxidizing archaea vs. ammonia oxidizing bacteria?

The following approaches were taken to address these questions:

1. 454 16S sequncing and community analysis at 9 depths

2. Functional gene (amoA) clone libraries

3. qPCR targeting archaeal and bacterial amoA

4. CARD-‐FISH and DAPI cell counts

Materials and Methods

Site description:

Salt Pond is a brackish, shallow, seasonally stratified pond located in

Falmouth, Massachusetts (Figure 2). It is a glacially formed kettle pond ranging 4.6

to 5.8 meters deep, with a surface area of 0.29 km2 (Simmons et al., 2004), and is

surrounded by 0.02 km2 of upstream salt marsh

(http://www.capecodcommission.org/tidalatlas/). The pond exhibits density

stratification due to permanent tidal exchange with Vineyard Sound and freshwater

inputs from runoff, precipitation, and groundwater. The average daily tidal

amplitude is ~0.6 m.

Sampling:

Sampling was conducted from an inflatable rowboat on July 8, 2011. A YSI

600R Water Quality Sampling Sonde (Yellow Springs, Inc., Yellow Springs, Ohio) was

used to collect salinity (ppt), temperature (°C), oxygen (% DO, mg/L) and pH data. A

profile was taken in two locations ~10 meters apart to determine the water depth

and location of the chemocline. The YSI probe was then attached to the intake of a

portable peristaltic pump to allow for simultaneous water collection and profiling

(Figure 3).

Water samples were collected from 9 depths spanning 0-‐4.0m. Water

samples for molecular work and FISH analysis were collected in acid rinsed 2L

polycarbonate bottles. In an attempt to measure sulfide concentrations 1 ml of

water was taken from the full 2 L with a tipless syringe and transferred into 25 ml of

zinc acetate solution (25 ml Milli-‐Q water + 1 ml 20% by volume zinc acetate). This

procedure did not adequately fix the sulfide or was too large a dilution as no sulfide

was detected using the Cline assay (Cline, 1969).

FISH:

Immediately on return to lab, water samples for FISH analysis were fixed

using 1% formaldehyde in 50 ml conical tubes. 2 and 5 ml aliquots were filtered

onto 0.2 µm membrane filters (Millipore GTTP, 25 mm). CARD-‐FISH was performed

using the following probes:

Eub338

Eub338 II

Bacterial

Eub338 III

Cren537

Cren554

Crenarchaeal

Eury806

Nutrient Analyses:

50 ml of water was sterile filtered and frozen for nutrient analysis. Ammonia

measurements were performed by Chris Algar at the MBL Ecosystem Center

according to (Scheiner, 1976). Nitrite measurements were performed

spectrophotometrically according to (Strickland J, 1968).

Community 16s RNA analysis:

Between 200-‐300 ml of water from each sampling depth was filtered onto 0.2 µm

membrane filters (Millipore GTTP, 47 mm) for DNA extraction. DNA for 454

sequencing was extracted using a PowerSoil DNA Isolation Kit and quantified using

a NanoDrop spectrophotometer (Thermo Scientific). SSU rRNA genes were

amplified with barcoded primers that also incorporated the Roche 454 Ti adapter

sequences. The barcode was on the forward primer. Each barcode was 9 nt long

and all the barcodes we used are in the mapping file for the 454 data.The primer

targets are 515F and 907R on the E. Coli 16S gene.

Forward primer (X denotes barcode, lowercase is the linker between barcode and

16S primer),

5'-‐CGTATCGCCTCCCTCGCGCCATCAGXXXXXXXXgaGTGYCAGCMGCCGCGGTAA-‐3'

Reverse primer (lowercase denotes linker between adapter and 16S primer),

5'-‐CTATGCGCCTTGCCAGCCCGCTCAGggCCGYCAATTCMTTTRAGTTT-‐3'

In the "AmplificationPlate" in the mapping file "plate1" was amplified for 32 cycles

and "plate4" in the "AmplificationPlate" field was amplified for 36 cycles. The PCR

program utilized a touchdown annealing temp for the first 10 cycles from 68-‐58C.

Then there were 12 cycles of three-‐step PCR (denaturation, annealing, elongation)

followed y 10-‐14 cycles of tow-‐step PCR (annealing and elongation at same temp). A

Phusion HF polymerase (2X mastermix) was used to amplify the gene with 8%

DMSO and 0.5 uM primers in the final reaction volume. The template was

normalized to 15 ng/uL (for DNA above 15 ng/uL) and 2 uL of each template was

used forPCR. Post amplification DNA was quantified using the PicoGreen assay and

then pooled ~125 ng of each PCR product. This pool was concentrated down to 100

uL using the vacufuge and gel purifed using the Montage Kit (Millipore). The gel-‐

purified pool was then shipped to Penn St. for sequencing.

The RDP classifier as implemented in the Qiime pipeline for community

microbial analysis tool was used to assign taxa to all sequences. Trees were built

using the GreenGenes NAST aligner tool to align sequnces with the core set of 16s

alignment templates. Sequences were aligned using the filter_alignment.py protocol

in Qiime with lane masking enabled (as described and referenced by Libusha Kelly,

course report, 2010).

Clone Libraries and qPCR:

DNA for all other molecular analyses was extracted using a phenol-‐

chloroform, freeze-‐thaw procedure in the Coolen Lab at WHOI. Total nucleic acid

extract was with a ladder in a 1% agarose and visualized with ethidium bromide

(Figure 4). DNA extracts were quantified using fluorometry and yielded the

following concentrations:

Sample Depth

ng/µl

DNA

PCR for archaeal and bacterial amoA clone libraries and qPCR were

performed using primers and thermocycling conditions previously described for

bacterial amoA (AmoA-‐1F*/AmoA-‐2R) specifically targeting the β-‐AOB (Rotthauwe

et al., 1997), and archaeal amoA (Arch-‐amoAF/Arch-‐amoAR) (Beman et al., 2008;

Francis et al., 2005a). An inhibition test was performed to check for inhibition due to

remaining extraction solvents, etc. Significant inhibition was not demonstrated in

any of the samples.

Clone libraries using archaeal primers as described above were constructed

for depths 0 m, 0.5 m, 1.5 m, and 4.0 m. PCR products were cloned into the TOPO2

TA Cloning Kit (Invitrogen). Sequences in the clone libraries were aligned against

each other using Muscle. Sequences in each library were locally BLASTed against the

a FunGene amoA database. A tree was constructed using FastTree and visualized in

iTOL.

Results and Discussion:

Salt Pond Depth Profile:

Seasonal density stratification in Salt Pond occurs during the summer

months– late June through September– and prevents vertical mixing (Simmons et

al., 2004). Sampling on July 8 revealed a distinct chemocline from 1.5 to 3 meters

(Figure 5), as reported in previous years (Saenz, 2008). The surface of the pond is

oxygenated freshwater with a salinity of 11.37 ppt, dissolved oxygen concentration

of 6.4-‐6.7, and a pH of 8.8. Respiratory oxygen consumption drives down dissolved

SP-A 0.0 m 178.5

SP-B 0.5 m 309.6

SP-C 1.0 m 239.6

SP-D 1.5 m 12.5

SP-E 2.0 m 168.3

SP-F 2.5 m 148.9

SP-G 3.0 m 121.6

SP-H 3.5 m 62.2

SP-I 4.0 m 116.7

oxygen that cannot be replenished by mixing and creates a chemocline. Below the

chemocline the salinity increases to 19 ppt, the dissolved oxygen drops to 4.1 mg/L,

and the pH decreases to 7. Sulfide concentrations likely increase to below the

oxycline to millimolar concentrations (Saenz, 2008).

16S Community Analysis by 454 sequencing:

The major phyla represented by 16S rRNA 454 sequencing are

Actinobacteria, Bacteroidetes, Chlorobi, Cyanobacteria, and Proteobacteria.

Cyanobacteria are the dominant phyla in water column down to 2.5 meters with the

exception of 0.5m where Proteobacteria (Figure 6). While photosynthetically active

radiation (PAR) was not measured, this abundance pattern reflects photosynthetic

activity in Cyanobacteria and Proteobacteria through the photic zone. At 3 meters

there is a slight relative increase in the abundance of Bacteroidetes followed by a

complete dominance of Chlorobi (97% of relative phyla counts at 4 meters). The

increase in Chlorobi at 3.5-‐4 meters reveals the spatial niche of low-‐light adapted

green sulfur bacteria in the euxinic bottom waters of Salt Pond. The time constraints

of this course did not allow for a very in-‐depth analysis of this dataset. For example,

breaking down the proteobacteria could provide information on the spatial niche

and relative representation of purple sulfur bacteria and ammonia oxidizing

bacteria.

In order to assess the difference in microbial communities at each depth,

weighted and unweighted dendrograms were produced using UniFrac (Figure 7).

The UniFrac method measures the phylogenetic distance between sets of taxa in a

phylogenetic tree as the fraction of the branch length of the tree that leads to

individual descendants from each water column depth (Lozupone & Knight, 2005).

Given the gradient of physiochemical conditions in Salt Pond, a logical hypothesis

would be that microbial communities at each depth would be the similar to the

communities at adjacent water depths. To a coarse degree this appears to be true in

the dendrograms. Samples, especially in the more closely chemically related surface

and deep water tend to cluster by depth, while samples in the more rapidly

changing chemocline are more spread out. In this case the 0.5 m sample may be

reflecting a photosynthetic community different from the otherwise dominant

cyanobacteria.

Rarefaction curves for the 16S community analysis do not show saturation at

any depth (Figure 8). This is unsurprising as each depth was only sampled once.

More notably, the grouping of the rarefaction curves mirrors patterns found in the

relative representation of phyla and the UniFrac dendograms, largely that the

sample from 0.5 meters was the only sample approaching a plateau.

qPCR:

qPCR was performed on the total nucleic acid extracts at each depth to look

at the relative abundance of putative ammonia-‐oxidizing archaea (AOA) and

bacteria (AOB) across the physiochemical gradient of the chemocline in Salt Pond.

Shifts in the relative abundance of AOA and AOB correlate closely with both

dissolved oxygen and ammonium concentration through the water column. In the

first ½ meter of the water column AOB dominate ~70% of amoA community.

Between ½ and 2 meters, in the oxic to suboxic water just above the chemocline,

AOA make up over 95% of the amoA community. Through the chemocline and into

the anoxic deep water, the abundance of AOB increases steadily to 66%. In the lower

3 meters of the water column the increase in the abundance of the AOB community

covaries with the increase in ammonium concentration.

In the surface water of Salt Pond, the high percentage of AOB may be due to

nutrient run off of surface water. In past years, high ammonium concentrations have

been observed in the upper meter of Salt Pond, so this may be a temporal variation

that was missed on the one sampling expedition. The location of a peak abundance

of AOA in the oxic to suboxic nitrification zone just above the chemocline has been

similarly observed in the Black Sea (Lam et al., 2007) associated with maximum

nitrification activity. In and below the chemocline with the decrease of dissolved

oxygen, the remineralization of organic matter and grodundwater inputs provide a

source of ammonium. As ammonium concentrations increase and dissolved oxygen

concentrations decrease, AOB become more prevalent than AOA. This suggests that

AOB outcompete AOA at high ammonia concentrations in a potentially

microaerophilic environment created by an influx of ground water from the

sediments.

Functional Gene Clone Libraries:

Clone libraries targeting the archaeal amoA gene were locally BLASTed

against the FunGene amoA database. While the results were not particularly

illuminating, they were the similar to uncultured and enrichment cultures of

ammonia oxidizing archaea and Crenarchaeota (Table 1).

Trees constructed from archaeal amoA clone libraries showed roughly three

clustering groups (Figure 10). There is a ‘very shallow’ depth (0-‐0.5m) cluster, a

‘shallow’ depth cluster, and a very distinct ‘deep’ cluster. This pattern appears in

both the tree ignoring branch lengths and the tree incorporating branch lengths. In

the tree incorporating branch lengths, the ‘deep’ clustering is more readily apparent

as the branch length of the cluster is distinct from both the ‘very shallow ‘ and

‘shallow’ clusters on either side.

CARD-FISH and DAPI Counts:

CARD-‐FISH counts with crenarchaeal and general bacterial probes revealed a

consistent ratio of bacteria to crenarchaea throughout the water column. The

crenarchaea generally make up ~1% of the total DAPI counts and the bacteria make

up between 70-‐80% of the counts. 20-‐30% of the DAPI counts were not

fluorescently labeled with either probe. Morphology in the crenarchaeal probe

resembled the peanut shape observed in (Könneke et al., 2005).

Conclusions:

• Ammonia-‐oxidizing crenarchaea and bacteria are both present in Salt Pond.

AOA are dominant in the oxic surface waters with low ammonia

concentrations while AOB increase through the chemocline and into the

suboxic, ammonia rich waters

• The putative ammonia-‐oxidizing archaea are closely related to cultured and

uncultured environmental ammonia-‐oxidizers in the FunGene amoA

database.

• CARD-‐FISH revealed a low overall abundance of crenarchaea compared to

bacteria throughout the water column.

• The microbial community changes significantly through water column in Salt

Pond, highlighting different ecological niches in a stratified marine system.

Future Directions and Remaining Questions:

• Phylogenetic comparison to known ammonia-‐oxidizing organisms: Given the

lack of specificity in the BLAST results from the FunGene amoA database, the

next step would be to build a tree with the clone libraries from Salt Pond to

compare to known organisms of interest such as Nitrosopumilus maritiums

and Crenarchaeota found in the Black Sea (Coolen et al., 2007).

• Nitrification rates: Rate measurements for ammonia oxidation and nitrite

oxidation would allow us to look at where in the water column, nitrification

is occurring relative to the numerical abundance of either AOA or AOB.

• Temporal variation: Salt Pond is a seasonally stratified pond. The chemocline

develops around late June, so sampling on July 8, 2011 is representative of an

early summer chemocline. The ammonia-‐oxidizing community may shift with

the evolution of the chemocline throughout the summer, and likely

redistributes with the vertical mixing in winter.

• Anammox: Below the chemocline Salt Pond becomes euxinic and could

potentially support an active anammox community. This could be

accomplished through similar methods used in this report or ladderane

lipids unique to anammox bacteria.

• Further 16S analysis: The 454 sequencing provided a depth of data, which

could not be fully explored during the limited time in this course. Further

probing of community structure at each depth would likely further our

understanding of spatial niches in the stratified marine environment of Salt

Pond.

Acknowledgements:

Thank you to course directors Dan Buckley and Steve Zinder for providing this truly

unique and fulfilling opportunity. Thank you to the course TAs (especially Chuck

and Lizzy) for your help and patience. Thank you to Cornelia Wuchter and Marco

Coolen for primers, probes, lab space, and scientific guidance. Thank you to the

waterbears for rocking the hole.

Funding provided by the Milston L. Shifman Endowed Scholarship. References: Beman, J.M., Popp, B.N., Francis, C.A., (2008) Molecular and biogeochemical evidence

for ammonia oxidation by marine Crenarchaeota in the Gulf of California. Isme Journal, 2(4), 429-‐441.

Cline, J.D., (1969) SPECTROPHOTOMETRIC DETERMINATION OF HYDROGEN SULFIDE IN NATURAL WATERS. Limnology and Oceanography, 14(3), 454-‐&.

Coolen, M.J.L., Abbas, B., van Bleijswijk, J., Hopmans, E.C., Kuypers, M.M.M., Wakeham, S.G., Damste, J.S.S., (2007) Putative ammonia-‐oxidizing Crenarchaeota in suboxic waters of the Black Sea: a basin-‐wide ecological study using 16S ribosomal and functional genes and membrane lipids. Environmental Microbiology, 9(4), 1001-‐1016.

Damste, J.S.S., Wakeham, S.G., Kohnen, M.E.L., Hayes, J.M., Deleeuw, J.W., (1993) A 6,000-‐YEAR SEDIMENTARY MOLECULAR RECORD OF CHEMOCLINE EXCURSIONS IN THE BLACK-‐SEA. Nature, 362(6423), 827-‐829.

Francis, C., Roberts, K., Beman…, J., (2005a) Ubiquity and diversity of ammonia-‐oxidizing archaea in water columns and sediments of the ocean. In: Proceedings of the ….

Francis, C.A., Beman, J.M., Kuypers, M.M.M., (2007) New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation. Isme Journal, 1(1), 19-‐27.

Francis, C.A., Roberts, K.J., Beman, J.M., Santoro, A.E., Oakley, B.B., (2005b) Ubiquity and diversity of ammonia-‐oxidizing archaea in water columns and sediments of the ocean. Proceedings of the National Academy of Sciences of the United States of America, 102(41), 14683-‐14688.

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Hallam, S.J., Mincer, T.J., Schleper, C., Preston, C.M., Roberts, K., Richardson, P.M., DeLong, E.F., (2006) Pathways of carbon assimilation and ammonia oxidation suggested by environmental genomic analyses of marine Crenarchaeota. Plos Biology, 4(4), 520-‐536.

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Hogslund, S., Revsbech, N.P., Cedhagen, T., Nielsen, L.P., Gallardo, V.A., (2008) Denitrification, nitrate turnover, and aerobic respiration by benthic foraminiferans in the oxygen minimum zone off Chile. Journal of Experimental Marine Biology and Ecology, 359(2), 85-‐91.

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Simmons, S.L., Sievert, S.M., Frankel, R.B., Bazylinski, D.A., Edwards, K.J., (2004) Spatiotemporal distribution of marine magnetotactic bacteria in a seasonally stratified coastal salt pond. Applied and Environmental Microbiology, 70(10), 6230-‐6239.

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Novel genes for nitrite reductase and Amo-‐related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling. Environmental Microbiology, 7(12), 1985-‐1995.

Ward, B.B., O'Mullan, G.D., (2002) Worldwide distribution of Nitrosococcus oceani, a marine ammonia-‐oxidizing gamma-‐proteobacterium, detected by PCR and sequencing of 16S rRNA and amoA genes. Applied and Environmental Microbiology, 68(8), 4153-‐4157.

Figures:

Figure 1. The marine nitrogen cycle highlighting the role of nitrification in connecting the remineraliztion of particulate organic nitrogren and nitrogen loss from the ocean. Modified from (Francis et al., 2007). discovered over 100 years ago (Winogradsky, 1890),

underlining just how rapidly these two majordiscoveries have taken place. In this review, wefocus on recent developments related to the micro-bial ecology of anaerobic and archaeal ammoniaoxidation. We expand upon existing reviews thatcover various aspects of the microbial N cycle(Kowalchuk and Stephen, 2001; Zehr and Ward,2002; Strous and Jetten, 2004; Arrigo, 2005; Kuyperset al., 2006; Nicol and Schleper, 2006; Revsbechet al., 2006), and focus particularly on archaealammonia oxidation, because this very recent dis-covery is an area of remarkably active research.

Anammox

Since the mid-1960s, oceanographers have recog-nized pervasive ammonium deficits in anoxic basinsthat hinted at the possible removal of ammonium byanaerobic microbial activity (Richards, 1965). Never-theless, for the remainder of the century, hetero-trophic denitrification was considered the only sinkfor fixed nitrogen under anoxic conditions innatural systems. The first evidence for anaerobicammonium oxidation (anammox) to N2 gas was

obtained from anoxic (denitrifying) bioreactors ofwastewater treatment plants (WWTPs) (Mulderet al., 1995), where it was eventually determinedthat novel organisms related to Planctomycetaleswere capable of oxidizing ammonium using nitrite(rather than O2) as the electron acceptor (Strouset al., 1999). Befitting micro-organisms capable ofsuch a novel metabolism, these ‘anammox’ bacteriahave a number of truly unique features, includingthe use of hydrazine (N2H4, i.e., rocket fuel) as a freecatabolic intermediate, the biosynthesis of ladder-ane lipids and the presence of an anammoxosome(intracytoplasmic compartment). All four currentlyrecognized genera of anammox bacteria – Candida-tus ‘Brocadia’, ‘Kuenenia’, ‘Scalindula’, and ‘Ana-mmoxoglobus’ – share these unique physiologicaland morphological features.

Owing to their distinct metabolism and physiol-ogy, anammox bacteria received considerable atten-tion in engineered systems, but were assumed to beminor players in the N cycle within naturalecosystems. However, in 2002, Thamdrup andDalsgaard found anammox to be responsible for24–67% of N loss in marine sediments (Thamdrupand Dalsgaard, 2002), and in 2003, two parallelstudies demonstrated that anammox was directly

Figure 1 Microbial nitrogen transformations above, below and across an oxic/anoxic interface in the marine environment (based in parton Arrigo, 2005). Nitrite is highlighted in red to emphasize the central role of this metabolic intermediate/product within and between N-cycling pathways. Key functional genes discussed in the text are shown in yellow: amo, ammonia mono-oxygenase; hao, bacterialhydroxylamine oxidoreductase (?¼unknown gene/enzyme in AOA); nir, nitrite reductase; and nor, nitric oxide reductase. For clarity,other functional genes and the process of nitrate/nitrite assimilation are not shown.

New processes and players in the nitrogen cycleCA Francis et al

20

The ISME Journal

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Figure 2. A) Location of Salt Pond in Falmouth, Massachusetts on Cape Cod. B) Location of depth profile taken in Salt Pond.

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Figure 5. Water column chemistry and dissolved oxygen in the Salt Pond water column.

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0.5

1

1.5

2

2.5

3

3.5

4

0 5 10 15 20 25 30

Depth (m)

Salt Pond Depth ProNile

pH

T (°C)

Salinity (ppt)

DO (mg/L)

Figure 6. 16S 454 sequence data normalized to sequence reads.

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Figure 7. A) Weighted dendrogram created through UNIFRAC analysis B) unweighted dendrogram.

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Figure 8. 16S 454 Sequencing rarefaction curves at each sampling depth.

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Figure 9. Nitrogen species in the water column (left) and quantification of archaeal and bacterial amoA copy number.

Table 1. Top BLAST hits when from the FunGene amoA database.

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Figure 10. Trees of archaeal amoA functional gene clone libraries created in FastTree and visualized in iTOL.

Legend:

Sample

SH B archaeal 26

SH B archaeal 52SH B archaeal 70

SH D archaeal 26SH B archaeal 59


SH D archaeal 13

SH D archaeal 33

SH D archaeal 34

SH D archaeal 40

SH D archaeal 47

SH D archaeal 67

SH B archaeal 38

SH D archaeal 5

SH B archaeal 5

SH B archaeal 28

SH B archaeal 78

SH D archaeal 20

SH D archaeal 23

SH A archaeal 6SH D archaeal 22 SH

B arch

aeal 64

SH B a

rchaea

l 32

SH D arch

aeal 48

SH D arch

aeal 58

SH B arch

aeal 42

SH B archa

eal 65

SH D ar

chaeal 2

SH B a

rchaeal

74

SH D ar

chaeal

35

SH A

archae

al 13

SH A a

rchaea

l 2SH A

archa

eal 30

SH B

archa

eal 81

SH D

archa

eal 4

9

SH A

arch

aeal

47

SH D

arch

aeal

69

SH D

arch

aeal

7

SH D

archa

eal 4

5

SH D archaea

l 24

SH D archaea

l 11

SH D archaeal 6

SH D archaeal 39

SH D archaeal 5

1

SH D archaeal 1

SH D archaeal 55

SH D archaeal 17

SH D archaeal 70

SH D archaeal 71

SH D archaeal 8


SH D archaeal 15

SH A archaeal 37

SH A archaeal 36

SH A archaeal 38

SH A archaeal 39

SH A archaeal 48

SH D archaeal 64SH D archaeal 21

SH D archaeal 36

SH A archaeal 34

SH A archaeal 9

SH I archaeal 12SH I archaeal 26



SH I archaeal 25

SH B archaeal 85

SH D

archaeal 61

SH D

archaeal 56

SH D

archaeal 62

SH A

arc

haea

l 19

SH A

arc

haea

l 42

SH A

arc

haea

l 17

SH A

arc

haea

l 49

SH B

arc

haea

l 79

SH B

arc

haea

l 3

SH A

arc

haea

l 1

SH B

arc

haea

l 15

SH B

arc

haea

l 18

SH D

arc

haea

l 37

SH B

arc

haea

l 25

SH D

arc

haea

l 25

SH A

arc

haea

l 31

SH A

arc

haea

l 7

SH A

arc

haea

l 35

SH A

arch

aeal

28

SH B

arch

aeal

10

SH B

arch

aeal

83

SH D

arch

aeal

42

SH A

archa

eal 1

2SH B

archae

al 62

SH A

archae

al 18

SH B a

rchaea

l 7

SH B a

rchaea

l 56SH

B arch

aeal 2

SH B a

rchaeal

9SH

D archa

eal 30SH

D archae

al 66

SH B

archa

eal 27

SH B

archa

eal 6

6

SH B archaeal 29

SH B archaeal 40

SH B archaeal 36

SH A archaeal 32

SH B archaeal 44

SH B archaeal 34

SH B archaeal 47

SH A archaeal 20

SH B archaeal 37

SH A archaeal 22

SH A archaeal 11

SH A archaeal 26

SH B archaeal 43

SH B archaeal 4

SH B arch

aeal 41

SH A archaeal 24

SH A archaeal 16

SH B archaeal 39

SH B archae

al 54SH B a

rchaeal 46

SH B archa

eal 13

SH B arch

aeal 6

SH B arch

aeal 12

SH A

arch

aeal

8

SH B

archa

eal 1

1

SH B

arc

haea

l 86

SH A

arc

haea

l 43

SH A

arc

haea

l 45

SH B archaeal 55

SH B archaeal 76

SH B archaeal 69

SH B archaeal 35

SH A archaeal 41

SH B archaeal 20SH

B a

rcha

eal 5

1

SH B

arc

haea

l 57

SH B archaeal 8

SH B archaeal 77

SH B archaeal 48

SH A archaeal 10

SH B archaeal 63

SH D archaeal 43

SH D archaeal 50SH A archaeal 15

SH D archaeal 57

SH D archaeal 54

SH B archaeal 17SH A archaeal 3

SH A archaeal 46SH D archaeal 3

SH D archaeal 9

SH D archaeal 52

SH D archaeal 60

SH D

arc

haea

l 63

SH A

arc

haea

l 44

SH A

arc

haea

l 21

SH A

arc

haea

l 4

SH A

arc

haea

l 40

SH D

arch

aeal

44

SH D

arch

aeal

53

SH D

arch

aeal

65SH D

arc

haea

l 31

SH D

arc

haea

l 27

SH A

arc

haea

l 14S

H B

arc

haea

l 53

SH B archaeal 80

SH B archaeal 45 SH

B a

rcha

eal 7

3

SH B

arc

haea

l 61

SH B

arc

haea

l 71

SH D

archaeal 18

SH D

arc

haea

l 4SH

A a

rcha

eal 2

3SH

D a

rcha

eal 2

9

SH A

arc

haea

l 5SH

A a

rcha

eal 3

3SH

A a

rcha

eal 2

7SH

B a

rcha

eal 8

7

SH D

arc

haea

l 41

SH B archaeal 68

SH D

archaeal 16SH

D archaeal 12

SH A archaeal 25

SH D

archaeal 46

SH D

archaeal 14SH

B archaeal 1

SH D

archaeal 19

SH D

archaeal 10

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Legend:

SampleSH B archaeal 26




SH D archaeal 13

SH D archaeal 33

SH D archaeal 34

SH D archaeal 40

SH D archaeal 47

SH D archaeal 67

SH B archaeal 38

SH D archaeal 5

SH B archaeal 5

SH B archaeal 28

SH B archaeal 78

SH D archaeal 20

SH D archaeal 23

SH A archaeal 6SH D archaeal 22 SH

B arch

aeal 64

SH B a

rchaea

l 32

SH D arch

aeal 48

SH D arch

aeal 58

SH B arch

aeal 42

SH B archa

eal 65

SH D ar

chaeal 2

SH B a

rchaeal

74

SH D ar

chaeal

35

SH A

archae

al 13

SH A a

rchaea

l 2SH A

archa

eal 30

SH B

archa

eal 81

SH D

archa

eal 4

9

SH A

arch

aeal

47

SH D

arch

aeal

69

SH D

arch

aeal

7

SH D

archa

eal 4

5

SH D archaea

l 24

SH D archaea

l 11

SH D archaeal 6

SH D archaeal 39

SH D archaeal 5

1

SH D archaeal 1

SH D archaeal 55

SH D archaeal 17

SH D archaeal 70

SH D archaeal 71

SH D archaeal 8


SH D archaeal 15

SH A archaeal 37

SH A archaeal 36

SH A archaeal 38

SH A archaeal 39

SH A archaeal 48

SH D archaeal 64SH D archaeal 21

SH D archaeal 36

SH A archaeal 34

SH A archaeal 9

SH I archaeal 12SH I archaeal 26SH I archaeal 3SH I archaeal 29


SH I archaeal 25

SH B archaeal 85

SH D

archaeal 61

SH D

archaeal 56

SH D

archaeal 62

SH A

arc

haea

l 19

SH A

arc

haea

l 42

SH A

arc

haea

l 17

SH A

arc

haea

l 49

SH B

arc

haea

l 79

SH B

arc

haea

l 3

SH A

arc

haea

l 1

SH B

arc

haea

l 15

SH B

arc

haea

l 18

SH D

arc

haea

l 37

SH B

arc

haea

l 25

SH D

arc

haea

l 25

SH A

arc

haea

l 31

SH A

arc

haea

l 7

SH A

arc

haea

l 35

SH A

arch

aeal

28

SH B

arch

aeal

10

SH B

arch

aeal

83

SH D

arch

aeal

42

SH A

archa

eal 1

2SH

B arc

haeal 6

2

SH A

archae

al 18

SH B a

rchaea

l 7

SH B a

rchaea

l 56SH

B arch

aeal 2

SH B a

rchaeal

9SH

D archa

eal 30SH

D archae

al 66

SH B

archa

eal 27

SH B

archa

eal 6

6

SH B archaeal 29

SH B archaeal 40

SH B archaeal 36

SH A archaeal 32

SH B archaeal 44

SH B archaeal 34

SH B archaeal 47

SH A archaeal 20

SH B archaeal 37

SH A archaeal 22

SH A archaeal 11

SH A archaeal 26

SH B archaeal 43

SH B archaeal 4

SH B arch

aeal 41

SH A archaeal 24

SH A archaeal 16

SH B archaeal 39

SH B archae

al 54SH B a

rchaeal 46

SH B archa

eal 13

SH B arch

aeal 6

SH B arch

aeal 12

SH A

arch

aeal

8

SH B

archa

eal 1

1

SH B

arc

haea

l 86

SH A

arc

haea

l 43

SH A

arc

haea

l 45

SH B archaeal 55

SH B archaeal 76

SH B archaeal 69

SH B archaeal 35

SH A archaeal 41

SH B archaeal 20SH

B a

rcha

eal 5

1

SH B

arc

haea

l 57

SH B archaeal 8

SH B archaeal 77

SH B archaeal 48

SH A archaeal 10

SH B archaeal 63

SH D archaeal 43

SH D archaeal 50SH A archaeal 15

SH D archaeal 57

SH D archaeal 54

SH B archaeal 17SH A archaeal 3

SH A archaeal 46SH D archaeal 3

SH D archaeal 9

SH D archaeal 52

SH D archaeal 60

SH D

arc

haea

l 63

SH A

arc

haea

l 44

SH A

arc

haea

l 21

SH A

arc

haea

l 4

SH A

arc

haea

l 40

SH D

arch

aeal

44

SH D

arch

aeal

53

SH D

arch

aeal

65

SH D

arc

haea

l 31

SH D

arc

haea

l 27

SH A

arc

haea

l 14S

H B

arc

haea

l 53

SH B archaeal 80

SH B archaeal 45 SH

B a

rcha

eal 7

3

SH B

arc

haea

l 61

SH B

arc

haea

l 71

SH D

archaeal 18

SH D

arc

haea

l 4SH

A a

rcha

eal 2

3SH

D a

rcha

eal 2

9

SH A

arc

haea

l 5SH

A a

rcha

eal 3

3SH

A a

rcha

eal 2

7SH

B a

rcha

eal 8

7

SH D

arc

haea

l 41

SH B archaeal 68

SH D

archaeal 16SH

D archaeal 12

SH A archaeal 25

SH D

archaeal 46

SH D

archaeal 14SH

B archaeal 1

SH D

archaeal 19

SH D

archaeal 10

Figure 11. CARD-‐FISH counts with bacterial and crenarchaeal probes.

0% 20% 40% 60% 80% 100%

0.5 m

1.5m

3.0 m

3.5 m

% of DAPI Counts

CARD-FISH Counts

% Cren

% Bacteria

% Other

Figure 12. Flourescence imgage of cells displaying peanut-‐like morphology of marine Crenarchaeota.

hurley final report - marine biological laboratory ·...

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