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Ammonia oxidation and ammonia-oxidizing Bacteria and
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Archaea from estuaries with differing histories of hypoxia
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Jane M. Caffrey1, Nasreen Bano2, Karen Kalanetra2, †, James T. Hollibaugh2
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11000 University Parkway, Pensacola, FL 32514, USA 2Department of Marine Science,
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University of Georgia, Athens, GA 30602,USA
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†
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95616, USA
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Supplemental Information
Center for Environmental Diagnostics and Bioremediation, University of West Florida,
current address: Department of Public Health Sciences, University of California, Davis, CA
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Methods
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Sampling design. Nine small sediment cores (4.6 cm ID) were collected at each site. Three
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cores were sectioned and the surficial 0-0.5 cm layer was used for potential nitrification rate
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measurements, community composition analysis, and determining porosity. Three cores
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were used for analysis of extractable NH4+ and were cut into 0-0.5, 0.5-1, 1-2 and 2-4 cm
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sections. Sediment chlorophyll a concentrations in the 0-0.5 cm layer were measured in
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separate samples collected at the same time using a cut-off 10 mL syringe. Three cores were
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sectioned under an N2 atmosphere into 0-1, 1-2 and 2-4 cm layers for porewater sulfide and
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reactive iron analysis. Values from the top layer are reported in Table 1.
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Potential Nitrification. Six replicates per site were incubated aerobically at room
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temperature on a shaker table. Approximately 0.5 g wet weight of sediment was added to
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filtered water from sites amended to a final concentration of 0.5 mM NH4Cl in a total volume
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of 50 ml (1). After 0.5 and 24 h, 15 mL aliquots were filtered through a GF/F filter and
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frozen for later NO2- and NO3- + NO2- analysis. Room temperature during individual
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incubations ranged from 20-26 °C, so rates were normalized to 21°C using the Arrhenius
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equation and assuming Q10=2.0.
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Sediment characteristics. Extractable NH4+ concentrations were determined by adding 1 M
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NaCl (10 mL/10 g wet sediment). Porewater sulfide was analyzed using an Ag/AgCl
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electrode. Sediment chlorophyll samples were extracted in 10 mL of 90% acetone, sonicated
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and read after 24 h on a Turner Designs™ fluorometer (2). Reactive Fe was measured in
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sediments following removal of pore waters using a 0.5 N HCl extraction. Ferrozine was used
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to measure Fe2+, hydroxylamine was used to reduce total iron to Fe2+. Fe3+ was calculated by
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difference.
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Nutrient analysis. NO3-, NH4+, and NO2- were analyzed on either a Bran and Lubbe AA3
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autoanalyzer or a Lachet FIA autoanalyzer (3,4). Sediment extracts from Pensacola Bay
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Shoal and from Apalachicola Bay Cat Point and Dry Bar sites were analyzed manually for
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NH4+ (5).
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Water quality data. Temperature, salinity, DO, pH, turbidity and depth were measured
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using YSI datasondes at all sites except Pensacola Bay channel. Instrument calibration and
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data post processing followed National Estuarine Research Reserve System Wide Monitoring
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Program protocols (6,7) for Apalachicola Bay, Elkhorn Slough, Rookery Bay, Sapelo Island,
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and Weeks Bay.
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Real-time quantitative PCR was used to estimate the relative abundance of Bacteria and
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Archaea amoA and 16S rRNA genes. Immediately following the collection of the samples,
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DNA was extracted from the top 0.5 cm layer of sediment and used as template for PCR
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amplifications. All samples were measured in triplicate using an iCycler real-time PCR
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system (Bio-Rad, Hercules, CA) as described previously (see references, Table 4) and an
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average value was calculated. Each sample was run for a total of 40 cycles and reactions were
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performed in a 25 µl volume with 1 µL of DNA template. Bacteria and Archaea 16S rRNA
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gene abundance was determined using TaqMan primers and probes. AOB and AOA amoA
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gene abundance was quantified using SYBRgreen to measure amplicon production. The
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primers and probes used in this study are listed in Table 4. TaqMan probes were 5' labeled
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with FAM as the reporter dye and 3' labeled with TAMRA as the quencher dye. TaqMan
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reaction mixtures contained 1.25 U Platinum Taq (Invitrogen), 0.25 U Amperase Uracil N-
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glycosylase (Applied Biosystems, Foster City, CA), 3 mM MgCl, and 200 µM dNTPs with
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400µM dUTP (Applied Biosystems) with primer and probe concentrations and PCR
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conditions as described previously (see references, Table 4). SYBRgreen reaction mixtures
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contained 12.5 µl iQ SYBR®Green Supermix (Bio-Rad) with 500 nM primer concentration
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and cycling conditions as described by Wuchter et al.(8). The plate read for AOB amoA
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quantification was carried out at 80ºC to avoid detection of nonspecific products. Product
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specificity was verified by melt curve analysis and by visualization in agarose gels of bands
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of the expected size; ca. 500 bp for AOB and 260 bp for AOA. Analysis of the sequences of
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DNA amplicons from randomly chosen samples further verified the specificity of the Q-PCR
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quantification.
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Standard curves were generated for each probe and/or primer set with serial dilutions of a
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standard containing a known number of the target sequences. Bacterial 16S rRNA gene
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standards used cloned 16S rRNA genes amplified from Escherichia coli K-12 ATCC 25196
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DNA. Standards for Archaea 16S rRNA and AOB and AOA amoA genes were generated
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from PCR amplicons cloned from environmental samples as follows. Target sequences were
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amplified with the same primer sets used for quantification, then purified by agarose gel
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electrophoresis. The PCR products were extracted with a QIAquick gel extraction kit
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(Qiagen, Valencia, CA), cloned using a TOPO TA cloning kit with TOP10 chemically
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competent cells (Invitrogen, Carlsbad, CA), then sequenced to verify specificity. A QIAprep
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Spin Miniprep kit (Qiagen) was used to extract the plasmids. Linearized plasmids were
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produced from cloned amplicons by digestion with NotI restriction enzyme (Promega,
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Madison, WI), then run on an agarose gel and extracted with a QIAquick gel extraction kit
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(Qiagen). DNA concentrations in the extracts were measured fluorometrically by PicoGreen
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(Molecular Probes, Eugene, OR) staining on a Shimadzu RF5000U spectrofluorophotometer.
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Gene abundance was calculated based on DNA concentration and plasmid plus insert
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sequence size. Dilution series ranging from 101 to 107 or 108 copies µl-1 were used for the
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standard curves. To eliminate any differences in extraction efficiency between environmental
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samples, we normalized by the 16S rRNA genes for the prokaryotic community in each
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sample. Relative abundance of amoA genes is expressed by normalizing amoA gene
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abundance to the number of copies of prokaryotic (Bacteria + Archaea) 16S rRNA genes in
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each sample.
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Statistics – Data were examined for normality. Potential nitrification, the relative abundance
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of AOB amoA and AOA amoA were log transformed. A paired t-test was done to compare
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AOB amoA and AOA amoA abundance. We performed a Pearson correlation analysis using
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environmental variables (temperature, salinity, bottom water DO, sediment chlorophyll a,
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extractable NH4+, reactive iron and pore water sulfide), potential nitrification rates,
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abundances of AOB amoA and AOA amoA. In addition, a multiple regression analysis was
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conducted examining the effect of salinity, bottom water DO, sediment chlorophyll a and
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AOA amoA abundance on potential nitrification rates. SYSTAT version 9 was used for
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statistical analysis.
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References
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1 Henriksen, K., Hansen, J.I., & Blackburn, T.H. Rates of nitrification, distribution of
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nitrifying bacteria, and nitrate fluxes in different types of sediment from Danish
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waters. Mar. Biol. 61,299-304 (1981).
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103
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2 Welschmeyer, N.A.. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b
and pheopigments. Limnol. and Oceanogr. 39, 1985-1992 (1994)
3 Diamond, D.H. Determination of ammonia in brackish or seawater by flow injection
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analysis colorimetry. QuikChem Method 31-107-06-1-C for Lachat Instruments.
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Milwaukee, WI. (1997a)
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4 Diamond, D.H. Determination of nitrate in brackish or seawater by flow injection analysis.
108
QuikChem Method 31-107-04-1-A for Lachat Instruments. Milwaukee, WI. (1997b)
109
5 Holmes, R.M., Aminot, A., Kerouel, R., Hooker, B.A., & Peterson, B.J. A simple and
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precise method for measuring ammonium in marine and freshwater ecosystems. Can.
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J. Fish. Aquat Sci. 56,1801–1808 (1999).
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6 Wenner, E.L., Holland, A.F., Arendt, M.D., Edwards, D., & Caffrey, J.M. A Synthesis of
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Water Quality Data from the National Estuarine Research Reserve System-Wide
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Monitoring Program. NOAA Grant NA97OR0209, MRD Contribution No. 459.
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NOAA. (2001).
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7 National Oceanic and Atmospheric Administration, Office of Ocean and Coastal Resource
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Management, National Estuarine Research Reserve System-wide Monitoring
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Program. Centralized Data Management Office, Baruch Marine Field Lab, University
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of South Carolina http://cdmo.baruch.sc.edu. (2004).
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8 Wuchter, C., Abbas, B., Coolen, M. J. L., Herfort, L., van Bleijswijk, J. et al. Archaeal
nitrification in the ocean. Proc. Natl. Acad. Sci. USA. 103,12317-12322 (2006).
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9 Suzuki, M. T., Taylor, L. T. & DeLong, E. F. Quantitative analysis of small-subunit rRNA
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genes in mixed microbial populations via 5'-nuclease assays. Appl. Environ.
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Microbiol. 66, 4605-4614 (2000).
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10 Rotthauwe, J.-H., Witzel, K.-P. & Liesack, W. The ammonia monooxygenase structural
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gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-
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oxidizing populations. Appl. Environ. Microbiol. 63, 4704-4712 (1997).
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11 Hornek, R., A. Pommerening-Röser, H.-P. Koops, A. H. Farnleitner, N. Kreuzinger, A.
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Kirschner, and R. L. Mach. Primers containing universal bases reduce multiple amoA
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gene specific DGGE band patterns when analyzing the diversity of beta-ammonia
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oxidizers in the environment. J. Microbiol. Methods. 66:147-155. (2006).
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Table 1 – Location of sampling sites, number of times they were sampled, bottom water salinity, bottom water dissolved oxygen (DO)
concentration (mg L-1), sediment chlorophyll a (µg cm-2), extractable NH4+ concentration (nmol cm-3), and porewater sulfide (mM).
Mean values and ranges for salinity and DO include data from the week preceding sampling date.
Estuary/site
Latitude
Longitude
Number Salinity
DO
Sediment
Extractable
Porewater
Chlorophyll a
NH4+
Sulfide
mg L-1
µg cm-2
nmol cm-3
mM
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2.1
1.9
148
0.1
22-28
0.4-4.1
18
7.6
4.0
1.7
0.2
5.4-24
0.4-9.4
10
5.5
1.1
218
1.6
0-19
0.2-10.9
8
3.6
1.9
158
1.4
of
times
sampled
PB Pensacola Bay, FL
Channel
30° 27.40’N
3
87° 7.94’W
Shoal
30° 27.53’N
4
87° 06.12’W
WKB Weeks Bay, AL
Mid Bay
30° 23.90’N
4
87° 49.65’ W
Fish River
30° 24.97'N
4
8
87° 49.37'W
0-17
0.0-9.0
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4.1
9.4-33
1.5-8.6
26
5.3
10.5-29
0.3-9.8
22
4.6
9.4-33
2.5-8.6
9
6.3
3.6-24
1.7-8.9
29
5.5
12.1-28
0.4-6.8
11
4.8
0.3-16
1.5-8.7
SAP Sapelo Island, GA
Dean Creek
31° 23.38N
2
81° 16.74W
Hunt Camp
31° 28.72'N,
2
81° 16.38'W
Marsh Landing
31° 25.07' N,
2
81° 17.77'W
3.5
70.9
0.2
0.6
51.2
0.5
0.9
49.2
0.5
3.4
66.8
0
16.2
12.5
0
1.7
329
0.1
APA Apalachicola Bay,
FL
Dry Bar
29º 40.48' N,
1
85º 3.50' W
Cat Point
2942.12N,
1
8452.81' W
East Bay
29º 47.15' N,
84º 52.52' W
1
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RKB Rookery Bay, FL
Fakahatchee Bay
25° 53.53’N
1
81° 28.62’W
Blackwater River
25° 56.06’N
1
81° 35.68’W
Henderson Creek
26° 1.54’N
1
81° 43.99’W
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5.2
19-32
3.2-9.9
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5.5
32-35
3.0-7.6
31
6.1
31-35
4.9-9.9
38
4.6
33-39
0.8-11.4
34
3.4
32-34
1.2-5.3
36
4.7
6.6
43.6
0.1
7.5
66.7
0.1
7.8
114
0.1
28.7
78.7
0
11.3
98.3
0
28.1
104
0.2
ELK Elkhorn Slough, CA
Azevedo Pond
36° 50.83'N
1
121° 45.27’W
South Marsh
36° 49.08’N
1
121° 44.30’W
North Marsh
3650.08’N,
12144.30’W
1
27-38
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Table 2- Correlation coefficients for AOB and AOA amoA gene abundances normalized to
total prokaryote rRNA gene abundance and environmental variables. Pearson correlation
coefficients, p values and n are listed.
AOB amoA AOA amoA
Temperature
Salinity
Porewater sulfide
AOB amoA
-0.55
-0.40
0.003
0.04
28
28
0.16
0.51
0.39
0.004
30
30
-0.21
-0.46
0.32
0.02
25
25
1
0.56
0.01
30
AOA amoA
0.56
0.001
30
1
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Table 3 – Multiple regression coefficients for the relationship between potential nitrification
and environmental variables (salinity, DO, sediment chlorophyll a,) and abundance of AOA
amoA genes. Standardized coefficients and levels of significance (p values) are listed.
Variable
Beta Standard
significance
Coefficient
Sediment chlorophyll a 0.89
0.001
Salinity
-0.56
0.008
DO
-0.48
0.004
AOA amoA abundance
0.32
0.05
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Table 4 – Primers and probes used for QPCR analysis
Target
Gene
Primer or
Name
Sequence (5' to 3')
Reference
Probe
Bacteria
Archaea
Ammonia oxidizing bacteria
Ammonia oxidizing archaea
16S rRNA
primer
BACT1369F
CGGTGAATACGTTCYCGG
9
16S rRNA
primer
PROK1492R
CGWTACCTTGTTACGACTT
9
16S rRNA
probe
Tm1389F
CTTGTACACACCGCCCGTC
9
16S rRNA
primer
ARCH1-1369F
CGGTGAATACGTCCCTGC
9
16S rRNA
primer
ARCH2-1369F
CGGTGAATATGCCCCTGC
9
16S rRNA
primer
PROK1541R
AAGGAGGTGATCCRGCCGCA
9
16S rRNA
probe
Tm1389F
CTTGTACACACCGCCCGTC
9
amoA
primer
amoA-1F
GGGGTTTCTACTGGTGGT
10
amoA
primer
amoAr NEW
CCCCTCBGSAAAVCCTTCTTC
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amoA
primer
Arch-amoA-for
CTGAYTGGGCYTGGACATC
8
amoA
primer
Arch-amoA-rev
TTCTTCTTTGTTGCCCAGTA
8