1
1
2
3 Jane M. Caffrey 1 , Nasreen Bano 2 , Karen Kalanetra 2, † , James T. Hollibaugh 2
4
5
6
1 Center for Environmental Diagnostics and Bioremediation, University of West Florida,
11000 University Parkway, Pensacola, FL 32514, USA 2 Department of Marine Science,
University of Georgia, Athens, GA 30602,USA
7
8
†
current address: Department of Public Health Sciences, University of California, Davis, CA
95616, USA
9 Supplemental Information
10 Methods
11 Sampling design.
Nine small sediment cores (4.6 cm ID) were collected at each site. Three
12 cores were sectioned and the surficial 0-0.5 cm layer was used for potential nitrification rate
13 measurements, community composition analysis, and determining porosity. Three cores
14 were used for analysis of extractable NH
4
+
and were cut into 0-0.5, 0.5-1, 1-2 and 2-4 cm
15 sections. Sediment chlorophyll a concentrations in the 0-0.5 cm layer were measured in
16 separate samples collected at the same time using a cut-off 10 mL syringe. Three cores were
17 sectioned under an N
2
atmosphere into 0-1, 1-2 and 2-4 cm layers for porewater sulfide and
18 reactive iron analysis. Values from the top layer are reported in Table 1.
19
20 Potential Nitrification . Six replicates per site were incubated aerobically at room
21 temperature on a shaker table. Approximately 0.5 g wet weight of sediment was added to
22 filtered water from sites amended to a final concentration of 0.5 mM NH
4
Cl in a total volume
23 of 50 ml (1). After 0.5 and 24 h, 15 mL aliquots were filtered through a GF/F filter and
24 frozen for later NO
2
-
and NO
3
-
+ NO
2
-
analysis. Room temperature during individual

2
25 incubations ranged from 20-26 °C, so rates were normalized to 21°C using the Arrhenius
26 equation and assuming Q
10
=2.0.
27 Sediment characteristics.
Extractable NH
4
+
concentrations were determined by adding 1 M
28 NaCl (10 mL/10 g wet sediment). Porewater sulfide was analyzed using an Ag/AgCl
29 electrode. Sediment chlorophyll samples were extracted in 10 mL of 90% acetone, sonicated
30 and read after 24 h on a Turner Designs™ fluorometer (2). Reactive Fe was measured in
31 sediments following removal of pore waters using a 0.5 N HCl extraction. Ferrozine was used
32 to measure Fe
2+
, hydroxylamine was used to reduce total iron to Fe
2+
. Fe
3+
was calculated by
33 difference.
34 Nutrient analysis. NO
3
-
, NH
4
+
, and NO
2
-
were analyzed on either a Bran and Lubbe AA3
35 autoanalyzer or a Lachet FIA autoanalyzer (3,4). Sediment extracts from Pensacola Bay
36 Shoal and from Apalachicola Bay Cat Point and Dry Bar sites were analyzed manually for
37 NH
4
+
(5).
38
39
40
41
42
Water quality data.
Temperature, salinity, DO, pH, turbidity and depth were measured using YSI datasondes at all sites except Pensacola Bay channel. Instrument calibration and data post processing followed National Estuarine Research Reserve System Wide Monitoring
Program protocols (6,7) for Apalachicola Bay, Elkhorn Slough, Rookery Bay, Sapelo Island, and Weeks Bay.
43 Real-time quantitative PCR was used to estimate the relative abundance of Bacteria and
44 Archaea amoA and 16S rRNA genes. Immediately following the collection of the samples,
45 DNA was extracted from the top 0.5 cm layer of sediment and used as template for PCR
46 amplifications. All samples were measured in triplicate using an iCycler real-time PCR
47 system (Bio-Rad, Hercules, CA) as described previously (see references, Table 4) and an
48 average value was calculated. Each sample was run for a total of 40 cycles and reactions were
49 performed in a 25 µl volume with 1 µL of DNA template. Bacteria and Archaea
16S rRNA
50 gene abundance was determined using TaqMan primers and probes. AOB and AOA amoA

51 gene abundance was quantified using SYBRgreen to measure amplicon production. The
52 primers and probes used in this study are listed in Table 4. TaqMan probes were 5' labeled
53 with FAM as the reporter dye and 3' labeled with TAMRA as the quencher dye. TaqMan
54 reaction mixtures contained 1.25 U Platinum Taq (Invitrogen), 0.25 U Amperase Uracil N-
55
56 glycosylase (Applied Biosystems, Foster City, CA), 3 mM MgCl, and 200 µM dNTPs with
400µM dUTP (Applied Biosystems) with primer and probe concentrations and PCR
57
58 conditions as described previously (see references, Table 4). SYBRgreen reaction mixtures contained 12.5 µl iQ SYBR ®
Green Supermix (Bio-Rad) with 500 nM primer concentration
59 and cycling conditions as described by Wuchter et al.(8). The plate read for AOB amoA
60 quantification was carried out at 80ºC to avoid detection of nonspecific products. Product
61 specificity was verified by melt curve analysis and by visualization in agarose gels of bands
62 of the expected size; ca. 500 bp for AOB and 260 bp for AOA. Analysis of the sequences of
63 DNA amplicons from randomly chosen samples further verified the specificity of the Q-PCR
64 quantification.
65
66 Standard curves were generated for each probe and/or primer set with serial dilutions of a
67 standard containing a known number of the target sequences. Bacterial 16S rRNA gene
68 standards used cloned 16S rRNA genes amplified from Escherichia coli K-12 ATCC 25196
69 DNA. Standards for Archaea 16S rRNA and AOB and AOA amoA genes were generated
70 from PCR amplicons cloned from environmental samples as follows. Target sequences were
71 amplified with the same primer sets used for quantification, then purified by agarose gel
72 electrophoresis. The PCR products were extracted with a QIAquick gel extraction kit
73 (Qiagen, Valencia, CA), cloned using a TOPO TA cloning kit with TOP10 chemically
74 competent cells (Invitrogen, Carlsbad, CA), then sequenced to verify specificity. A QIAprep
75 Spin Miniprep kit (Qiagen) was used to extract the plasmids. Linearized plasmids were
3

4
76 produced from cloned amplicons by digestion with Not I restriction enzyme (Promega,
77 Madison, WI), then run on an agarose gel and extracted with a QIAquick gel extraction kit
78 (Qiagen). DNA concentrations in the extracts were measured fluorometrically by PicoGreen
79 (Molecular Probes, Eugene, OR) staining on a Shimadzu RF5000U spectrofluorophotometer.
80 Gene abundance was calculated based on DNA concentration and plasmid plus insert
81 sequence size. Dilution series ranging from 10
1
to 10
7
or 10
8
copies µl
-1
were used for the
82 standard curves. To eliminate any differences in extraction efficiency between environmental
83 samples, we normalized by the 16S rRNA genes for the prokaryotic community in each
84 sample. Relative abundance of amoA genes is expressed by normalizing amoA gene
85 abundance to the number of copies of prokaryotic (Bacteria + Archaea) 16S rRNA genes in
86 each sample.
87
88 Statistics – Data were examined for normality. Potential nitrification, the relative abundance
89 of AOB amoA and AOA amoA were log transformed. A paired t-test was done to compare
90 AOB amoA and AOA amoA abundance. We performed a Pearson correlation analysis using
91 environmental variables (temperature, salinity, bottom water DO, sediment chlorophyll a ,
92 extractable NH
4
+ , reactive iron and pore water sulfide), potential nitrification rates,
93 abundances of AOB amoA and AOA amoA . In addition, a multiple regression analysis was
94 conducted examining the effect of salinity, bottom water DO, sediment chlorophyll a and
95 AOA amoA abundance on potential nitrification rates. SYSTAT version 9 was used for
96 statistical analysis.
97
98 References

5
99 1 Henriksen, K., Hansen, J.I., & Blackburn, T.H. Rates of nitrification, distribution of
100 nitrifying bacteria, and nitrate fluxes in different types of sediment from Danish
101 waters. Mar. Biol.
61 ,299-304 (1981).
102 2 Welschmeyer, N.A.. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b
103
105 and pheopigments. Limnol. and Oceanogr.
39 , 1985-1992 (1994)
104 3 Diamond, D.H. Determination of ammonia in brackish or seawater by flow injection analysis colorimetry. QuikChem Method 31-107-06-1-C for Lachat Instruments.
106 Milwaukee, WI. (1997a)
107 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
110 precise method for measuring ammonium in marine and freshwater ecosystems. Can.
111 J. Fish. Aquat Sci.
56 ,1801–1808 (1999).
112 6 Wenner, E.L., Holland, A.F., Arendt, M.D., Edwards, D., & Caffrey, J.M. A Synthesis of
113
114
115
Water Quality Data from the National Estuarine Research Reserve System-Wide
Monitoring Program. NOAA Grant NA97OR0209, MRD Contribution No. 459.
NOAA. (2001).
116 7 National Oceanic and Atmospheric Administration, Office of Ocean and Coastal Resource
117 Management, National Estuarine Research Reserve System-wide Monitoring
118
119
Program. Centralized Data Management Office, Baruch Marine Field Lab, University of South Carolina http://cdmo.baruch.sc.edu
. (2004).
120 8 Wuchter, C., Abbas, B., Coolen, M. J. L., Herfort, L., van Bleijswijk, J. et al. Archaeal
121 nitrification in the ocean. Proc. Natl. Acad. Sci. USA . 103 ,12317-12322 (2006).

122 9 Suzuki, M. T., Taylor, L. T. & DeLong, E. F. Quantitative analysis of small-subunit rRNA
123 genes in mixed microbial populations via 5'-nuclease assays. Appl. Environ.
124 Microbiol.
66 , 4605-4614 (2000).
125 10 Rotthauwe, J.-H., Witzel, K.-P. & Liesack, W. The ammonia monooxygenase structural
126
127 gene amoA as a functional marker: molecular fine-scale analysis of natural ammoniaoxidizing populations. Appl. Environ. Microbiol.
63 , 4704-4712 (1997).
128 11 Hornek, R., A. Pommerening-Röser, H.-P. Koops, A. H. Farnleitner, N. Kreuzinger, A.
129
130
131
132
Kirschner, and R. L. Mach. Primers containing universal bases reduce multiple amoA gene specific DGGE band patterns when analyzing the diversity of beta-ammonia oxidizers in the environment. J. Microbiol. Methods. 66:147-155. (2006).
6

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 NH
4
+ 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 Porewater
Longitude
Number of times
Salinity DO mg L
-1
Sediment
Chlorophyll a
µg cm -2
Extractable
NH
4
+ nmol cm
-3
Sulfide mM sampled
PB Pensacola Bay, FL
Channel 30° 27.40’N
87° 7.94’W
3 26
22-28
2.1
0.4-4.1
1.9 148 0.1
Shoal
WKB Weeks Bay, AL
Mid Bay
Fish River
30° 27.53’N
87° 06.12’W
30° 23.90’N
87° 49.65’ W
30° 24.97'N
4
4
4
18 7.6
5.4-24 0.4-9.4
10
0-19
8
5.5
0.2-10.9
3.6
4.0
1.1
1.9
1.7
218
158
0.2
1.6
1.4
7

SAP Sapelo Island, GA
Dean Creek
Hunt Camp
Marsh Landing
APA Apalachicola Bay,
FL
Dry Bar
Cat Point
East Bay
87° 49.37'W
31° 23.38N
81° 16.74W
31° 28.72'N,
81° 16.38'W
31° 25.07' N,
81° 17.77'W
29º 40.48' N,
85º 3.50' W
29

42.12N,
84

52.81' W
29º 47.15' N,
84º 52.52' W
2
2
2
0-17 0.0-9.0
26 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
1
1
1
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
3.5
0.6
0.9
3.4
16.2
1.7
51.2
49.2
70.9
66.8
12.5
329
0.5
0.5
0.2
0
0
0.1
8

RKB Rookery Bay, FL
Fakahatchee Bay
Blackwater River
Henderson Creek
25° 53.53’N
81° 28.62’W
25° 56.06’N
81° 35.68’W
26° 1.54’N
81° 43.99’W
ELK Elkhorn Slough, CA
Azevedo Pond
South Marsh
36° 50.83'N
121° 45.27’W
36° 49.08’N
North Marsh
121° 44.30’W
36

50.08’N,
121

44.30’W
1
1
1
1
1
1
31
19-32
5.2
3.2-9.9
34
32-35
5.5
3.0-7.6
31
31-35
6.1
4.9-9.9
38 4.6
33-39 0.8-11.4
34
32-34
36
27-38
3.4
1.2-5.3
4.7
6.6
7.5
7.8
28.7
11.3
28.1
0
0
0.2
0.1
0.1
0.1
78.7
98.3
104
43.6
66.7
114
9

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.
Temperature
AOB amoA
-0.55
0.003
28
AOA amoA
-0.40
0.04
28
Salinity
Porewater sulfide
AOB amoA
0.16
0.39
30
-0.21
0.32
25
1
0.51
0.004
30
-0.46
0.02
25
AOA amoA
0.56
0.01
30
1 0.56
0.001
30
10

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 significance Beta Standard
Coefficient
Sediment chlorophyll a 0.89
Salinity -0.56
DO -0.48
AOA amoA abundance 0.32
0.001
0.008
0.004
0.05
11

Table 4 – Primers and probes used for QPCR analysis
Name Target
Bacteria
Archaea
Ammonia oxidizing bacteria
Ammonia oxidizing archaea
Gene Primer or
Probe
16S rRNA primer
16S rRNA primer
16S rRNA probe
16S rRNA primer
16S rRNA primer
16S rRNA primer
16S rRNA probe amoA amoA primer primer amoA amoA primer primer
Sequence (5' to 3') Reference
BACT1369F CGGTGAATACGTTCYCGG
PROK1492R
Tm1389F
CGWTACCTTGTTACGACTT
CTTGTACACACCGCCCGTC
ARCH1-1369F CGGTGAATACGTCCCTGC
ARCH2-1369F CGGTGAATATGCCCCTGC
PROK1541R
Tm1389F
AAGGAGGTGATCCRGCCGCA
CTTGTACACACCGCCCGTC
9
9
9
9
9
9
9 amoA -1F GGGGTTTCTACTGGTGGT 10 amoAr NEW CCCCTCBGSAAAVCCTTCTTC 11
ArchamoA -for CTGAYTGGGCYTGGACATC 8
ArchamoA -rev TTCTTCTTTGTTGCCCAGTA 8
12