1
Supporting Information
1)
Method of NH4+-N Isotopic Analysis
5
The technique used for the isolation of NH4+ for N stable isotope analysis is a
modified version of the acidified disk diffusion method (Brooks et al., 1989; Sørensen
and Jensen, 1991). In brief, dissolved NH4+ in the sample was converted to NH3 gas in a
sealed bottle. The NH3 was subsequently trapped on an acidified quartz disk enclosed in
10
a gas permeable, polytetrafluoroethylene (PTFE) membrane. PTFE traps were prepared
using pre-combusted, 5mm quartz disks punched from Whatman QMA filters. The disks
were acidified with 10μL of 0.5N H2SO4 immediately before placing onto a clean 1.25”
piece of ½” wide PTFE tape, which is folded over and sealed around the disk. PTFE
traps were stored in an air-tight container until used. The required sample volume
15
containing 12-30μg of NH4+-N was pipetted into 50mL serum bottles. A magnetic stir
bar and Nanopure DI was added to each bottle so the total volume was approximately
20mL. In order to prevent the PTFE traps from absorbing water and bursting, KCl was
added to each diffusion bottle to bring the KCl concentration of the sample solution to
4M. Phenolphthalein pH indicator solution was added to each diffusion bottle and the
20
samples are made basic by the drop-wise addition of a NaOH solution until they turn pink
(pH = 8-9). Immediately after the solution turned pink, 2mL of a tetraborate pH buffer
solution was added to stabilize the pH at about 9.5. A PTFE trap was then placed in the
diffusion bottle and the bottle stoppered. Diffusion bottles were placed on a magnetic
stirrer for 10 days to ensure complete diffusion of the NH3 onto the acidified disks.
1
2
25
Three internal
15
NH4+ isotope standards (15N = +0.6‰, +20.9‰, +35.2‰) were
included in each diffusion run.
All standards and samples are run in duplicate or
triplicate. Once diffusion was complete, the acidified disks were frozen, freeze-dried
overnight and then stored in air-tight vials.
The N isotopic composition of the diffusion disks containing the NH4+ as
30
(NH4)2SO4 were analyzed using a Carlo Erba 1108 Elemental Analyzer interfaced with a
Thermo Instruments Deltaplus – IRMS (Thermo Fisher Scientific, Milan Italy). NH4+-N
isotope ratios are reported in delta (δ) notation relative to atmospheric N2. The precision
associated with the 15N-NH4+ analysis was generally better than ±0.3‰.
35
2) Method of NO3--N Isotopic Analysis
The isolation of nitrate for isotopic analysis followed a modified version of the
ion exchange method described by Silva et al. (2000). Details of the modifications used
for nitrate isolation and isotopic analysis are described in Spoelstra et al. (2001, 2004).
40
Briefly, sulfate and phosphate were removed by precipitation with barium chloride.
Nitrate was collected by passing samples through a column containing BioRad AG 1-X8
anion exchange resin. Nitrate was then eluted using hydrochloric acid and the resulting
solution subsequently neutralized with silver oxide. After filtering to remove the AgCl
precipitate, the solution was freeze-dried to produce AgNO3.
45
Nitrate 15N values were determined by loading 0.4mg of silver nitrate with 12mg of sucrose into tin capsules for analysis on the same instrument used for the 15NNH4+ determination. All samples were run with sets of internal silver nitrate standards
2
3
that were previously calibrated against international 15N standards. A precision of
0.3‰ was attained for repeat analysis of standards and samples.
50
The N isotopic analysis of NH4+ disks and AgNO3 by isotope ratio mass
spectrometry was conducted by the Environmental Isotope Laboratory, University of
Waterloo. Results are reported in the standard δ-notation relative to the reference
standard (air).
55
3) Method of Bacterial Community Assessment
DNA analysis was determined on particulate material filtered from a 240 mL
ground water sample filtered through 0.22-μm Sterivex filters (Millipore). Nucleic acids
were extracted using a phenol chloroform extraction technique as described previously
(Neufeld et al., 2007). General bacterial 16S rRNA gene primers for denaturing gradient
60
gel electrophoresis (DGGE; GC-341f and 518r; Muyzer et al., 1993) and anammoxspecific 16S rRNA gene primers (An7f and An1388r; Penton et al., 2006) were used for
PCR. General bacterial 16S rRNA gene profiles were generated using DGGE primers.
Anammox-specific 16S rRNA gene profiles were generated using a ‘nested’ PCR
technique: PCR products from amplification with An7f and An1388r were diluted (10-2)
65
in water, and products were used as template for PCR with bacterial DGGE primers. PCR
amplification components (25 μl volume): 1.5 μl bovine serum albumin (10 mg ml-1;
Kreader, 1996), 2.5 μl Thermal Polymerase Buffer (10x; NEB), 0.05 μl dNTPs (100 mM;
NEB), 0.05 μl each forward and reverse primer (100 μM), 0.1 μl Taq DNA polymerase (5
U μl-1), and 1 μl extracted environmental nucleic acids (1 to 10 ng μl-1). PCR thermal
70
program for An7f and An1388r included an initial denaturation of 5 minutes at 95oC,
3
4
then 30 cycles of 60 seconds at 95 oC, 60 seconds at 63 oC, and 60 seconds at 72 oC, with
a final extension step of 7 minutes at 72 oC. For general bacterial 16S rRNA profiles (GC341f and 518r), initial denaturation was 5 minutes at 95 oC, then 30 cycles of 60 seconds
at 95 oC, 30 seconds at 55 oC, and 60 seconds at 72 oC, with a final extension step of 7
75
minutes at 72 oC. For anammox-specific profiles, the bacterial DGGE PCR was
conducted as above, but with only 20 cycles for the second (nested) amplification.
DGGE used a 30% to 70% denaturing gradient in 10% acrylamide gels; gels were
run for 14 hours at 85 V using the CBS system according to a previously published
protocol (Green et al. 2010). Bands were cut from the gel and sequenced at the Toronto
80
Centre for Applied Genomics (TCAG). DGGE band DNA sequences were manually
edited to correct base mis-calls, and primer sequences were removed. NCBI BLAST was
used to identify representative homologues. Sequenced DGGE bands were submitted to
Genbank with accession numbers HQ287903 - HQ287914.
85
90
4
5
95
Fig. S1. Long Point study site showing plan view of Tile bed 2 and location of multi-level
bundled piezometers,
100
105
5
6
NO3--N (mg/L)
100
Proximal shallow, LP100/122-2.0 m
Proximal deep, LP123-5.5 m
Distal mid-depth, LP124-3.6 m
Distal deep, LP124-4.5 m
75
50
25
0
1990
2000
2010
Date
110
Fig. S2. Long term trends of NO3- -N concentrations at selected monitoring points along
the plume core.
115
120
6
7
125
Fig. S3. Bromide concentrations (mg/L) in the Tile Bed 2 plume, 42 days after injecting
NaBr into the septic tank on September 9, 1990 (top) and 68 (middle) and 250 (bottom)
days after injecting NaBr on July 4, 2008.
7
8
130
Fig. S4. Distribution of: a) NO3--N, b) NH4+-N, c) δ15N-NH4+ and d) total inorganic
nitrogen (TIN) along the plume centerline, March 2, 2010
8
9
135
Nest 122
Tile Bed
Nest 138
7 m downgradient
Nest 124
17 m downgradient
9
N2O
NO 2
Elevation (m)
8
7
6
5
4
3
0
140
200 400 600 800
N (ug/L)
0
200 400 600 800
N (ug/L)
0
200 400 600 800
N (ug/L)
Fig. S5. Depth profiles of reaction intermediates NO2- and N2O, below the centre of the
tile bed (nest 122), 7 m down gradient (nest 138) and 17 m down gradient (nest 124).
N2O sampled June, 2008 (N2O data from Li, 2010); NO2- sampled April 1, 2010.
9