1 A horizontal permeable reactive barrier stimulates nitrate removal

2 and shifts microbial ecology during rapid infiltration for managed

3 recharge

4 Sarah Beganskasa*, Galen Gorskia, Tess Weathersa, Andrew T. Fishera, Calla Schmidtc, Chad 5 Saltikovb, Kaitlyn Redfordb, Brendon Stoneburnerb, Ryan Harmona1, Walker Weira2 6 7 aEarth and Planetary Sciences, University of California Santa Cruz, Santa Cruz, California, 95064 8 bMicrobiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, 9 California, 95064 10 cEnvironmental Science, University of San Francisco, San Francisco, California, 94117 11 1Present address: Hydrologic Science and Engineering, Colorado School of Mines, Golden, 12 Colorado, 80401 13 2Present address: Hydrologic Sciences, University of Nevada Reno, Reno, NV, 89557 14 *Corresponding author, [email protected] 15 16 SUPPORTING INFORMATION 17 29 pages; 5 figures; 9 tables 18 19 DETAILED MATERIALS AND METHODS 20 21 Infiltration testing. 22 The infiltration capacity of soils has been measured using a variety of tools and methods in the 23 last century (Alagna et al., 2016; Burgy and Luthin, 1956; Youngs, 1991). The de-facto standard 24 for such measurements remains the single- or double-ring infiltrometer (Ahmed et al., 2014; 25 ASTM Standard D3385, 2009; Fatehnia et al., 2016; Ruggenthaler et al., 2016), despite the well- 26 known issue of lateral flow in soils below the instruments (Bouwer et al., 1999; Marshall and 27 Stirk, 1950). For studies that seek to resolve fundamental soils properties such as saturated 28 hydraulic conductivity or saturation parameters, this requires application of analytical corrections 29 to account for lateral flow, and/or specialized instrumentation that establishes boundary 30 conditions needed to focus on parameters of interest (Alagna et al., 2016; Bagarello et al., 2016; 31 Bouwer et al., 1999). 32 33 The issue of lateral flow is related to infiltration measurement scale: for a larger area of testing, 34 there is a smaller ratio of test site circumference to test area. In addition, all else being equal, 35 larger test areas are more likely to encounter the fastest infiltration pathways in heterogeneous 36 soils; this helps explain why tests of larger areas tend to yield higher infiltration rates compared 37 to tests of the same sites using smaller areas, and why tests of smaller areas tend to indicate high 38 heterogeneity (Burgy and Luthin, 1956; Fatehnia et al., 2016; Heeren et al., 2015, 2014; 39 Khodaverdiloo et al., 2017; Lucke et al., 2014). Larger test areas also present practical 40 challenges, generally requiring greater construction effort, higher cost for supplies, and a larger 41 water supply. The latter is a particular limitation for tests run for more than a few hours. 42 43 In this study, we were mainly interested in determining the total and vertical infiltration rate over 44 a 10–15-day experimental period, rather than transient response during early time, and were 45 limited as to the rate at which water could be supplied, conveyed, and applied to the test plots. 46 Given expected rapid infiltration rates in coarse and vegetated soils, we elected to construct 1 m2 47 plots to measure total infiltration by mass balance and vertical infiltration using heat as a tracer 48 (Hatch et al., 2006; Racz et al., 2011). Past experience with the test plot configuration suggested 49 that we could expect a ratio of total to vertical infiltration of 8:1 to 12:1 (as was subsequently 50 observed). This plot size is larger than that commonly applied to field infiltration testing using 51 rings or disk infiltrometers and is consistent with plot sizes used in other field studies (ASTM 52 Standard D3385, 2009; Heeren et al., 2015, 2014; Khodaverdiloo et al., 2017; Reynolds et al., 53 2000; Youngs, 1991). In addition, temperature probes placed near the center of these plots to 54 assess vertical infiltration should be far enough from the plot margins to avoid being influenced 55 by them: the characteristic length scale of measurement for these probes can be estimated as L = 56 (α t)1/2, where α = thermal diffusivity (~10-7 m2/s), and t = time (86,400 s for diel temperature 57 variations). For this experimental configuration, L ~10 cm. 58 59 Plot construction, operation, and sampling. 60 We hand-excavated four 1 × 1 m plots for the tests. The native soil plots were ~60 cm deep, 61 whereas the PRB plots were ~90 cm deep to allow for installation of a 30-cm-thick layer of 62 redwood chips at the bottom. The redwood chips were sourced from a local landscape supply 63 store. The plots were lined with fiberglass siding to prevent water from flowing sideways above 64 the base of the plots. We assembled the four plot walls as a mechanical unit and lowered them 65 into position using rope handles. We filled in gaps around the outsides of the walls with 66 bentonite chips at the base, activated with water to form a seal, and topped with native soil. 67 68 We installed two thermal probes in each plot, one attached to a real-time data telemetry system, 69 and one autonomous (recording internally). For each probe, a pair of thermal sensors (resolution 70 ± 0.015°C) were installed in polyvinyl chloride tubes, placed at 5 and 20 cm below the base of 71 the plot. The probes were placed in hand-augered holes (7-cm diameter) and surrounded by silica 72 slurry to ensure good thermal contact with the soil and prevent fluid from flowing vertically 73 along the annulus around the probes. Data with a 15-minute sampling rate from these probes was 74 processed to calculate vertical infiltration rates. Filtered thermal records from these probes were 75 compared to assess the magnitude of amplitude reduction with depth over time, using a band- 76 pass filter to isolate the frequency of variation with the greatest power (Hatch et al., 2006). 77 Greater downward infiltration is associated with a smaller amplitude reduction in a monotonic 78 but non-linear relationship. This method is based on a transient equation for conductive- 79 advective-dispersive heat transport during steady-state fluid flow, allowing assessment across a 80 wide range of infiltration rates (cm/d to m/d). We used physical and thermal sediment properties 81 from a previous analysis at this field site (Racz et al., 2011). Autonomous probes were recovered 82 after each experiment; real-time probes telemetered data using a dedicated logger and cellular 83 modem, with hourly uploads. 84 85 We installed two piezometer nests in each plot to allow sampling at depths of 30, 55, and 80 cm 86 below the base of the plot. Each fluid sampling piezometer was constructed from 1-cm diameter 87 rigid polycarbonate tubing (6 mm inner diameter, ID), perforated at the base and wrapped with a 88 fine mesh nylon screen. A piezometer was also installed within the redwood chip layer for the 89 PRB plots. Nylon tubing with a 6 mm ID extended from the piezometers to the side of the plot 90 for fluid sampling. Each piezometer was assembled and acid-washed prior to deployment. The 91 piezometers were installed at targeted depth into a 7-cm outer diameter (OD) hand-augered hole 92 in the base of the plot. The walls of these holes were gently scoured to break up a “skin” that 93 may have developed during augering. A coarse, well-rounded quartz sand/gravel filter (10–20 94 mesh) was installed around each screen, followed by a 15 cm-thick bentonite seal topped with 95 native soil. Piezometers were developed after installation (and after allowing time for the 96 bentonite seals to set) to ensure a good connection with the formation. We used a peristaltic 97 pump to flush de-ionized (MilliQ) water back and forth (gently) through the piezometer screens 98 and the surrounding sand/gravel pack. 99 100 The same peristaltic pump was used with a manifold to collect fluid samples daily from the 101 piezometers during plot operation. We pumped sampled fluid through a 0.45 µm cellulose 102 acetate filter into acid-washed polyethylene sample bottles. The bottles were pre-rinsed with 103 sample water prior to filling. The pump, manifold, and filter system were flushed with de-ionized 104 (>12 MOhm) water between samples. We collected individual replicate fluid samples to be 105 analyzed for nitrogen species, dissolved organic carbon (DOC), and nitrogen and oxygen 106 isotopes. The samples were put on ice in the field immediately and frozen upon return until 107 analysis. Samples could only be collected if the soil surrounding the piezometer was saturated. 108 To verify the accuracy of our internal results, twice during each test we collected additional fluid 109 samples to be run at Monterey Bay Analytical Services (MBAS, ELAP #2385). These samples 110 were collected, placed on ice in the field, delivered directly to MBAS, and analyzed within 48 111 hours. 112 113 Before and after the two-week infiltration test, we collected 1-m soil cores using a hand auger. 114 We photographed and sampled the cores at 10–20 cm intervals. We collected separate samples 115 for grain size analysis, total C and N, and microbiological analysis. Soil samples were put on ice 116 immediately and frozen until analysis. 117 118 For the PRB plots, after the subsurface instruments were installed, we added a 30-cm thick layer 119 of redwood chips. An additional sample tube terminated like a mini piezometer was installed in 120 each PRB. We put a nylon mesh screen (18x18 thread per cm2) on top of the woodchips and 121 weighted it down with washed river rocks from a local garden supply. For consistency, we 122 placed identical nylon mesh and river rocks above the native soil plots. 123 124 A hose delivered water to the plots mainly from a shallow groundwater well, recovering water 125 from the Harkins Slough MAR system. For most of the time when these experiments were run, 126 the water applied was composed entirely of shallow groundwater. But because the experimental 127 water supply fitting was connected to a distribution pipe for agency “delivered water,” which 128 mixes MAR recovery, groundwater from farther back in the basin, and recycled water 129 (tertiary treated and disinfected wastewater),a component of delivered water was applied on 130 some days. As noted in the main text, this was readily detectable because of elevated ammonium 131 concentrations. Sample data were not used for denitrification calculations from days when this 132 mixing occurred. 133 134 The inflow hose was anchored at the plot corner, and an electronic flow meter recorded flow 135 with a pulse logger. A float valve connected to a solenoid valve at the end of the hose controlled 136 water delivery, stopping inflow when the water level reached a desired height, and restarting 137 flow when the water level dropped below a second height. Each fill cycle lasted 10–20 minutes. 138 This system was powered using batteries, trickle charged with a solar panel. We operated each 139 test for 13–15 days. 140 141 We installed an autonomous pressure gauge (2-minute sampling rate) in a PVC stilling well at 142 the base of each plot. We converted pressure to water depth, correcting for barometric pressure, 143 measured simultaneously on site with an atmospheric gauge. We converted pressure gauge 144 readings to water level using a staff plate positioned on the side of the plot and monitored 145 throughout the tests. For each fill-empty cycle, we divided the change in water level by the time 146 elapsed to calculate the bulk infiltration rate (see inset diagram in Figure 1C). 147 148 Phylogenetic sequencing: Primer specifications, amplification protocols, and additional method 149 details. 150 Partial 16S rRNA gene (V4 and V5 variable regions) was amplified using primers (Parada et al., 151 2016) modified to contain 5’ sequencing adapters (underlined) for barcoding and sequencing 152 using the Illumina MiSeq platform: 153 154 515F-Y-MS 155 (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA-3’) 156 926R-MS 157 (5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCGYCAATTYMTTTRAGTTT-3’) 158 159 PCR methods were based on Klindworth et al. (2013) and were further optimized for Illumina 160 MiSeq sequencing platform (Illumina Inc., 2013). PCRs consisted of 3 ng DNA extract, 0.4 µM 161 each forward and reverse primer, 1 µL Titanium Taq DNA polymerase (Clonetech), 2.5µL 25 162 mM dNTP mix (New England BioLabs), 5 µL 10X MasterAmp PCR Enhancer (Illumina), 5 µL

163 10X Titanium Taq DNA polymerase buffer (Clonetech), 3.5µL 25 mM MgCl2 (New England 164 BioLabs) and was adjusted to 50 µL with nuclease-free water. The following thermocycler 165 profile was used: 95˚C 3 min initial denaturation, 25 cycles of 95˚C 45 s, 50˚C 45 s, 68˚C 90 s, 166 and a final extension at 68˚C for 5 min followed by a 4˚C hold step. Samples were analyzed by 167 agarose gel electrophoresis to confirm the presence of ~550 bp amplicons. A PCR amplicon 168 sequencing pipeline was adapted from the Illumina MiSeq platform sequencing protocol for 16S 169 metagenomic sequencing libraries (Illumina Inc., 2013). 170 171 After the primary PCR described above, the amplicons were cleaned up using AMPure XP 172 magnetic beads (Agencourt) (0.8X final concentration). The Illumina sequencing indices were 173 added to the cleaned-up PCR amplicons using a PCR step similar to the one described above 174 except 5 µL of each Nextera XT index primer 1 and index primer 2 were used in place of the 175 forward and reverse 16S rRNA gene primers. The index primers anneal to the 5’ adapter 176 sequences. Each PCR amplicon received a unique combination of index primer 1 and 2. The 177 thermocycle protocol for Index PCR consisted of: 95˚C for 3 min, 8 cycles of 95˚C for 30 s, 178 55˚C for 30 s, and 72˚C for 30 s, and a final extension at 72˚C for 5 min with a 4˚C hold. 179 180 Indexed PCR amplicons were purified using AMPure XT magnetic beads (1.12X final 181 concentration) and eluted in a final volume of 25 µL. Library preparation was validated by 182 analyzing indexed PCR amplicons using a DNA 100 High Sensitivity Bioanalyzer chip. This 183 step verified the correct size and the removal of primers. The final steps before sequencing 184 included: quantify the DNA concentration for each library using a Qubit 4 Fluorometer 185 (Invitrogen); normalizing each library to 4 nM using 10 mM Tris pH 8.5, and pooling each 186 normalized library (5 µL each) into one tube. A PhiX control-spike (20%) was added to the 187 pooled library as a technical control to improve base-calling during the sequencing run, as 188 recommended by Illumina if the DNA libraries have a low sequence diversity.(Illumina Inc., 189 2013) The pooled library was sequenced on the Illumina MiSeq (600 cycles v3 PE300 flow cell 190 kit) at the University of California, Davis Genome Center. 191 192 Plugins for taxonomy visualization, alignment, tree generation, diversity, differential abundance 193 Taxonomy was tabulated and visualized with the metadata and taxa plugins, respectively 194 (https://github.com/qiime2/q2-metadata, https://github.com/qiime2/q2-taxa). Alignment and 195 masking of highly variable regions was performed with alignment (Katoh and Standley, 2013). 196 Trees were generated using FastTree 2 (Price et al., 2010), community difference metrics were 197 calculated with Faith’s Phylogenetic Diversity (Faith and Baker, 2006) and Kruskal-Wallis 198 pairwise correlations using the diversity plugin. Weighted, non-rarefied beta diversity (Chang et 199 al., 2011) was visualized with EMPeror (Vazquez-Baeza et al., 2013). To determine the 200 dominant factors explaining community variance, simplicial linear regressions were performed 201 with gneiss (Morton et al., 2017); a 10-fold cross validation confirmed model overfitting did not 202 occur. Analysis of Composition of Microbes (ANCOM) was used to evaluate differential 203 abundance between samples (Mandal et al., 2015). 204 205 References.

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Figure S1. Location of Harkins Slough MAR field site. The Pajaro Valley Groundwater Basin is located within the lower Pajaro River watershed (shown above) along California’s Central Coast. Experimental plots for the present study were installed in a part of the infiltration basin that was not instrumented in previous studies (Racz et al., 2011; Schmidt et al., 2011).

Figure S2. The water level record for test NS2 indicates several wetting and drying cycles due to intermittent water supply. As saturated conditions were not maintained in the shallow subsurface, this test provided limited useful hydrologic or geochemical data.

Figure S3. Soil conditions were similar for all experimental plots. Average TN, TOC, d10

(grain size for which 10% of grains are finer) d50 (grain size for which 50% of grains are finer), and d90 (grain size for which 90% of grains are finer) with depth for all plots. Error bars show range among samples from the four experimental plots. For TN and TOC data, solid symbols represent data from before the experiments and open symbols represent data from the end of the experiment.

Figure S4. Vertical infiltration rates calculated from different thermal probes indicate similar infiltration rates within the central instrumented area within each plot. Vertical infiltration rates for tests NS1, PRB1, and PRB2 were calculated from two thermal probes (one telemetering data in real time, the other autonomous) at two different locations in the test plot. The rates indicated by these two probes generally matched closely.

Figure S5. PCoA results for 16S rRNA gene sequencing demonstrate that infiltration through woodchips shifted soil microbial communities; shallower samples had a larger shift than deeper samples. A. Samples collected before (open symbols) and after (filled symbols) infiltration for tests with (diamonds) and without (squares) a PRB plotted along the first two principal components. B. The primary principal component plotted against sample depth below the plot base.

Table S1. Total organic carbon (TOC), total nitrogen (TN), and grain size data for all soil samples collected.

Depth below plot TOC TOC TN TN d10 d50 d90 Test # base (cm) before after before after (µm) (µm) (µm) 1 0 0.04 0.04 0.003 0.005 149 273 505 1 20 0.03 0.04 0.003 0.003 151 269 501 1 40 0.04 0.03 0.003 0.004 152 287 534 1 60 0.03 0.03 0.002 0.006 162 276 523 1 80 0.03 0.03 0.003 0.004 157 279 493 1 100 0.03 0.03 0.004 0.004 159 311 554 1 120 0.03 0.03 0.003 0.004 152 283 500 1 140 0.05 0.03 0.003 0.006 154 299 556 2 0 0.03 — 0.003 — 154 281 453 2 20 0.03 0.05 0.003 0.007 140 274 446 2 40 0.03 0.04 0.003 0.006 130 249 462 2 60 0.02 0.03 0.003 0.007 158 269 465 2 80 0.07 0.03 0.003 0.006 149 253 421 2 100 0.03 0.03 0.004 0.002 153 254 413 3 0 0.05 0.03 0.007 0.004 115 274 449 3 10 0.04 0.03 0.006 0.004 127 270 438 3 20 0.04 0.03 0.006 0.004 148 269 454 3 30 0.04 0.03 0.006 0.004 139 260 439 3 50 0.04 0.05 0.006 0.006 149 279 480 3 70 0.04 0.04 0.004 0.002 161 282 458 3 90 0.06 0.02 0.005 0.002 163 267 459 3 110 0.05 0.03 0.004 0.004 162 322 538 3 130 0.05 0.03 0.005 0.005 155 293 488 4 0 0.03 — 0.003 — 149 247 407 4 10 0.06 — 0.004 — 144 272 462 4 20 0.04 — 0.005 — 151 265 445 4 40 0.03 — 0.003 — 137 252 423 4 60 0.03 — 0.003 — 146 269 471 4 80 0.04 — 0.004 — 134 264 428 4 100 0.04 — 0.004 — 102 258 427

Table S2. Water chemistry data for test NS1.

Depth [NO3] [NO2] [NH4] [DOC] [Cl] [SO4] Date Day Piez. (cm) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 150817 4 0 22.0 0.19 0.26 27.7 192 198 150817 4 A 30 23.9 0.49 4.24 29.2 135 144 150817 4 B 30 23.5 0.38 2.45 28.1 179 222 150818 5 0 23.2 0.00 0.06 25.5 129 125 150818 5 A 30 24.0 0.00 0.28 27.0 150 152 150818 5 B 30 23.2 0.22 0.32 25.7 117 99.4 150820 7 0 22.3 0.00 0.09 26.6 127 135 150820 7 A 30 22.4 0.00 0.19 26.5 145 140 150820 7 B 30 22.4 0.00 0.12 26.5 124 92.7 150820 7 A 55 21.9 0.59 0.85 26.9 129 127 150820 7 B 55 22.7 0.00 0.78 26.2 142 162 150821 8 0 24.5 0.15 0.12 27.3 139 138 150821 8 A 30 23.1 0.11 5.69 27.4 118 122 150821 8 B 30 24.0 0.00 3.28 14.4 121 128 150823 10 0 23.1 0.00 0.25 25.4 150 169 150823 10 A 30 24.0 0.12 3.49 26.3 117 114 150823 10 B 30 24.2 1.26 2.34 26.3 122 147 150823 10 A 55 22.4 0.11 2.57 26.6 125 118 150823 10 B 55 23.1 0.87 3.51 27.2 132 134 150825 12 0 24.7 0.00 0.02 27.7 121 111 150825 12 A 30 24.7 0.32 0.03 27.8 119 108 150825 12 B 30 23.8 0.12 0.02 27.7 121 111 150825 12 A 55 23.6 0.65 0.07 27.2 133 144 150825 12 B 55 22.9 0.00 0.04 24.8 130 133 150826 13 0 23.3 0.00 0.00 25.2 136 137 150826 13 A 30 22.6 0.00 0.00 27.3 163 190 150826 13 B 30 23.7 0.00 0.00 27.4 143 150 150826 13 A 55 23.2 0.00 0.00 23.6 136 138 150826 13 B 55 22.6 0.00 0.03 26.1 114 122

Table S3. Water chemistry data for test NS2.

Depth [NO3] [NO2] [NH4] [DOC] [Cl] [SO4] Date Day Piez. (cm) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 150628 6 0 21.4 0.00 0.03 28.6 94.9 68.5 150628 6 A 30 21.4 0.00 0.04 24.5 76.2 56.2 150628 6 B 30 21.1 0.00 0.04 24.5 91.2 65.7 150629 7 0 23.2 0.00 0.03 20.2 141 164 150629 7 A 30 23.6 0.00 0.08 22.0 127 133 150629 7 B 30 22.5 0.00 0.04 24.0 131 140 150630 8 0 21.9 0.00 0.05 28.3 137 150 150630 8 A 30 20.9 0.00 0.04 27.7 89.2 58.7 150630 8 B 30 21.0 0.00 0.03 25.8 85.3 65.6 150701 9 0 21.9 0.00 0.03 30.0 99.2 80.6 150701 9 A 30 21.4 0.00 0.02 26.6 138 117 150701 9 B 30 21.5 0.00 0.03 26.7 129 107 150702 10 0 23.3 0.00 0.04 29.5 121 118 150702 10 A 30 23.9 0.00 0.05 27.8 137 130 150702 10 B 30 23.3 0.00 0.03 29.2 640 602 150703 11 0 21.8 0.00 0.01 128.7 123 150703 11 A 30 21.7 0.00 0.03 82.2 64.3 150703 11 B 30 21.4 0.00 0.04 105.8 79.0

Table S4. Water chemistry data for test PRB1.

Depth [NO3] [NO2] [NH4] [DOC] [Cl] [SO4] Date Day Piez. (cm) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 150828 2 0 23.5 0.00 0.23 29.5 167 181 150828 2 PRB 24.7 0.17 0.14 31.0 130 123 150828 2 A 30 24.5 0.29 3.70 32.1 168 194 150828 2 B 30 24.5 0.37 1.66 36.1 130 128 150828 2 A 55 23.6 0.00 5.86 35.7 140 146 150828 2 B 55 25.0 0.51 5.17 37.0 115 177 150830 4 0 23.5 0.00 0.00 27.5 127 116 150830 4 PRB 23.6 0.00 0.01 26.4 122 115 150830 4 A 30 23.5 0.00 0.28 28.4 121 118 150830 4 B 30 23.2 0.00 0.21 27.5 191 207 150830 4 A 55 23.8 0.00 0.08 26.6 127 119 150830 4 B 55 23.7 0.00 0.23 30.0 126 117 150831 5 0 24.6 0.00 0.00 28.8 175 200 150831 5 PRB 24.9 0.00 0.00 31.5 152 160 150831 5 A 30 24.6 0.14 0.37 32.9 129 126 150831 5 B 30 24.6 0.00 0.57 32.6 150 163 150831 5 A 55 22.5 0.24 0.05 34.9 160 184 150831 5 B 55 23.7 0.14 0.10 31.2 129 127 150831 5 A 80 19.8 0.27 0.03 32.9 113 116 150901 6 0 23.0 0.00 0.05 26.9 118 115 150901 6 PRB 23.6 0.00 0.05 27.6 139 126 150901 6 A 30 21.4 0.60 4.29 33.9 133 130 150901 6 B 30 23.1 0.27 2.47 30.5 111 120 150901 6 A 55 21.4 0.70 0.82 33.6 135 135 150901 6 B 55 23.4 0.27 4.06 30.8 144 138 150901 6 A 80 19.7 0.97 0.20 34.7 129 146 150902 7 0 23.0 0.00 0.00 25.1 127 111 150902 7 PRB 23.2 0.00 0.03 26.6 125 114 150902 7 A 30 22.7 0.00 0.94 27.9 117 116 150902 7 B 30 21.8 0.00 0.55 27.6 129 114 150902 7 A 55 20.2 0.25 0.78 29.2 116 109 150902 7 B 55 22.8 0.00 1.28 25.5 129 116 150903 8 0 23.0 0.00 0.08 26.8 127 116 150903 8 PRB 24.3 0.00 0.06 26.6 129 121 150903 8 A 30 21.5 0.20 4.75 32.9 142 134 150903 8 B 30 23.8 0.00 1.46 28.6 125 117 150903 8 A 55 21.5 0.17 3.08 33.2 119 126 150903 8 B 55 23.8 0.00 2.97 28.4 114 123 150903 8 A 80 21.1 0.19 1.76 34.4 118 117 150904 9 0 24.3 0.00 0.05 25.7 111 113 150904 9 PRB 24.1 0.00 0.00 25.8 107 130

Depth [NO3] [NO2] [NH4] [DOC] [Cl] [SO4] Date Day Piez. (cm) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 150904 9 A 30 23.1 0.12 2.51 30.4 91.4 104 150904 9 B 30 24.5 0.00 0.76 27.1 138 137 150904 9 A 55 22.1 0.15 1.05 32.1 133 134 150904 9 B 55 24.9 0.00 1.45 27.0 125 121 150904 9 A 80 21.8 0.15 0.90 32.1 90.7 124 150907 12 0 25.6 0.00 0.00 26.8 115 132 150907 12 PRB 25.3 0.00 0.00 27.2 105 132 150907 12 A 30 24.8 0.00 2.52 30.3 116 125 150907 12 B 30 25.0 0.00 0.99 28.3 145 146 150907 12 A 55 24.9 0.00 2.97 27.4 138 139 150907 12 B 55 25.2 0.00 1.62 28.1 141 142 150907 12 A 80 24.1 0.13 3.47 32.7 135 138 150908 13 0 24.6 0.00 0.00 27.4 91.7 101 150908 13 PRB 23.8 0.00 0.00 26.7 105 132 150908 13 A 30 24.7 0.00 2.72 27.7 117 121 150908 13 B 30 24.6 0.00 1.00 27.3 91.2 120 150908 13 A 55 24.9 0.00 3.80 28.5 121 127 150908 13 B 55 24.9 0.00 1.89 26.7 138 135 150908 13 A 80 25.0 0.00 4.22 27.4 136 137 150909 14 0 23.5 0.00 0.00 27.0 117 112 150909 14 PRB 23.6 0.00 0.00 27.5 115 112 150909 14 A 30 23.8 0.00 0.94 26.4 120 120 150909 14 B 30 24.2 0.00 0.24 28.3 131 124 150909 14 A 55 24.1 0.00 1.81 24.7 116 122 150909 14 B 55 23.8 0.00 0.53 27.6 133 128 150909 14 A 80 24.0 0.00 2.22 28.4 113 122 150910 15 0 23.4 0.00 0.01 21.8 128 122 150910 15 PRB 22.4 0.00 0.02 27.4 119 117 150910 15 A 30 23.6 0.00 0.16 27.5 106 115 150910 15 B 30 23.4 0.00 0.07 26.7 126 122 150910 15 A 55 23.5 0.00 0.30 28.4 122 135 150910 15 B 55 23.8 0.00 0.07 28.0 129 123 150910 15 A 80 23.0 0.00 0.29 27.7 105 105

Table S5. Water chemistry data for test PRB2.

Depth [NO3] [NO2] [NH4] [DOC] [Cl] [SO4] Date Day Piez. (cm) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 150708 2 0 23.1 0.00 0.03 101 78.9 150708 2 PRB 22.9 0.00 0.02 94.8 92.1 150708 2 A 30 23.5 0.00 0.13 119 116 150708 2 B 30 23.3 0.00 0.13 115 96.6 150709 3 0 22.2 0.00 0.02 85.6 57.1 150709 3 PRB 22.0 0.00 0.04 127 123 150709 3 A 30 22.2 0.00 0.12 81.0 72.8 150709 3 B 30 22.1 0.00 0.08 78.6 77.6 150709 3 A 60 22.2 0.00 0.07 109 94.5 150709 3 B 60 21.9 0.00 0.06 122 117 150709 3 A 90 22.2 0.00 0.10 121 117 150710 4 0 22.6 0.00 0.02 119 97.8 150710 4 PRB 21.8 0.00 0.04 124 118 150710 4 A 30 21.8 0.00 0.14 122 108 150710 4 B 30 22.2 0.00 0.05 121 108 150710 4 A 60 22.7 0.00 0.05 124 110 150710 4 B 60 22.2 0.00 0.05 133 124 150710 4 A 90 21.4 0.00 0.04 114 101 150710 4 B 90 22.0 0.00 0.03 123 110 150712 6 0 22.0 0.00 0.04 116 105 150712 6 PRB 22.2 0.00 0.04 146 140 150712 6 A 30 21.9 0.00 0.06 133 124 150712 6 B 30 21.8 0.00 0.08 109 88.2 150712 6 A 60 21.7 0.00 0.06 107 78.0 150712 6 B 60 22.1 0.00 0.00 143 134 150712 6 A 90 21.2 0.00 0.06 96.3 73.3 150713 7 0 22.7 0.00 0.03 180 170 150713 7 PRB 22.6 0.00 0.07 193 185 150713 7 A 30 23.9 0.00 0.06 199 186 150713 7 B 30 23.6 0.00 0.08 237 223 150713 7 A 60 22.9 0.16 0.96 195 185 150713 7 B 60 24.4 0.00 0.88 200 187 150713 7 A 90 24.9 0.00 0.48 192 176 150714 8 0 22.3 0.00 0.00 145 131 150714 8 PRB 22.5 0.00 0.05 148 131 150714 8 A 30 22.3 0.00 0.07 97.8 86.7 150714 8 B 30 22.2 0.00 0.04 106 93.7 150714 8 A 60 22.5 0.00 0.24 192 174 150714 8 B 60 22.2 0.00 0.43 179 164 150714 8 A 90 22.8 0.00 0.98 134 117 150715 9 0 24.2 0.00 0.06 193 174

Depth [NO3] [NO2] [NH4] [DOC] [Cl] [SO4] Date Day Piez. (cm) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 150715 9 PRB 23.8 0.00 0.02 132 119 150715 9 A 30 23.8 0.11 0.04 145 132 150715 9 B 30 23.3 0.21 0.08 129 120 150715 9 A 60 23.7 0.00 0.45 140 139 150715 9 B 60 25.1 0.00 0.84 128 125 150715 9 A 90 24.1 0.75 0.53 151 165 150716 10 0 22.9 0.00 0.00 150716 10 PRB 23.2 0.00 0.02 123 110 150716 10 A 30 23.0 0.18 0.00 120 110 150716 10 B 30 23.2 0.00 0.00 120 108 150716 10 A 60 23.4 0.15 0.05 120 108 150716 10 B 60 20.5 0.24 0.12 115 106 150716 10 A 90 23.0 0.22 0.06 115 108 150717 11 0 23.5 0.00 0.02 137 142 150717 11 PRB 25.2 0.10 0.01 124 117 150717 11 A 30 24.5 0.00 0.05 144 158 150717 11 B 30 23.3 0.00 0.03 123 130 150717 11 A 60 24.0 0.34 2.36 144 170 150717 11 B 60 23.7 0.25 0.42 129 119 150717 11 A 90 23.4 0.52 3.72 133 134 150720 14 0 25.4 0.00 0.07 161 170 150720 14 PRB 25.3 0.00 0.03 165 154 150720 14 A 30 24.9 0.00 0.26 98.9 85.3 150720 14 B 30 22.5 0.40 0.79 100 112 150720 14 A 60 25.1 0.64 0.72 178 173 150720 14 B 60 23.6 0.75 2.55 127 117 150720 14 A 90 24.9 1.69 4.27 179 185 150721 15 0 22.7 0.00 0.28 137 152 150721 15 PRB 22.7 0.00 0.04 121 121 150721 15 A 30 23.8 0.00 0.15 120 120 150721 15 B 30 23.7 0.00 0.00 126 131 150721 15 A 60 24.7 0.00 0.06 129 138 150721 15 B 60 24.1 0.31 0.08 119 117 150721 15 A 90 23.0 0.62 0.09

Table S6. Nitrate isotope data for a subset of samples from tests NS1 and PRB1.

d18O Depth d15N VSMOW Day Piez. (cm) Air (‰) (‰) Test NS1 6 0 6.45 5.56 6 A 30 6.57 5.21 7 0 5.54 5.32 7 B 30 5.56 5.36 7 B 55 5.49 5.31 10 0 5.62 5.18 10 A 30 5.82 5.50 10 A 55 5.65 5.41 13 0 5.66 5.41 13 A 30 5.50 5.27 13 A 55 5.59 5.49 Test PRB1 5 0 5.99 5.90 5 PRB 5.99 5.82 5 A 30 6.10 6.10 5 A 55 6.37 6.59 5 A 80 6.36 6.57 6 0 5.69 5.53 6 PRB 5.85 5.57 6 A 30 7.41 5.25 6 A 55 7.77 5.38 6 A 80 8.99 5.08 8 0 5.77 5.62 8 PRB 5.76 5.61 8 A 30 6.88 5.93 8 A 55 6.88 5.90 8 A 80 7.10 5.91 9 0 5.69 5.57 9 PRB 5.71 5.27 9 A 30 6.30 5.65 9 A 55 6.52 5.90 9 A 80 6.61 5.61 14 0 5.60 5.45 14 PRB 5.59 5.40 14 A 30 5.65 6.05 14 A 55 5.84 5.44 14 A 80 5.92 5.53

Table S7. Result of pairwise statistical testing of microbiological sequencing data divided into different groups. A high p-value (p > 0.05) indicates that there is not a statistically significant difference between the two groups; a low p-value (p < 0.05) indicates a statistically significant difference between the two groups. Generated using Faith’s Phylogenetic Diversity metric (Faith and Baker, 2006).

Number of Kruskall-Wallis Sample group samples Divided by p-value All 20 Treatment 0.058 All 20 Time 0.003 All 20 Depth 0.650 After infiltration 10 Treatment 0.030 Before infiltration 10 Treatment 0.087 Native soil 6 Time 0.050 Native soil 6 Depth 0.643 PRB 14 Time 0.014 PRB 14 Depth 0.841 Table S8. List of OTUs present at >0.1% summed across all samples.

Kingdom Phylum Class Order Family Genus Species Archaea Crenarchaeota Thaumarchaeota Cenarchaeales Cenarchaeaceae – – Archaea Crenarchaeota Thaumarchaeota Cenarchaeales Cenarchaeaceae Nitrosopumilus – Archaea Crenarchaeota Thaumarchaeota Cenarchaeales SAGMA-X – – Archaea Crenarchaeota Thaumarchaeota Nitrososphaerales Nitrososphaeraceae Candidatus Nitrososphaera gargensis Archaea Euryarchaeota Thermoplasmata E2 [Methanomassiliicoccaceae] – – Bacteria – – – – – – Bacteria Acidobacteria – – – – – Bacteria Acidobacteria Acidobacteria-6 iii1-15 – – – Bacteria Acidobacteria Acidobacteria-6 iii1-15 mb2424 – – Bacteria Acidobacteria Acidobacteriia Acidobacteriales Koribacteraceae – – Bacteria Acidobacteria DA052 Ellin6513 – – – Bacteria Acidobacteria Solibacteres Solibacterales [Bryobacteraceae] – – Bacteria Acidobacteria Sva0725 Sva0725 – – – Bacteria Actinobacteria Acidimicrobiia Acidimicrobiales – – – Bacteria Actinobacteria Acidimicrobiia Acidimicrobiales – – Bacteria Actinobacteria Acidimicrobiia Acidimicrobiales EB1017 – – Bacteria Actinobacteria Actinobacteria Actinomycetales Microbacteriaceae Microbacterium – Bacteria Actinobacteria Actinobacteria Actinomycetales Micrococcaceae – – Bacteria Actinobacteria Actinobacteria Actinomycetales Micromonosporaceae – – Bacteria Actinobacteria Actinobacteria Actinomycetales Nocardioidaceae Nocardioides – Bacteria Actinobacteria Actinobacteria Actinomycetales Streptomycetaceae Streptomyces – Bacteria Actinobacteria Actinobacteria Actinomycetales Streptomycetaceae Streptomyces – Bacteria Actinobacteria MB-A2-108 0319-7L14 – – – Bacteria Actinobacteria MB-A2-108 – – – – Bacteria Actinobacteria Thermoleophilia Gaiellales Gaiellaceae – – Bacteria Bacteroidetes Cytophagia Cytophagales Cytophagaceae – – Bacteria Chlorobi BSV26 A89 – – – Bacteria Chlorobi BSV26 C20 – – – Bacteria Chloroflexi Anaerolineae GCA004 – – – Bacteria Chloroflexi Anaerolineae SB-34 – – – Bacteria Chloroflexi Anaerolineae envOPS12 – – – Bacteria Chloroflexi Ellin6529 – – – – Bacteria Chloroflexi Gitt-GS-136 – – – – Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus flexus Kingdom Phylum Class Order Family Genus Species Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus muralis Bacteria GAL15 – – – – – Bacteria GN04 MSB-5A5 – – – – Bacteria Gemmatimonadetes Gemm-1 – – – – Bacteria NC10 12-24 MIZ17 – – – Bacteria Nitrospirae Nitrospira Nitrospirales Nitrospiraceae – – Bacteria Nitrospirae Nitrospira Nitrospirales Nitrospiraceae Nitrospira – Bacteria Planctomycetes Planctomycetia Pirellulales Pirellulaceae – – Bacteria Planctomycetes Planctomycetia Planctomycetales Planctomycetaceae Planctomyces – Bacteria Proteobacteria – – – – – Bacteria Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae Asticcacaulis biprosthecium Bacteria Proteobacteria Alphaproteobacteria Ellin329 – – – Bacteria Proteobacteria Alphaproteobacteria Rhizobiales – – – Bacteria Proteobacteria Alphaproteobacteria Rhizobiales Bradyrhizobiaceae Bradyrhizobium – Bacteria Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Devosia – Bacteria Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Hyphomicrobium – Bacteria Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Rhodoplanes – Bacteria Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae – Bacteria Proteobacteria Alphaproteobacteria Rhodospirillales Rhodospirillaceae – Bacteria Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae – Bacteria Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Lutibacterium – Bacteria Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Kaistobacter – Bacteria Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Novosphingobium – Bacteria Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingobium – Bacteria Proteobacteria Betaproteobacteria – Bacteria Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae – Bacteria Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Polaromonas – Bacteria Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae Janthinobacterium – Bacteria Proteobacteria Betaproteobacteria Ellin6067 – – – Bacteria Proteobacteria Betaproteobacteria IS-44 – – – Bacteria Proteobacteria Betaproteobacteria MND1 – – – Bacteria Proteobacteria Betaproteobacteria Methylophilales Methylophilaceae – – Bacteria Proteobacteria Betaproteobacteria Methylophilales Methylophilaceae Methylotenera mobilis Bacteria Proteobacteria Deltaproteobacteria – – – Bacteria Proteobacteria Deltaproteobacteria Myxococcales OM27 – – Kingdom Phylum Class Order Family Genus Species Bacteria Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophobacteraceae – – Bacteria Proteobacteria Deltaproteobacteria [Entotheonellales] [Entotheonellaceae] – – Bacteria Proteobacteria Gammaproteobacteria – – – Bacteria Proteobacteria Gammaproteobacteria Xanthomonadales Sinobacteraceae – – Bacteria Verrucomicrobia Opitutae Opitutales Opitutaceae – – Bacteria Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobiaceae – Bacteria Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobiaceae Prosthecobacter debontii Bacteria Verrucomicrobia [Pedosphaerae] [Pedosphaerales] – – – Bacteria WS3 PRR-12 Sediment-1 – – –

Table S9. 16S rRNA gene sequencing results. The average relative abundance for groups NSB (native soil, before infiltration), NSA (native soil, after infiltration), PRBB (PRB, before infiltration), and PRBA (PRB, after infiltration). Only OTUs present at >0.1% summed across all samples are shown (see complete taxonomy for each OTU in Table S8). Log2 fold-changes comparing NSB and NSA (column NS) and comparing PRBB and PRBA (column PRB) quantify enhancement of OTUs in native soil and below the PRB. Differential abundance test results highlight the differences between sample groups. W-values are shown as a metric of significance as calculated by ANCOM (Analysis of Composition of Microbes (Mandal et al., 2015) with samples grouped by PRB and native soil (column “Treatment”), and with samples grouped by PRBB, PRBA, NSB, and NSA (column “Treatment, Time”). A * indicates a significant W-score for these comparisons. A ~ indicates significant W-scores based on collection time; ^ based on treatment and depth; and # based on treatment, depth, and time.

Average % relative Log2 fold- Differential abundance W- Standard deviation (%) Lowest assigned taxonomy abundance changes value

PRBB PRBA NSB NSA PRBB PRBA NSB NSA PRB NS Treatment Treatment, time Cenarchaeaceae (f) 0.98 0.22 0.17 0.31 0.51 0.19 0.07 0.09 -2.2 0.7 31 48 Nitrosopumilus (g) 0.69 0.45 0.16 0.35 0.20 0.55 0.12 0.23 -0.6 -0.8 20 18 SAGMA-X (f) 3.72 2.63 9.20 12.15 0.67 1.51 0.68 1.45 -0.5 -4.2 16-102* 27-101* N. gargensis (s) 0.65 0.15 1.67 0.79 0.34 0.08 1.26 0.29 -2.1 -1.2 18-21 17-29 Methanomassiliicoccaceae (f) 2.64 0.48 2.03 6.50 2.24 0.50 0.49 0.96 -2.5 -1.5 6-20 17-31 Bacteria (k) 0.00 1.59 0.00 0.00 0.00 1.52 0.00 0.00 – – 5 36 Acidobacteria (p) 0.34 0.25 2.35 0.71 0.21 0.40 0.53 0.20 -0.5 -1.8 21 30 iii1-15 (o) 5.38 3.29 5.53 4.67 0.47 1.35 0.27 0.75 -0.7 -3.3 17-23 26-33 mb2424 (f) 1.10 0.45 1.51 1.14 0.69 0.24 1.02 0.40 -1.3 -0.7 7-18 10-31 Koribacteraceae (f) 0.22 0.15 0.71 0.62 0.07 0.07 0.11 0.05 -0.6 -3.0 16 25 Ellin6513 (o) 0.71 0.43 0.38 0.55 0.27 0.37 0.14 0.14 -0.7 -1.0 17 26 Bryobacteraceae (f) 0.25 1.00 0.01 0.25 0.13 0.90 0.02 0.07 2.0 -0.9 14 11 Sva0725 (o) 1.61 0.78 0.56 0.75 0.57 0.51 0.15 0.30 -1.0 -0.4 20-23 18-28 Acidimicrobiales (o) 2.10 0.68 2.18 2.47 0.91 0.35 0.37 0.32 -1.6 -1.4 7-33 15-49 EB1017 (f) 0.97 0.36 0.78 0.66 0.40 0.22 0.32 0.25 -1.4 -0.7 17 24 Microbacterium (g) 0.03 1.55 0.01 0.03 0.04 0.81 0.03 0.02 5.6 0.4 7 112* Micrococcaceae (f) 3.47 5.09 3.59 3.21 2.91 7.28 1.53 0.72 0.6 -0.1 10 16 Micromonosporaceae (f) 0.14 0.00 1.90 0.00 0.20 0.00 2.68 0.00 – – 1 62 Nocardioides (g) 0.63 0.06 0.90 0.10 0.47 0.03 0.30 0.04 -3.5 2.2 6 24 Streptomyces (g) 0.91 0.20 1.45 0.52 0.48 0.09 0.34 0.06 -2.2 -0.1 5-22 24-34 MB-A2-108 (c) 0.20 0.18 3.72 2.44 0.09 0.11 1.13 0.74 -0.2 -4.8 34-121* 39-120* ^# 0319-7L14 (o) 1.84 0.52 2.01 1.47 0.76 0.19 0.64 0.22 -1.8 -0.9 14-17 27-28 Gaiellaceae (f) 3.12 1.16 4.21 3.39 0.96 0.43 1.20 0.35 -1.4 -1.8 5-101* 24-37 Cytophagaceae (f) 2.03 0.89 0.39 0.87 1.05 0.87 0.09 0.24 -1.2 0.3 10-13 21-28 A89 (o) 0.76 0.38 1.46 2.55 0.47 0.43 0.54 0.52 -1.0 -2.4 5-111* 8-104* ^ C20 (o) 0.66 0.19 0.54 0.42 0.33 0.12 0.13 0.09 -1.8 -0.4 17 28 envOPS12 (o) 2.29 1.21 1.03 0.79 0.84 0.53 0.12 0.28 -0.9 0.1 25-57 32-52 GCA004 (o) 0.35 0.10 0.85 0.79 0.26 0.09 0.31 0.47 -1.8 -1.6 4 19 SB-34 (o) 0.05 0.00 0.66 1.15 0.09 0.00 0.60 0.59 – -3.6 60 34 Ellin6529 (c) 2.93 1.29 4.50 3.59 0.87 0.56 0.90 0.65 -1.2 -2.1 18-114* 28-108* ^ Gitt-GS-136 (c) 0.22 0.15 1.05 0.59 0.14 0.12 0.19 0.20 -0.6 -2.0 29 34 B. flexus (s) 0.97 0.17 0.48 0.15 0.32 0.10 0.08 0.06 -2.5 1.1 26 63 B. muralis (s) 0.65 0.18 0.49 0.12 0.26 0.14 0.17 0.01 -1.9 1.0 15 30 GAL15 (p) 7.85 3.06 3.91 8.00 3.22 2.80 1.42 0.96 -1.4 -1.3 4-114* 3-91 Gemm-1 (c) 3.64 1.49 6.32 3.12 2.24 0.95 2.14 0.25 -1.3 -0.5 2-35 9-91 MSB-5A5 (c) 1.61 0.74 0.01 0.00 2.91 0.77 0.01 0.00 -1.1 – 6 2 MIZ17 (o) 1.40 1.00 0.96 1.06 1.06 0.86 0.39 0.44 -0.5 0.0 9 18 Nitrospiraceae (f) 0.44 0.21 0.41 0.29 0.16 0.20 0.04 0.08 -1.1 -0.8 9 20 Nitrospira (g) 2.27 1.20 2.76 3.19 0.62 0.66 0.49 0.84 -0.9 -2.4 13-17 22-32 Pirellulaceae (f) 0.70 0.23 0.17 0.47 0.30 0.20 0.08 0.12 -1.6 -0.7 15 29 Planctomyces (g) 0.18 2.70 0.13 0.43 0.07 2.83 0.05 0.32 3.9 -2.7 4-5 6-7 Proteobacteria (p) 3.38 0.82 0.11 0.15 3.08 0.71 0.02 0.03 -2.0 4.4 41 24 A. biprosthecium (s) 0.00 1.66 0.00 0.00 0.01 3.13 0.00 0.00 8.9 – 16 81 Ellin329 (o) 0.09 0.17 0.41 0.68 0.07 0.15 0.09 0.09 0.9 -3.3 11 21 Rhizobiales (o) 6.44 1.77 3.83 4.92 1.05 0.74 1.12 0.75 -1.9 -2.2 16-108* 15-107* ^ Bradyrhizobium (g) 0.54 0.39 0.65 0.10 0.61 0.49 0.33 0.02 -0.5 2.6 2 6 Devosia (g) 0.09 1.09 0.00 0.02 0.10 1.01 0.00 0.01 3.6 2.3 17 103* Hyphomicrobium (g) 0.88 0.27 0.36 0.16 0.39 0.13 0.16 0.07 -1.7 1.3 34 31 Rhodoplanes (g) 1.56 1.00 2.34 1.99 0.41 0.31 0.56 0.52 -0.6 -2.3 25-37 28-36 Rhodobacteraceae (f) 0.00 6.97 0.00 0.03 0.01 6.19 0.00 0.04 11.0 -2.1 8 122* ~# Rhodospirillaceae (f) 1.05 0.58 1.02 0.91 0.36 0.24 0.21 0.16 -0.9 -1.3 15-19 27-33 Erythrobacteraceae (f) 0.00 1.73 0.00 0.00 0.00 1.61 0.00 0.00 – – 15 112* Lutibacterium (g) 0.00 3.30 0.00 0.00 0.00 4.57 0.00 0.00 – – 9 123* # Kaistobacter (g) 0.63 0.53 0.63 1.06 0.30 0.38 0.09 0.10 -0.3 -1.8 18 31 Novosphingobium (g) 0.00 13.90 0.01 0.00 0.01 9.99 0.02 0.00 11.8 – 9-14 121-127* # Sphingobium (g) 0.00 1.91 0.00 0.00 0.00 2.68 0.00 0.00 – – 7 113* Betaproteobacteria (c) 2.02 1.11 1.56 1.51 0.25 0.74 0.04 0.13 -0.9 -2.6 10-27 20-32

Comamonadaceae (f) 0.20 3.14 0.40 0.04 0.24 2.98 0.09 0.01 4.0 2.5 13-15 33-115* Polaromonas (g) 0.02 2.72 0.06 0.05 0.02 1.31 0.06 0.03 7.2 -1.0 4 114* ~ Janthinobacterium (g) 0.15 0.66 0.71 0.05 0.15 0.62 0.39 0.01 2.1 1.5 2 9 Ellin6067 (o) 2.97 1.68 1.06 1.30 1.90 0.89 0.08 0.16 -0.8 0.5 12-47 27-29 IS-44 (o) 4.14 1.54 2.22 2.35 1.00 1.44 0.31 0.41 -1.4 -1.2 8-15 20-28 Methylophilaceae (f) 0.03 1.40 0.03 0.03 0.03 1.33 0.03 0.02 5.4 0.4 11 86 M. mobilis (s) 0.07 2.19 0.03 0.00 0.05 1.65 0.05 0.00 4.9 – 25 110* MND1 (o) 8.10 2.87 7.26 7.81 1.01 1.79 0.38 0.94 -1.5 -2.9 11-113* 18-135* ^ Deltaproteobacteria (c) 0.11 0.06 1.59 1.25 0.18 0.12 0.66 0.56 -0.8 -2.8 71 22 Entotheonellaceae (f) 0.94 0.22 0.48 0.43 2.21 0.48 0.22 0.12 -2.1 2.4 2 7 OM27 (f) 0.01 1.80 0.00 0.03 0.02 1.62 0.00 0.03 7.9 -0.6 8 108* Syntrophobacteraceae (f) 1.85 1.60 1.63 1.89 0.23 1.03 0.44 0.04 -0.2 -3.0 8-18 2-34 Gammaproteobacteria (c) 0.49 0.52 0.09 0.08 0.41 0.41 0.11 0.03 0.1 2.4 28 21 Sinobacteraceae (f) 1.13 0.46 0.01 0.00 1.13 0.89 0.01 0.00 -1.3 – 84 36 Pedosphaerales (o) 0.65 0.57 0.20 0.50 0.31 0.59 0.06 0.16 -0.2 -0.7 18 25 Opitutaceae (f) 0.95 0.37 0.12 0.40 0.33 0.22 0.07 0.17 -1.4 -0.3 43 52 Verrucomicrobiaceae (f) 0.19 2.61 0.04 0.27 0.12 3.54 0.03 0.11 3.7 -1.2 18 60 P. debontii (s) 0.00 1.27 0.00 0.00 0.00 1.56 0.00 0.00 – – 16 120* # Sediment-1 (o) 0.61 0.28 2.02 1.32 0.43 0.30 0.51 0.62 -1.1 -1.6 19 29