Supplementary Information
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for
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Response of microbial community function to fluctuating
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geochemical conditions within a legacy radioactive waste trench
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environment
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Xabier Vazquez-Campos1, Andrew S. Kinsela1,2, Mark W. Bligh1,2, Jennifer J. Harrison2,
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Timothy E. Payne2 & T. David Waite1*
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UNSW Water Research Centre and School of Civil and Environmental Engineering, The
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University of New South Wales, Sydney, New South Wales 2052, Australia
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Organisation, Locked Bag 2001, Kirrawee DC, New South Wales 2232, Australia
Institute for Environmental Research, Australian Nuclear Science and Technology
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ISME Journal
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(Submitted September 2016)
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*Corresponding author: Professor T.D. Waite; Email: [email protected]
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Material and Methods
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Sample collection
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Trench water samples were collected on five separate occasions across a two month
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period from LFLS. They were obtained from a screened PVC pipe which extends 1.55 m
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below the ground surface. This pipe provides the only point of access into the legacy trenches
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and was opportunistically installed during the partial collapse of the trench surface. Further
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details of the trench sampler have been described by Payne et al. (2013) and Ikeda-Ohno et
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al. (2014). Briefly, a peristaltic pump operating under low flow rate was used to purge the
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borehole until chemical parameters, particularly pH and oxidation/reduction potential (ORP),
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became stable (Supplementary File 1). Measurements were made using a multi-probe system
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(YSI 556 MPS) coupled to a 100 mL flow cell. Stability usually took between 30 and 60 min,
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after which sample collection began. Chemical parameters, along with the water depth, were
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monitored over the sampling period (typically some hours) on the day of sampling to ensure
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the water was representative of that particular period. The stability of flow cell parameters
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and the fact that the water level did not decrease during the collection of 4–5 L of sample
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material provides assurance that the samples were representative of the particular event rather
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than anomalous local conditions.
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The sample collection period commenced (on 23rd April 2015) immediately after a
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substantial rainfall event, during which ~220 mm of rain fell over 3 days (Figure S1). Trench
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water samples were subsequently collected on the 27th and 29th April, 14th May and 9th
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June (or days 0, 4, 6, 21 and 47, post-rain, Figure S1).
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Samples for chemical analyses were collected both with and without an in-line filtration
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membrane (0.45 µm PES, Waterra FHT-Groundwater) in pre-washed and trench water rinsed
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HDPE bottles except samples for organic carbon analysis which were collected in glass
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bottles. Samples for radiochemical and cation analyses were acidified using double-distilled
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HNO3. All samples were then stored at 4 °C until analysis.
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Samples for microbial analyses were collected by directly filtering the trench water
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using a sterile PES 0.22 µm syringe filter (Sterivex-GP, Merck Millipore) with the volumes
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of filtrate passing through the membranes recorded. The filters were capped on site before
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being transported back to the laboratory and stored at -18 °C until analysis. Microbial
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sampling at all time points was performed in triplicate to assess the inherent variation of the
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technique.
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It is important to note that due to protocols governing sampling at the site, no solid
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phase material could be extracted from the trenches. All sampling within the trenches pertains
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to the extraction of soluble or suspended solid-phases from the screened borehole.
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Chemical analyses of trench waters
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The primary radiochemical contaminants,
239+240
Pu and
241
Am, were separated from
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other radiochemical fractions using TEVA™ and TRU™ resin cartridges (Harrison et al.,
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2011) before measuring activity by alpha spectroscopy using a Canberra Alpha Analyst
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system coupled with Passive Implanted Planar Silicon detectors (Canberra).
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Cations (including Si, S and P) were measured by either ICP-AES or ICP-MS
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depending on relative concentration. Anions (F-, Cl-, Br-, I-, PO43-and NO3-) were determined
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by ion chromatography (Dionex DX-600 IC System).
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Non-purgeable dissolved organic carbon (0.45 µm filtered) was measured on freshly-
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collected replicate samples by combustion catalytic oxidation (TOC-5000A, Shimadzu) with
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concentration determined by a five point calibration using newly-prepared potassium
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hydrogen phthalate solutions.
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Ferrous iron concentrations from 0.45 µm filtered samples were preserved in the field
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with ammonium acetate-buffered phenanthroline solution (APHA et al., 1998). The
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absorbance of Fe(II) was measured at 510 nm (USB4000, Ocean Optics) calibrated against a
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freshly-prepared ammonium ferrous sulfate solution.
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In field measurements of dissolved sulfide were attempted using methylene blue
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colorimetry, but were consistently below detection limits (~1 µM) at all sampling time points.
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Results and Discussion
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Reactive oxygen species detoxification
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The rapid trench water redox cycling involving the production of large quantities of
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Fe(II), along with elevated concentrations of organic compounds, creates conditions
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conducive to reactive oxygen species production (Page et al., 2013; Minella et al., 2015;
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Klüpfel et al., 2014; Page et al., 2012; Tong et al., 2016). Superoxide dismutase (SOD,
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SUPEROX-DISMUT-RXN, EC:1.15.1.1) was found to be the predominant RXN related to
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reactive oxygen species (ROS) detoxification at all time points, with a maximum at day 47
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(Figure 5F). Catalase peaked at day 4 and superoxide reductases (SOR, 1.15.1.2-RXN) at day
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47, both exhibiting similar relative abundances at the lowest values (days 0 and 4).
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The general classic concept of strict anaerobes being unable to cope with O2 and
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reactive oxygen species has been long obsolete (Imlay, 2002). While SODs are well
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distributed amongst aerobic and anaerobic organisms, catalases are more commonly found in
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aerobes, and SORs in anaerobes (Sheng et al., 2014). This is consistent with the results
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presented above.
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Levels of SOD have been correlated with the aerotolerance of anaerobes (Hassan, 1989;
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Tally et al., 1977). Gene copy numbers have been correlated with expression levels for
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numerous proteins. The high relative abundance of SOD during the anaerobic phase could
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relate to the physiological needs of the anaerobes and aerotolerant microbes thriving in the
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trenches to deal with transient high oxygen concentrations (Brioukhanov et al., 2002; Sheng
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et al., 2014).
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References
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APHA, AWWA, WEF. (1998). Standard methods for the examination of water and
wastewater. 20th ed. APHA-AWWA-WEF: Washington, D.C.
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Brioukhanov AL, Thauer RK, Netrusov AI. (2002). Catalase and superoxide dismutase in the
cells of strictly anaerobic microorganisms. Microbiology 71: 281–285.
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Harrison JJ, Zawadzki A, Chisari R, Wong HKY. (2011). Separation and measurement of
thorium, plutonium, americium, uranium and strontium in environmental matrices. J Environ
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Hassan HM. (1989). Microbial superoxide dismutases. In: Scandalios JG (ed) Vol. 26.
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Ikeda-Ohno A, Harrison JJ, Thiruvoth S, Wilsher K, Wong HKY, Johansen MP, et al. (2014).
Solution speciation of plutonium and americium at an Australian legacy radioactive waste
disposal site. Environ Sci Technol 48: 10045–10053.
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Imlay JA. (2002). How oxygen damages microbes: Oxygen tolerance and obligate
anaerobiosis. In: Physiology B-A in M (ed) Advances in Microbial Phisiology Vol. 46.
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Klüpfel L, Piepenbrock A, Kappler A, Sander M. (2014). Humic substances as fully
regenerable electron acceptors in recurrently anoxic environments. Nature Geosci 7: 195–
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Minella M, De Laurentiis E, Maurino V, Minero C, Vione D. (2015). Dark production of
hydroxyl radicals by aeration of anoxic lake water. Sci Total Environ 527–528: 322–327.
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Page SE, Kling GW, Sander M, Harrold KH, Logan JR, McNeill K, et al. (2013). Dark
formation of hydroxyl radical in arctic soil and surface waters. Environ Sci Technol 47:
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Page SE, Sander M, Arnold WA, McNeill K. (2012). Hydroxyl radical formation upon
oxidation of reduced humic acids by oxygen in the dark. Environ Sci Technol 46: 1590–1597.
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Payne TE, Harrison JJ, Hughes CE, Johansen MP, Thiruvoth S, Wilsher KL, et al. (2013).
Trench ‘bathtubbing’ and surface plutonium contamination at a legacy radioactive waste site.
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Sheng Y, Abreu IA, Cabelli DE, Maroney MJ, Miller A-F, Teixeira M, et al. (2014).
Superoxide Dismutases and Superoxide Reductases. Chem Rev 114: 3854–3918.
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Tally FP, Goldin BR, Jacobus NV, Gorbach SL. (1977). Superoxide dismutase in anaerobic
bacteria of clinical significance. Infect Immun 16: 20–25.
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Tong M, Yuan S, Ma S, Jin M, Liu D, Cheng D, et al. (2016). Production of abundant
hydroxyl radicals from oxygenation of subsurface sediments. Environ Sci Technol 50: 214–
221.
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Supplementary Figures
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Figure S1. Daily rainfall at Lucas Heights (ANSTO) Meteorological Station from April to July
2015 (bottom). Corresponding trench water levels across the sampling period, along with the
ground surface elevation shown by the dashed line (top). Circles depict dates of chemical and
microbial sampling. Rainfall data is courtesy of the Australian Government, Bureau of Meteorology.
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Figure S2. Chitobiase. Marker for the degradation of chitobiose and/or chitin.
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Figure S3. ISOCIT-CLEAV-RXN. Marker for the glyoxylate pathway.
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Figure S4. COENZYME-F420-HYDROGENASE-RXN. Indicator of methanogenesis from H2 and CO2.
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Figure S5. SULFITE-DEHYDROGENASE-RXN (EC:1.8.2.1). Assimilatory sulfate reduction.
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Figure S6. Relevant RXNs related to the nitrogen cycle.
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Figure S7. PHOSPHOKETOLASE-RXN and RXN0-310. Markers for heterolactic and propionate
fermentation respectively.
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Figure S8. Bray-Curtis similarity tree of the functional profiles for the individual sampling
replicates.
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Supplementary Files
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Supplementary file 1. Spreadsheet with YSI data.
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Supplementary file 2. Krona HTML file with the taxonomy.
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Supplementary file 3. Spreadsheet with the relative abundances of all the RXNs.
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Supplementary file 4. Table with the raw HUMAnN2 output.
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All supplementary files can be viewed at: https://dx.doi.org/10.6084/m9.figshare.3817356
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