Total Phosphate Influences the Rate of Hydrocarbon Degradation but

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Total phosphate influences the rate of hydrocarbon degradation but phosphate mineralogy shapes microbial community composition in cold-region calcareous soils. Steven D Siciliano, Tingting Chen, Courtney Leigh Phillips, Jordan Graeme Hamilton, David M Hilger, Blaine Chartrand, Jay Grosskleg, Kris Bradshaw, Trevor Carlson, and Derek Peak Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05911 • Publication Date (Web): 15 Apr 2016 Downloaded from http://pubs.acs.org on April 15, 2016

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Environmental Science & Technology

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Total phosphate influences the rate of hydrocarbon degradation but phosphate mineralogy shapes

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microbial community composition in cold-region calcareous soils.

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Steven D Siciliano1, Tingting Chen1, Courtney Phillips1, Jordan Hamilton1, David Hilger1,

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Blaine Chartrand2, Jay Grosskleg3, Kris Bradshaw3, Trevor Carlson3 and Derek Peak1*.

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Department of Soil Science, University of Saskatchewan, Saskatoon, Canada 2

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Saskatchewan Polytechnique, Saskatoon, Canada

Federated Cooperatives Limited, Saskatoon, Canada

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Title Running Head

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Phosphate mineralogy controls microbial hydrocarbon degradation *Corresponding Author

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Contact Information

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Derek Peak

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51 Campus Drive, Department of Soil Science, Agriculture Bldg

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University of Saskatchewan

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Saskatoon, SK, Canada, S7N 5A8

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Phone: 306-966-6806. Fax: 306-966-6881

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Email: [email protected]

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Key words: contaminated soil, bioremediation, mineralogy, hydrocarbons, phosphate,

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XANES, catabolic genes Limit of 7000: Current = 4598 words: 2 small figures = 600, 3 large figures = 1500: One table = 300 Total = 6998 words

1 ACS Paragon Plus Environment


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Abstract

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Managing phosphorus bioaccessibility is critical for the bioremediation of hydrocarbons in

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calcareous soils. This paper explores how soil mineralogy interacts with a novel biostimulatory

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solution to both control phosphorus bioavailability and influence bioremediation. Two large bore

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infiltrators (1 m diameter) were installed at a PHC contaminated site and continuously supplied

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with a solution containing nutrients and an electron acceptor. Soils from 8 contaminated sites

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were prepared and pretreated, analyzed pre-trial, spiked with diesel, placed into nylon bags into

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the infiltrators, and removed after 3 months. From XAS, we learned that 3 principal phosphate

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phases had formed: adsorbed phosphate, brushite, and newberyite. All measures of

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biodegradation in the samples (in-situ degradation estimates, mineralization assays, culturable

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bacteria, catabolic genes) varied depending upon the soil’s phosphate speciation. Notably,

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adsorbed phosphate increased anaerobic phenanthrene degradation and bzdN catabolic gene

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prevalence. The dominant mineralogical constraints on community composition were the relative

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amounts of adsorbed phosphate, brushite, and newberyite. Overall, this study finds that total

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phosphate influences microbial community phenotypes whereas relative percentages of

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phosphate minerals influences microbial community genotype composition.

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Introduction

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Retail fuel distribution releases diesel and gasoline into soil. In North America’s calcareous

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glacial till soils, natural attenuation of hydrocarbons can be limited because these soils are near

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freezing for half the year and soil water moves through preferential flow paths1-4. One

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remediation approach for these soils is to stimulate soil organisms to degrade hydrocarbons5 by

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adding nitrogen, phosphorus, and electron acceptors (e.g., nitrate, ferric iron or ferric sulfate).

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This approach works ex-situ6, 7 but often fails in-situ, perhaps due to limited microorganisms,

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electron acceptors, or nutrients8, 9. In particular, bioavailability of phosphorus can limit

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subsurface remediation efforts10.

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Phosphate in solution is the most bio-accessible form of P. However, phosphate has a

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strong tendency to adsorb to Fe, Al, and Ca minerals resulting in low solution concentrations 11,

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12

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Determining phosphate speciation in soils can be done using synchrotron-based X-ray absorption

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near edge spectroscopy (XANES) to probe the average local bonding environment of phosphate

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13-20

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components using linear combination fitting and a reference library of phosphate standard

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compounds21, 22, although this approach has difficulties distinguishing components with similar

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spectral signatures17 and may need improved baseline correction algorithms23 to be quantitative.

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. Therefore, identifying phosphate speciation is an important step in the remediation process.

. It is possible to semi-quantitatively deconvolute a heterogeneous sample into its

Carbonate mineralogy also plays an important role in the chemical fate of phosphate in

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calcareous soils. The potential for adsorption and subsequent precipitation of calcium phosphates

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onto carbonate minerals can limit the accessible fraction of soil phosphate16. However,

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identifying minerals using laboratory X-ray diffraction (XRD) often requires sample pre-

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treatments to remove organic matter, carbonates, and sand/silt coarse soil fractions24-26.



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Synchrotron powder XRD eliminates the need for sample pre-treatments27, 28, and enables clay

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mineralogy to be determined without affecting soil carbonates.

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Investigators usually use a range of approaches to demonstrate biodegradation, including

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characterizing community composition29; conducting catabolic gene assays8, viability assays30,

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and mineralization assays31; and applying environmental fate models of hydrocarbon

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biodegradation32. Likewise, we used a range of approaches to evaluate how phosphorus

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mineralogy is linked to hydrocarbon degradation. To characterize 16S rRNA composition, we

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used the EMIRGE approach, which applies universal primers to amplify almost the entire 16S

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rRNA gene and then fragments, sequences, and assembles these amplicons in silico33. EMIRGE

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avoids some primer biases associated with amplifying a small target area and is best used to

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assess dominant members of the microbial community34.

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Measuring catabolic gene levels helps determine an increase in organisms containing the

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catabolic pathway of interest. However, because primers do not amplify all organisms containing

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the catabolic gene, PCR-based approaches are susceptible to false negatives. Using multiple gene

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targets reduces this false negative risk. A key step in anaerobic degradation of alkanes is adding

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fumarate to an alkane to form 1-methylalkyl-succinate, which is catalyzed by 1-methylalkyl-

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succinate synthase35 and assessed by QPCR primers targeting the assA gene36. We assessed

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anaerobic aromatic degradation using the following genes: (i) bssA, which encodes the α-subunit

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of benzylsuccinate synthase that detects degraders using fumarate to activate the aromatic

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substrate37 and form benzylsuccinate, which is further activated to produce benzylsuccinyl-CoA;

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(ii) bzd or bcr, which encode class I benzoyl-CoA reductases to catalyze reduction of benzoyl-

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CoA38, 39 by Azoarcus or Thauera strains, respectively40; and (iii) bamA, found in facultative and

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obligate anaerobes41, which catalyzes the hydrolysis of 1-hydroxy-benzoyl-CoA.


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Gene presence is not necessarily linked to activity; therefore, assays demonstrating

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mineralization and increases in culturable degraders are needed to confirm biodegradation30, 31.

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Assays using 13C hydrocarbons demonstrate mineralization by detecting enriched 13CO2 in the

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microcosm42 headspace. Most probable number (MPN) estimates of culturable organisms avoid

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biases associated with DNA extraction and amplification and thus can confirm catabolic gene

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prevalence estimates. Environmental fate models can estimate in-situ degradation rates32,

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confirming mineralization assays and MPN estimates.

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We hypothesized that biostimulatory solutions worked at some sites but not others due to

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variation in phosphate speciation, whereas site-specific effects not linked to phosphate are

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secondary. Soil samples were derived from the treated and untreated soils from 8 hydrocarbon

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contaminated sites plus 2 common end-members of phosphorus mineralogy, hydroxyapatite and

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phosphate absorbed on hematite. Treatment consisted of chemical extraction to remove Ca (and

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add Mg) to force a greater range of phosphate solubility. These 18 samples were then duplicated

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and incubated in duplicate large bore infiltrators at an active field site undergoing N and

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phosphorus biostimulation with a solution we designed to stimulate bioremediation. At 0, 3, and

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6 months, we removed the soils and assessed phosphate speciation (via laboratory methods and

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XANES), solid-state mineralogy (via synchrotron powder XRD), in-situ degradation activity,

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hydrocarbon mineralization potential, culturable degraders, catabolic gene prevalence, and

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bacterial community composition. Our intent was to evaluate our hypothesis by using molecular

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tools to link soil mineralogy, nutrient bioaccessibility, and hydrocarbon remediation.

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Materials and Methods

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Contaminated Sites



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Using a push core drill, soil from 8 (Colonsay, Young, 33rd Sand Lens, 33rd Street Clay

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Lens, Broadway South Site, Broadway West Side, Meadow Lake, Winnipeg Street) retail

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gasoline and diesel outlets in Alberta and Saskatchewan were collected from 2-4 m below

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ground between May and August, assessed for hydrocarbon contamination, and stored at -20°C.

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Prior to collection, all soils had been exposed to on-site gasoline and diesel leaks for a least a

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year and had been frozen for 4-6 months per year.

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Large Bore Injection System

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In late 2013, two 5.5 m deep infiltrators (1 m diameter) were installed by a rotary bucket

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caisson auger at an urban site and backfilled with coarse-grained silica sand. Each infiltrator had

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1 infiltration well (5 cm), 1 overflow well connected to the infiltration well (30 cm), 3 wells (10

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cm) in which we hung in situ reactors, and 1 standard monitoring well (5 cm). Submersible

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pumps in the underground storage tank were connected to infiltrator lines, with floats and auto

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shutoff switches installed in the tanks and infiltrators. Amendment delivery began August 14,

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2014. The tank solution was pH 6.5 and contained 11 mM MgSO4, 1 mM H3PO4, and 0.08 mM

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HNO3 in municipal potable water. These concentrations were selected to: (i) match local

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groundwater chemistry for sulfate, (ii) be at the maximum allowed groundwater nitrogen based

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on local guidelines, and (iii) be below solubility for any phosphate mineral formation in both the

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storage tanks and the groundwater of the site based upon PHREEQC modeling of both tap and

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groundwater. This experimental design provided a constant nutrient solution level to the in-situ

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reactors over a relatively long time scale to satisfy our research needs, while also respecting the

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industrial partner’s remediation requirements. It was pumped into the infiltrators 0.5 m below

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ground at a flow rate of ~5000 L/day.

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In-Situ Reactors


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Samples were air-dried, sieved to 850 µm, and divided into 2 treatment groups (in

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duplicate): control and Ca-depleted. The control soil was not treated. The Ca-depleted soil was

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prepared by adding deionized water to a 1:1 soil slurry, adjusting soil pH to 6.5 with 1.0 M

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H2SO4, centrifuging, and washing with deionized water 3 times to remove excess ions as well as

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CaSO4(s). The depleted, washed soil was added to 1.0 M MgCl2 in 1:4 ratio, stirred with a

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magnetic bar overnight, centrifuged, and washed with deionized water 3 times. This entire

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procedure was repeated 3 times to exchange bound Ca2+ for Mg2+. Hematite (#310050, Sigma-

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Aldrich) and hydroxyapatite (CAS No.12167-74-7, Alfa Aesar) were used for the mineral control

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groups.

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Soils (15 g) were placed in an in-situ reactor (12.5 cm × 4 cm 60-micron mesh Nitex

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membrane [Aqua Dynamic] bag) and spiked with retail diesel (100 µl) because the process of

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altering mineralogy removed most volatile hydrocarbons. Reactors were zip-tied onto Tygon

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tube (Fisher Scientific) and suspended 3 m underground (2.5 m below the water level) in the

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research wells. The in-situ reactors were placed into the infiltrators on August 22, 2014. On

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August 22 and December 9, 2014, and February 23, 2015, the reactors were removed and soil

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was extracted from the in-situ reactors in an anaerobic chamber then immediately tested for

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DNA, hydrocarbons, phosphate, and MPNs.

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Chemical and Mineralogical Speciation

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Synchrotron powder diffraction measurements were collected at the Canadian Light

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Source (CLS) CMCF-BM beamline (08B1-1) using a wide-area detector. The data was

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processed using GSAS-II43, with phase identification performed using X'pert Highscore Plus

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software v. 3.0 (PANalytical, Inc.).



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Phosphorus speciation was performed in laboratory with a sequential chemical extraction

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scheme modified from the Hedley extraction14. Extract analyses were performed via colorimetry

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using Folio Instruments AA1 autoanalyzer. All P K-edge XANES were collected at the CLS

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SXRMB beamline (08-B1-1) in fluorescence mode, using a 4-element solid-state detector

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(Bruker). Multiple scans were collected to ensure adequate signal-to-noise for analysis; all data

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was processed and analyzed using the Demeter software package44. A library of phosphate

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standard compounds (pure and mixed Ca and Mg phosphates), organic phosphate compounds,

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iron and aluminum phosphate minerals, and adsorption complexes of phosphate on mineral

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surfaces were analyzed; standards were diluted to 1%wt phosphorus in boron nitride powder if

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necessary.

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DNA Extraction and Microbial Community Analysis

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Soil DNA was extracted with the FastDNATM SPIN Kit for Soil (MP Biomedicals), and

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DNA concentration and purity determined by Qubit® Fluorometer (Life Technologies). A 16S

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rDNA PCR was performed with primers 27F and 1492R across a gradient of 8 temperatures (48-

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54°C)33. Generation and sequencing of amplicons was as detailed by Handley et al. (2014),

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except a Nextera DNA Sample Prep Kit was used to shear the DNA, and DNA was sequenced on

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an Illumina Miseq. The supplemental information details library preparation and bioinformatics.

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Catabolic Gene Prevalence

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Quantitative PCR (qPCR) was performed on the 7500 Real-Time PCR System (Applied

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Biosystems). The qPCR mixtures contained 10 µl SYBR Green master mix (Qiagen), 1 µl of 10

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μM primer pairs, 6 µl water, and 2 µl template DNA, with a final volume of 20 µl. Primers were

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ordered from Integrated DNA Technologies (Supplemental Table 1). For alkB, a conventional

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qPCR was performed as follows: 95°C × 10’ for 1 cycle; 95°C × 1’, 50°C × 1’, and 72°C× 1’ for


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35 cycles; followed by a melt curve. For nah, rpoB, assA, bzdN, bcrC, and bamA, touchdown

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qPCR reactions were performed as follows: 95°C × 10’ for 1 cycle; 95°C × 1’, 65°C × 1’

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(decreasing 1°C per cycle), and 72°C × 1’ for 11 cycles; 95°C × 1’, 47-65°C (47°C for nah, 49°C

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for rpoB, 56°C for assA and bamA, 58°C for bzdN, 65°C for bcrC) × 1’, and 72°C× 1’ for 35

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cycles; followed by a melt curve. Standard strain for alkB was Pseudomonas putida ATCC 2934,

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and for nah, Pseudomonas putida ATCC 17484. For other genes, clone libraries were created

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with E. coli cells using the TOPO TA Cloning Kit (Invitrogen) from environmental DNA. DNA

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was then extracted from clones and standardized to 109 copies.

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Aerobic and Anaerobic Mineralization Assays

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For aerobic assays, soil (2.5 g) from the in-situ bioreactor was transferred to 2 dram vials

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and stored at 4°C for

Total Phosphate Influences the Rate of Hydrocarbon Degradation but

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