AEM Accepted Manuscript Posted Online 19 June 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.01470-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Microbial community composition, functions and activities in the Gulf of Mexico, one

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year after the Deepwater Horizon accident.

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Etienne Yergeau1, Christine Maynard1, Sylvie Sanschagrin1, Julie Champagne1, David Juck1,

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Kenneth Lee2,3 and Charles W. Greer1*

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Canada

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2

National Research Council Canada, Energy Mining and Environment, Montréal, Quebec,

Centre for Offshore Oil, Gas and Energy Research (COOGER), Bedford Institute of

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Oceanography, Fisheries and Oceans Canada, Dartmouth, Nova Scotia, Canada

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3

12

Research Centre, Kensington, WA, Australia

Commonwealth Scientific and Industrial Research Organization (CSIRO), Australian Resources

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Keywords: Deepwater Horizon, microbial functions, microbial activities, Gulf of Mexico,

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metagenomics, metatranscriptomics

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Running title: Microbiology of the GOM one year after the DWH spill

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*

Corresponding author: [email protected]; Tel: 514-496-6182

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Abstract

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Several studies have assessed the effects of the released oil on microbes, either during or

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immediately after the Deepwater Horizon accident. However, little is known about the potential

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longer-term persistent effects on microbial communities and their functions. In this study, one

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water column station near the wellhead (3.78 km SW of the wellhead), one water column

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reference station outside of the affected area (37.77 km SE of the wellhead), and deep-sea

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sediments near the wellhead (3.66 km SE of the wellhead) were sampled one year after the

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capping of the well. In order to analyze microbial community composition, function and activity,

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we used metagenomics, metatranscriptomics and mineralization assays. Mineralization of

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hexadecane was significantly higher at the wellhead station at a depth of ~1200 m as compared

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to the reference station. Community composition based on taxonomical or functional data

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showed that the samples taken at a depth of ~1200 m were significantly more dissimilar

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between the stations than at other depths (surface, 100 m, 750 m and >1500 m). Both Bacteria

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and Archaea showed reduced activity at depths of ~1200 m when comparing the wellhead

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station to the reference station, and their activity was significantly higher in surficial sediments

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as compared to 10 cm sediments. Surficial sediments also harbored significantly different active

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genera when compared to 5 and 10 cm sediments. For the remaining microbial parameters

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assessed, no significant differences could be observed between the wellhead and reference

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stations and between surface and 5-10 cm deep sediments.

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Introduction

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Following the explosion and sinking of the Deepwater Horizon (DWH) oil rig in the Gulf

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of Mexico, an estimated 3.26 to 4.9 million barrels of light crude oil was released at a depth of

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1544 m from April 20 to July 15, 2010, making it the largest and deepest offshore spill in United

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States history (3, 22). When including gaseous hydrocarbons, like methane, the total discharge

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was 40% higher than the abovementioned estimates (12). During the spill, a deep water oil

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plume was detected at depths of 1000-1200 m (4, 10), but this plume was no longer detectable

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after a few months (25), in agreement with the very high degradation rates observed in

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laboratory incubations (10). However, most microbiological research to date has focused on the

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effects of the oil spill with samples taken during the contamination event or shortly thereafter (2,

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10, 16, 17, 20, 24, 27, 33, 34, 39), and only one study reported on the bacterial communities at

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plume depth 1 year after the spill (41). In view of the high degradation rates observed and slow

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mixing of deep water, it was suggested that oxygen depletion at plume depth might persist for

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several years (1, 12, 26, 35). The cause, extent and duration of this oxygen depletion was

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subject to debate (11, 15, 16), and it is not clear how, and if, it would impact the microbial

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communities in the long term. Recent work also indicated that significant quantities of oil sank to

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the sea floor (38), potentially affecting microbial communities in the sediments.

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The microbial characterization of the water column shortly after the beginning of the spill

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identified Oceanospirillales as a dominant group of hydrocarbon degrading organisms making

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up as much as 90% of the of the 16S rRNA gene clone libraries (6, 10, 27, 33, 41). Shortly after

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this, other Gammaproteobacteria affiliated with Colwellia and Cycloclasticus appeared,

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indicative of a succession from alkane to aromatic degrading bacteria (6, 33, 39, 41). In

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addition, other phyla of bacteria (Alteromonas, Halomonas, Pseudoalteromonas) were observed

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in the water column (10, 39). A recent DNA-Stable Isotope Probing (SIP) study provided direct

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evidence that most of the abovementioned taxa were in fact capable of degrading various 3

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hydrocarbons (9). Following the spill, after the flow of hydrocarbons had been arrested,

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methylotrophs including known methane oxidizers, became dominant in the region of the plume

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(16). Microbial communities are at the base of several crucial biogeochemical processes in

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marine environments, including hydrocarbon degradation. Full ecosystem recovery is intimately

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linked to microbial community recovery. Microorganisms might also serve as highly sensitive

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bioindicators (37), as they have been shown to be sensitive to very low concentration of

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pollutants especially with regard to their transcriptome (43-45). For these reasons,

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microorganisms could be used as indicators of pollution and ecosystem recovery through the

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examination of their gene content and gene expression patterns.

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Two approaches were used to determine the potential effects of the DWH blowout on

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microbial communities more than one year after the event: 1) comparison of two water column

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stations, one very close to the well and the second 38 km away, outside the plume area; 2)

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depth profile of deep-sea sediment cores taken in the proximity of the Macondo well. We used a

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shotgun metagenomic and metatranscriptomic approach and compared the microbial functions,

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community composition and activities of the different stations with depth.

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Material and methods

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Sampling sites

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A map of the sampling sites is provided as Fig. 1. The wellhead water column station (BM-57,

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28.7051°, -88.4016°) was located at a distance of 3.78 km SW from the actual Deepwater

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Horizon wellhead and corresponded to the plume station BM-57 used by Hazen and colleagues

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(10). The reference water column station (A6, 28.6632°, -88.0095°) was located 37.77 km SE

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from the Deepwater Horizon wellhead, but in the same “dome” area and was outside the plume

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area during the spill. A series of 6 deep-sea sediment cores were collected on Nov. 16, 2011

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during a second cruise. The cores were collected from the vicinity of the Deepwater Horizon

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wellhead (around 28.715011°; -88.358703°, 3.66 km SE from the wellhead) at a depth of

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approximately 1600 m.

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Water and sediment sampling

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Water samples were collected between Sept. 9-16, 2011 using either a large bailing bucket

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(surface samples) or a CTD Niskin rosette equipped with 20 L bottles. For each depth, three

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replicate water samples were taken. Samples were returned to the boat and immediately

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transferred to 4 L carboys which were previously rinsed with 70% ethanol and sterile distilled

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water. Sample filtration was started immediately after transfer using Millipore GSWP (0.22 µm

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pore size, 47 mm diameter) filters and glass filter supports. Each 4 L water sample was filtered

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on two filters, resulting in a total of six filters per depth. The filters were then transferred to ice

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and then stored at -80˚C. Between samples, the glass filter supports were rinsed with 70%

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ethanol and sterile distilled water. For the shipping of filtered samples, coolers with dry ice were

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used and upon arrival at the lab, filters were stored at -80˚C until nucleic acid extraction was

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performed. Water samples destined for mineralization analysis were collected from the same 5

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carboys as used for filtration. Samples were placed in sterile 50 ml Falcon tubes and stored at

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4˚C. Samples were shipped on ice and upon arrival at the lab were placed immediately at 4˚C.

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Microcosms were started as soon as possible after arrival into the lab (within 24 h). Water for

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chemical analysis was also taken and kept at 4°C until processing.

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Sediments were frozen on-board the sampling vessel. Samples were shipped and

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received frozen and stored at -20˚C until processing. Sample processing was performed at -

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20˚C based on a protocol modified from Juck et al. (13). In brief, an approximately 5 cm strip of

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the core sample plastic sleeve was cut and removed (from top to bottom of the core) and a

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‘clean’ area of the core (i.e. not contacted by the sample sleeve) was exposed using a sterile

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chisel. Once this flat clean area was exposed, a sterile 1.4 cm drill bit was used to slowly drill

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into the core sample, parallel to the core surface. The drilled core sub-sample was then

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transferred to a sterile 50 ml Falcon tube and stored at -80˚C until extraction of nucleic acids

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was performed. Each core was sampled at 3 different depths – ‘0 cm’ was from the surface of

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the sediment to 1.4 cm, ‘5 cm’ was from approximately 4.3 to 5.7 cm from core surface and ’10

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cm’ was from approximately 9.3 to 10.7 cm from the sediment surface. From the remaining core

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samples, the material remaining at 0, 5 and 10 cm was sampled and used for hydrocarbon

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analysis as described below.

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Water microcosm mineralization assays

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Mineralization assays using microcosms were set up using 15 ml of seawater and

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(100,000 dpm) hexadecane (2.5 ppm), as sole carbon source, with no amendments added. The

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sealed bottles containing seawater from all the different depths for both water column stations

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and the spiked substrate were all incubated at 15˚C (the range of sample temperatures at the

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time of collection was 4˚C (bottom samples) to 30˚C (surface samples) with orbital shaking (140 6

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C labeled

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rpm) and a microcosm KOH trap (1 ml of 1.0 M KOH in a test tube). Sampling was performed at

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T=3, 7, 14, 22, 28, 35, 42, 49, 56 and 63 days. The amount of radioactive

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to the complete mineralization of the added carbon source (hexadecane) was determined by

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scintillation counting of the KOH solution recovered from the microcosm flasks and is presented

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as a percentage of

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During sampling of the KOH traps, atmospheric oxygen was introduced (through a 0.22 µm

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filter) into the microcosms to ensure sufficient aeration of the samples. The 700 m depth sample

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from the reference station was also used as a sterile abiotic control by autoclaving for 20 min

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and cooling to room temperature before addition of the radioactive spike.

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CO2 produced, due

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CO2 produced from the known quantity of carbon source added at T=0.

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Hydrocarbon analyses

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Water column samples were extracted for C10-C50 and PAH analyses using liquid-liquid

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extraction

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http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3510c.pdf). Extracts of water were

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analyzed using high resolution gas chromatography (Agilent 6890 GC) coupled to a mass

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selective detector (Agilent 5973N) (Willmington, DE, USA) operated in the selective ion

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monitoring mode (SIM) using the following GC (MDN-5S column 30 m x 0.25mm id 0.25 μm film

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thickness, Supelco Canada) conditions: cool on-column injection with oven track mode (track 3

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°C higher that the oven temperature program) 80 °C hold 2 min ramp at 4 °C/min to 280 °C hold

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10 min. Deep-sea sediments were processed according to King and Lee (19) and the GC-MS

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conditions outlined for the water extracts were applied to sediment extracts.

(US

EPA

Method

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Total DNA extraction from filters (seawater)

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3510

C,

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From each water sample (2 stations X 5 depths X 3 replicates = 30 water samples), one filter

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was used and treated for DNA extraction, resulting in 30 DNA extracts. In the 50-ml Falcon

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tube containing the filter, 1.7 ml of Tris-EDTA (TE) pH 8.0 buffer was added with 45 µl of 20%

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(w/v) sodium dodecyl sulfate (SDS) and 9 µl of 20mg/ml proteinase K. The tube was incubated

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with gentle inversion at 37°C for one hour. At the end of the incubation, 300 µl of 5M NaCl was

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added in addition to 240 µl of a 10% (w/v) cetyl trimethylammonium bromide (CTAB) and 0.7M

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NaCl solution. The tube was incubated at 65°C for 10-min. The total DNA was extracted with 1

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volume of 24:1 chloroform/isoamyl alcohol. After centrifugation for 10 min at 3,000 x g, the

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upper phase was transferred and mixed with one volume of 25:24:1 phenol/chloroform/isoamyl

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alcohol. Following centrifugation at 16,000 x g for 10min at 4°C, the supernatant was

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precipitated by mixing 0.6 volume of isopropanol and 1/50 volume of glycogen (5 mg/mL),

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incubating one hour at -80°C and centrifuging at 12,000 x g for 30 min at 4°C. The DNA pellets

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were washed using 1ml of 80% (v/v) ice-cold ethanol and dried using a SpeedVac. The DNA

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was re-suspended in 50µl of nuclease-free water and treated with RNase If (NEB, Ipswich, MA)

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according to the manufacturer’s instructions. After the reaction was complete and the enzyme

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was inactivated, the DNA was purified with the QIAEX II Kit (QIAgen, Valencia, CA) and

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quantified using the PicoGreen assay (Invitrogen).

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Total RNA extraction from filters (seawater)

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For RNA extraction, one replicate seawater sample was used (5 depths X 2 stations = 10

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samples). All solutions were RNase free. In the 50-ml Falcon tube containing the filter, 1.6 mL of

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freshly prepared lysozyme (10 mg/ml in TE pH 8.0) and 80 uL of 20% SDS were added and the

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tube was then incubated at 64°C for 5 min. At the end of the incubation, 176 µl of 3M sodium

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acetate pH 5.2 and 1.6 mL of pre-warmed acid phenol was added to the lysate incubated at

8

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64°C for 6 min, with mixing every minute. The tube was transferred on ice for 2 min and then

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centrifuged at 16,000 x g for 10 min at 4°C. The upper phase was transferred and mixed with

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1.6 ml of chloroform before centrifugating at 16,000 x g for 2 min at 4°C. The upper aqueous

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phase was transferred, mixed with 20 µl of glycogen (5 mg/ml), 160 µL of 3M sodium acetate

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pH 5.2 and 4 mL of 100% ice-cold ethanol and incubated for 30 min on dry ice before

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centrifuging at 12,000 x g for 30 min at 4°C. The pellet was washed with 1 ml of 80% ice-cold

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ethanol and dried using a SpeedVac. The RNA was re-suspended in 400 µL of nuclease-free

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water (Ambion, Life Technologies, Burlington, Ontario, Canada) and pooled together in the

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same tube. The extracted total RNA was treated with Turbo DNase I (Ambion) and purified with

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RNeasy MinElute Cleanup Kit (Qiagen).

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DNA/RNA extraction (deep-sea sediments)

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DNA and RNA were extracted simultaneously from 2 g of sediment using the MoBio RNA

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PowerSoil Total RNA Isolation Kit with the RNA PowerSoil DNA Elution Accessory kit (MoBio

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Laboratories, Carlsbad, CA).

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Metagenomic sequencing

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Each DNA library was prepared for sequencing from 50-100 ng of DNA using the Ion Xpress

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Plus Fragment Library Kit (Life Technologies) with the Ion Xpress Barcode Adapters 1-16 (Life

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Technologies), using the Ion Shear Plus Reagents and a Pippin Prep instrument (SAGE

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Science, Beverly, MA) for size-selection. Barcoded libraries were pooled in an equimolar ratio

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three by three. A total of 3.50 x 107 molecules were used in an emulsion PCR using the Ion

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OneTouch 200 Template Kit (Life Technologies) and the OneTouch instrument (Life

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Technologies). The sequencing of the pooled libraries was performed using the Personal

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Genome Machine (PGM) system with the Ion Sequencing 200 kit and 316 chips (Life

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Technologies). Sequencing statistics are shown in Table S1.

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Metatranscriptomic sequencing

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In order to get enough RNA for library preparation, RNA samples were amplified using the

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MessageAmp II-Bacteria Kit (Ambion) according to the manufacturer's protocol. The antisense

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RNA (aRNA) obtained was subjected to ribosomal RNA subtraction following the procedure of

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Stewart et al. (36) with the exception that the T7 promoter was coupled to the forward primer

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instead of the reverse primer. After subtraction, a 227 bp control RNA transcribed from the

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pSPT18 vector (positions 2867-3104 and 1-70) was added in a 1:1000 ratio (on a nanogram

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basis) to the total rRNA-subtracted RNA. This mixture was then reverse-transcribed using the

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SuperScript III kit (Invitrogen, Life Technologies). Illumina libraries were prepared following the

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protocol of Meyer and Kircher (30), with tags 1 to 34. The indexed libraries were pooled in an

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equimolar ratio and sent for eight lanes of Illumina HiSeq 2000 paired-end 2x100 bp sequencing

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at

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Canada).Sequencing statistics are shown in Table S2.

the

McGill

University

and

Genome

Quebec

Innovation

Centre

(Montreal,

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Bioinformatics

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Metagenomic sequences were submitted to MG-RAST where they were de-replicated using the

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method of Gomez-Alvarez et al. (8) and trimmed using the dynamic trimming method of Cox et

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al. (5) in a way that each individual sequence would contain a maximum of 5 bases below a

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Phred score of 15. Within MG-RAST, significant matches were defined as having 60%

10

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sequence identity over at least 15 aa or 50 bp and with an e-value below 10-5. Metagenomic

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data was used as relative abundance by dividing the abundance of sequences for a particular

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organisms/gene by the total number of sequence retrieved from the sample. Metatranscriptomic

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data resulted in 544 files (34 samples x 2 reads x 8 lanes). Data from the different lanes were

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pooled together and the resulting 68 files were filtered using a custom-made Perl script, as

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follows. Paired-end reads were processed in parallel. Reads were first trimmed at the first

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occurrence of a low quality base (Phred score below 20) or when the adapter sequence was

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encountered. Following this step, sequences of less than 75 bp were removed from further

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analyses. If only one of the paired reads was filtered out, then the remaining read was also

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removed. The filtered reads were then submitted to MG-RAST 3.0 (29) where mate-paired

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reads were joined using the fastq-join utility. Mate-paired reads that did not overlap were kept

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for downstream analyses. Within MG-RAST, significant matches were defined as having 60%

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sequence identity over at least 15 aa or 50 bp and with an e-value below 10-5. The number of

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sequences related to the pSPT18 vector in the filtered metatranscriptomic datasets was

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obtained by Blast using an e-value cutoff of 10-25 and this number was used to normalize the

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number of transcripts using the method of Moran et al. (31).

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Statistical analyses

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All statistical analyses were carried out in R (v. 2.13.2, The R foundation for statistical

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computing, Vienna, Austria). Normal distribution of the data was tested using the “shapiro.test”

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function. If necessary, data was then transformed using log or square root transformations.

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Analysis of variance (ANOVA) was performed using the “aov” function while post-hoc Tukey

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honestly significant difference tests were carried out using the “TukeyHSD” function. If these

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transformations failed to normalize the data, a non-parametric Kruskal-Wallis test was carried

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out in lieu of ANOVA (function “kruskal.test”). Correlation analyses were carried out based on

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Spearman correlation using the “cor” function. Bray-Curtis dissimilarities were calculated using

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the “vegdist” function of the “vegan” library.

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Data deposition

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Raw sequence reads were submitted to the NCBI Sequence Read Archive (SRA) under the

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BioProject accession PRJN0000 (pending) and the SRA project accession SRP0000 (pending).

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Annotated metagenomes (MG) and metatranscriptomes (MT) are available in MG-RAST under

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accessions 4494020.3-4494048.3 and 4494917.3 (MG, water, project 1012) 4500695.3-

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4500711.3 (MG, sediments, project 1891), 4508873.3-4508882.3 (MT, water, project 2384) and

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4508988.3-4509004.3 (MT, sediments, project 2866).

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Results

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The goal of this study was to observe the effects of the DWH spill approximately one year after

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the successful capping of the well. In order to do this, water column samples from a reference

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station that was outside the spill area were compared to water column samples taken at similar

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depths at a station that was directly in the spill area. In addition, deep-sea sediment cores were

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taken in the direct vicinity of the well, and the surface, 5 cm and 10 cm sediment layers were

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compared.

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Chemical analyses and mineralization assays

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The chemical analyses of the water and sediments revealed very low concentration of alkanes

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mostly in the ng per liter of water or ng per g of sediment range (Fig. 2). At these

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concentrations, near the detection limit, variation between replicates was quite high, and the

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only significant difference between the reference and affected water column was between the

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surface water samples (t-test: t=3.53, P

Microbial Community Composition, Functions, and Activities in the Gulf of Mexico 1 Year after the Deepwater Horizon Accident.

Several studies have assessed the effects of the released oil on microbes, either during or immediately after the Deepwater Horizon accident. However,...
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