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Assessing the abundance of genes involved in the denitrification pathways collected in marsh and subtidal unvegetated sediments at the Chandeleur Islands, July 2015 to February 2016

Alabama Center for Ecological Resilience (ACER)

DOI:
10.7266/N7154F44
 
UDI:
R4.x262.000:0021
Last Update:
Aug 14 2017 16:20 UTC
 
Dataset Author(s):
Flournoy, Nikaela
Point of Contact:
Sobecky, Patricia
The University of Alabama / Department of Biological Sciences
Box 870344
Tuscaloosa, Alabama  35487
USA
psobecky@ua.edu
Funding Source:
RFP-IV
Data Collection Period:
2015-07-09 to 2016-02-22

Identified Submitted Review Available
3 3 3 3

Abstract:

Selected genes involved in nitrogen cycling were enumerated from sediment collected on the marsh platform as well as from nearby subtidal unvegetated sediments in the Chandeleur Islands. Data were collected seasonally from July 2015 to February 2016.

Purpose:

The purpose of this study was to enumerate genes involved in denitrification in marsh and subtidal habitats that were previously exposed to moderately levels of hydrocarbons.

Theme Keywords:

sediments, denitrification, nitrogen cycling

File Format:

csv

Dataset Downloads:

3

Assessing the abundance of genes involved in the denitrification pathways collected in marsh and subtidal unvegetated sediments at the Chandeleur Islands, July 2015 to February 2016



Identification Information
Distribution Information
Metadata Maintenance Information

Metadata: 
  File identifier: 
      R4.x262.000-0021-metadata.xml
  Language: 
      eng; USA
  Character set: 
    Character set code: 
      utf8
  Hierarchy level: 
    Scope code: 
      dataset
  Metadata author: 
    Responsible party: 
      Individual name: 
          Lei Hu
      Organisation name: 
          Dauphin Island Sea Lab (DISL) / University Programs
      Position name: 
          Data Manager
      Contact info: 
        Contact: 
          Phone: 
            Telephone: 
              Voice: 
                  2518617521
              Facsimile: 
          Address: 
            Address: 
              Delivery point: 
                  101 Bienville Blvd
              City: 
                  Dauphin Island
              Administrative area: 
                  Alabama
              Postal code: 
                  36528
              Country: 
                  USA
              Electronic mail address: 
                  lhu@disl.org
      Role: 
        Role code: 
          pointOfContact
  Date stamp: 
      2018-08-11T13:38:49+00:00
  Metadata standard name: 
      ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
  Metadata standard version: 
      ISO 19115-2:2009(E)
  Dataset URI: 
      https://data.gulfresearchinitiative.org/metadata/R4.x262.000:0021
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Identification info: Data identification: Citation: Citation: Title: Assessing the abundance of genes involved in the denitrification pathways collected in marsh and subtidal unvegetated sediments at the Chandeleur Islands, July 2015 to February 2016 Alternate title: Date: Date: Date: 2017-06-23 Date type: Date type code: publication Abstract: Selected genes involved in nitrogen cycling were enumerated from sediment collected on the marsh platform as well as from nearby subtidal unvegetated sediments in the Chandeleur Islands. Data were collected seasonally from July 2015 to February 2016. Purpose: The purpose of this study was to enumerate genes involved in denitrification in marsh and subtidal habitats that were previously exposed to moderately levels of hydrocarbons. Status: Progress code: completed Point of contact: Responsible party: Individual name: Patricia Sobecky Organisation name: The University of Alabama / Department of Biological Sciences Position name: Professor Contact info: Contact: Phone: Telephone: Voice: 2053481409 Facsimile: Address: Address: Delivery point: Box 870344 City: Tuscaloosa Administrative area: Alabama Postal code: 35487 Country: USA Electronic mail address: psobecky@ua.edu Role: Role code: pointOfContact Descriptive keywords: Keywords: Keyword: sediments Keyword: denitrification Keyword: nitrogen cycling Type: Keyword type code: theme Descriptive keywords: Keywords: Keyword: Chandeleur Islands Keyword: Red Fish Point Keyword: Gulf of Mexico Keyword: Louisiana Type: Keyword type code: place Language: eng; USA Topic category: Topic category code: environment Topic category: Topic category code: oceans Extent: Extent: Geographic element: Geographic bounding box: West bound longitude: -88.841188 East bound longitude: -88.841188 South bound latitude: 29.862799 North bound latitude: 29.862799 Geographic element: BoundingPolygon: Polygon: gml:MultiPoint: gml:pointMember: Point: gml:pos: 29.862799 -88.841188 Temporal element: Temporal extent: Extent: Time period: Description: ground condition Begin date: 2015-07-09 End date: 2016-02-22 Supplemental Information: Date: month, day and year, Location: Location on the Map, Habitat: Marsh platform or subtidal unvegetated sediments, Cores: replicate cores that were collected from each site, 16S (gene copies per gram): 16S ribosomal (rRNA) in copies per gram sediment, napA (gene copies per gram): napA (nitrate reductase) gene copies per gram sediment, norB (gene copies per gram): norB (nitric oxide reductase) gene copies per gram sediment, nirS (gene copies per gram): nirS (nitrite reductase) gene copies per gram sediment, Latitude Degrees: measured in degrees, Latitude Minutes: measured in minutes, Latitude Seconds: measured in seconds, Longitude Degrees: measured in degrees, Longitude Minutes: measured in minutes, Longitude Seconds: measured in seconds|Replicate cores were collected in the field and brought back to the laboratory where they were set up for measurements of denitrification rates with the isotope pairing technique. At the termination of the rate measurements the top 10cm of the cores were homogenized and frozen at -80oC for later analysis. DNA was extracted in triplicate from 1 g sediment from each core with the FastDNATM Spin Kit for Soil (MP Biomedicals) per the manufacturer’s protocol with the addition of two-minute ice incubations after the homogenization and 4oC centrifugation steps. The triplicate extractions were pooled and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research) and eluted with sterile milliQ water (total volume 50 µl). DNA concentrations were measured via absorption at 260 nm using a NanoDrop ND-1000. Quantitative PCR (qPCR) was performed to assess the abundance of three genes in the denitrification pathway: napA (nitrate reductase), nirS (nitrite reductase), and norB (nitric oxide reductase). Total community abundance was determined by qPCR of 16S ribosomal RNA (rRNA). Each reaction included Platinum SYBR Green qPCR supermix-UDG with ROX (12.5 µL) (Invitrogen), 1 µL of forward and reverse primer (5 µM, IDT DNA technology), 0.5 µL MgCl2 (50 mM), and 5 ng of DNA template. The total reaction volume was adjusted to 25 µL with PCR grade water (ThermoFisher). Following 2 min at 50ºC and activation of Platinum SYBR Green qPCR supermix-UDG with ROX at 95ºC for 10 min, thermal cycles consisted of (napA and nirS) 40 cycles of: 15 s at 95ºC (denaturation), (norB) 30 cycles 15 s at 95ºC, 1 min at 60ºC elongation step, (16S) 30 cycles 15 s at 95oC, and 30 s at 72oC. Samples were run in duplicate on a 7000 Sequence Detection System (ABI Prism) with the primer combinations, optimal annealing temperatures, and qPCR conditions detailed below. Primers and thermal profiles (* denotes modified annealing temperature) used for PCR to generate standards and qPCR quantification of different functional genes. Target gene: napA (qPCR) Primers: napA-1F; napA-1R Sequence(5’ 3’): GTY ATG GAR GAA AAA TTC AA; GAR CCG AAC ATG CCR AC References: Smith et al. (2007) Optimal Annealing Temp.: 55 Celsius - 60 s Target gene: napA (PCR) Primers: napA V67 F; napA V67 R Sequence(5’ 3’): TAY TTY YTN HSN AAR ATH ATG TAY GG; DAT NGG RTG CAT YTC NGC CAT RTT References: Flanagan et al. (1999) Optimal Annealing Temp.: 55 Celsius - 30 s* Target gene: nirS (qPCR and PCR) Primers: nirScd3aF; nirS R3cd Sequence(5’ 3’): GTS AAC GTS AAG GAR ACS GG; GAS TTC GGR TGS GTC TTG A References: Throback et al. (2004) Optimal Annealing Temp.: 51 Celsius - 60 s Target gene: norB (qPCR and PCR) Primers: cnorB2F; cnorB6R Sequence(5’ 3’): GAC AAG NNN TAC TGG TGG T; GAA NCC CCA NAC NCC NGC References: Geets et al. (2007) Optimal Annealing Temp.: 50 Celsius - 60 s Target gene: 16S (PCR) Primers: 341F; 534R Sequence(5’ 3’): CCT ACG GGA GGC AGC AG; ATT ACC GCG GCT GCT GGC A References: Bru et al. (2008) Optimal Annealing Temp.: 55 Celsius - 45 s Target gene: (q-PCR) Primers: N/A Sequence(5’ 3’): N/A References: López-Gutiérrez et al. (2004) Optimal Annealing Temp.: 60 Celsius - 30 s Bru D, Martin-Laurent F, Philippot L. 2008. Quantification of the Detrimental Effect of a Single Primer-Template Mismatch by Real-Time PCR Using the 16S rRNA Gene as an Example. Applied and Environmental Microbiology 74:1660-1663. Flanagan DA, Gregory LG, Carter JP, Karakas-Sen A, Richardson DJ & Spiro S. 1999. Detection of genes for periplasmic nitrate reductase in nitrate respiring bacteria and in community DNA. Fems Microbiology Letters 177: 263-270. Geets J, de Cooman M, Wittebolle L, Heylen K, Vanparys B, De Vos P, Verstraete W, & Boon N. 2007. Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge. Applied Microbology and Biotechnology 75:211-221. López-Gutiérrez JC, Henry S, Hallet S, Martin-Laurent F, Catroux G & Philippot L. 2004. Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR. J Microbiol Meth 57: 399-407. Smith, C. J., D.B. Nedwell, L.F. Dong, and A.M. Osborn. 2007. Diversity and abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA), and their transcripts in estuarine sediments. App. Environ. Microbiol. 73: 3612-3622. Throbäck IN, Enwall K, Jarvis A, Hallin S. 2004. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiology Ecology 49:401-417.||||
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Distribution info: Distribution: Distributor: Distributor: Distributor contact: Responsible party: Organisation name: Gulf of Mexico Research Initiative Information and Data Cooperative (GRIIDC) Contact info: Contact: Phone: Telephone: Voice: 3618253604 Address: Address: Delivery point: 6300 Ocean Drive City: Corpus Christi Administrative area: TX Postal code: 78412 Country: USA Electronic mail address: griidc@gomri.org Online Resource: Online Resource: Linkage: URL: https://data.gulfresearchinitiative.org Role: Role code: distributor Distributor format: Format: Name: csv Version: inapplicable File decompression technique: Distributor transfer options: Digital transfer options: Transfer size: 0.0065 Online: Online Resource: Linkage: URL: https://data.gulfresearchinitiative.org/data/R4.x262.000:0021 Protocol: https
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Metadata maintenance: Maintenance information: Maintenance and update frequency: unknown Maintenance note: This ISO metadata record was automatically generated from information provided to GRIIDC for dataset: R4.x262.000:0021 on 2018-09-26T13:17:13+00:00
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