Abstract:

The dataset provides bacterial 16S rDNA sequencing data to show the composition changes in bacterial communities resulting from the effect of bioturbation and oil exposure. Another part of this dataset illustrates the effect of bioturbation and oil exposure on a growth increase of bacterial population, which could degrade PAH more efficiently. To characterize that a quantitative PCR assay was developed to calculate the gene copy number for the key PAH degrading enzyme (PAH ring hydroxylating dioxygenase). The number of genes equals the number of cell possessing this enzyme assuming that there is one gene copy per cell. This dataset provides information on microbial community composition in the FASTA file titled “RazorClam2014.fasta” and the gene-copy number in the spreadsheet titled “Dataset-RazorClam2014”.

Suggested Citation:

Chistoserdov, Andrei.. 2017. The stout razor clam mesocosm experiment with microbial community composition data and PAH dioxygenase gene copy number data. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7R78C6X

Purpose:

This experiment aims to determine whether animal, oil and/or both animal and oil enhances the PAH degradation rate in sediment.

Data Parameters and Units:

For the calculation of gene copy number the obtained value from qPCR was normalized and expressed as per gram of sediment.

Methods:

After greenhouse mesocosm experiment (see R2.x226.000:0001), the Mobio PowerLyzer® PowerSoil® DNA isolation kit was used to isolate microbial genomic DNA from the sediment samples with 40µl final elution volume. Portions of this DNA was sent to a sequencing provider (Mr. DNA, Shallowater, TX) for sequencing and rest are used for other experiments like quantifying PAH degrading gene PAH dioxygenase. Bacterial 16S rDNA gene was sequenced using ion torrent PGMTM technology and the resulting raw data were submitted herein. Many Gram-positive and Gram negative bacteria possess the PAH-ring hydroxylating dioxygenase gene and, therefore, are able to degrade PAH efficiently. Typically, most of these microorganisms carry only one PAH dioxygenase gene copy per cell and, therefore, to determine the number of PAH degrading organisms, enumeration of PAH dioxygenase gene copies by qPCR was performed. The resulted gene copy number is expressed per gram of sediment and represents the number of PAH degrading bacterial cells present. 1µl DNA was used in for qPCR reactions, which were performed in an Applied Biosystems StepOne™ StepOnePlus™ Real-Time thermocycler. The qPCR reaction consisted of: 4.5 l of GoTaq qPCR Master Mix (Promega, Madison, WI), 1l of DNA mix, 0.5l of each reverse and direct primer, and 3.5l of ddH2O. PCR reaction conditions were: 5 min. at 95oC; 50 cycles of 30 sec. at 95oC, 30 sec. at 54oC, 30 sec. at 72oC and 10 sec at 80oC; followed by primer extension for 7 min at 72oC. Dilutions of known genome numbers from Pseudomonas putida ATCC17484 and Rhodococcus sp. NCIMB 12038 were used as quantification standards for Gram–negative and Gram-positive bacteria, respectively. To calculate a total gene-copy number, the numbers of copies returned by the thermocycler software were multiplied by elution volume and then divided by the sediment weight.

Instruments:

Applied Biosystems StepOne™ StepOnePlus™ Real-Time thermocycler.

Error Analysis:

The instrument was calibrated prior to use. Data entry into digital files was al least in triplicates and gene copy numbers averaged.

Provenance and Historical References:

None

Individual Files: