DOI: 10.5285/df829b9f-c4c5-4e53-9217-c9c1e5bd078d
title: Molecular analysis of freshwater bacterial biofilm communities under experimentally manipulated dissolved organic carbon regimes at Llyn Brianne (2014)

description: These data consist of raw 16S rRNA gene sequences for the bacterial communities in three upland Welsh river sites under different treatments. A mapping file with metadata for each sample is provided and a operational taxonomic unit (OTU) table. These sites were situated in three streams from the Llyn Brianne Stream Observatory, Powys, Wales, UK (52°08' N, 3°45' W). The catchments cover approximately 300 square kilometres of upland Wales in the upper Afon Tywi. These first to third order experimental streams rise in either rough, sheep-grazed moorland (named as L6 and L7) or plantations of Sitka spruce Picea sitchensis with lodgepole pine Pinus contorta (named as L3). Some reductions of forest cover have occurred in L3 with normal logging operations. A 24-hour experiment was conducted at the Diversity in Upland Rivers for Ecosystem Service Sustainability (DURESS) cascading flumes at these streams during September 2014. Each flume consisted of 3 channels, each assigned a different treatment: control, sugar addition and peat addition. Sugar (sucrose) and peat were added to channels to represent a simple and complex form of dissolved organic carbon (DOC) respectively. Five biofilm samples were collected from random locations in each experimental channel. Samples were taken at 0.5, 3, 15 and 24 hours after the start of the experiment. Epilithon were taken from unglazed ceramic tiles that had been colonised by epilithon in the river. After amplification, the 16S rRNA fragments were sequenced on the Illumina MiSeq next generation sequencing platform.

The main goal of this survey was to characterise bacterial diversity, the chemical and biological consequences of elevated DOC inputs, and to investigate the role of bacterial organisms in controlling organic carbon flux. Prof Andy Weightman and Dr Isabelle Durance were responsible for organising the experiments. Sampling was carried out by Dr. Isa-Rita Russo and a team of Post Doctoral Research Assistants (PDRA's)/students. The work was carried out under the Diversity in Upland Rivers for Ecosystem Service Sustainability (DURESS) project (Grant reference NERC NE/J014818/1). DURESS was a project funded by the Natural Environment Research Council (NERC) Biodiversity and Ecosystem Service Sustainability (BESS) programme.
description: Dissolved organic carbon (DOC) additions were made to replicate recirculating flumes at three upland streams in mid-Wales. Water and biofilm samples were taken at regular intervals over the following 24-hour period to monitor the effects of carbon addition. Temperature and water oxygen content were also monitored over the course of the experiment. The water samples were analysed for total carbon, inorganic carbon and organic carbon content. Epilithon were taken from unglazed ceramic tiles that had been colonised by epilithon in the river using the tooth-brush method. Samples were frozen immediately after collection. On return to the laboratory, samples were stored at -70 degrees Celsius prior to DNA extractions. Total genomic DNA was extracted from epilithon using the FastDNA (registered trademark) SPIN Kit for soil, incorporating a bead beating step. An extraction kit negative control was included in the sequencing run.

To test for the presence of DNA, amplifications were carried out in a total volume of 50 microlitre containing 10 microlitre of Polymerase chain reaction P(CR) Mix (Buffer), 0.2 micromolar of each of the forward and reverse primers, 1.5 Uracil (U) of Taq DNA polymerase and 1 microlitre of DNA (approximately 50 nanogram/microliter). Primers 357F (Turner et al. 1999) and 518R (Muyzer et al. 1993) were used for amplification. In addition (these amplifications were carried out by a commercial company - GeneTiCA), the bacterial 16S region was amplified with primers S-D-Bact-0341-b-S-17: CCTACGGGNGGCWGCAG, S-D-Bact-0785-a-A-21: GACTACHVGGGTATCTAATCC (Klindworth et al. 2013). Triplicate Polymerase chain reactions (PCRs) for each sample were cleaned with the AMPure bead method, pooled and sequenced on an Illumina MiSeq v3 (2 x 300 base pairs) (Illumina, Inc., San Diego, USA) with Nextera XT assay chemistry. Polymerase chain reaction (PCR) and sequencing were done by GeneTiCA (Prague, Czech Republic). Samples were double-barcoded to allow 192 samples to be run together; the sequencing run was shared with samples from another experiment.

Sequences were quality filtered and grouped into operational taxonomic units (OTUs) using USEARCH and QIIME. Taxonomy was assigned from the Greengenes database. Each row in the OTU table corresponds to an OTU. Column 1 contains the OTU identifiers, columns 2-98 are headed as per the sample IDs in the mapping file; columns 99-105 contain the taxonomic information.