Stream biofilm 15N DNA stable isotope probing experiment

This resource contains the results of a 15N DNA stable isotope probing (DNA-SIP) experiment that identified and compared biofilm bacterial assemblages that assimilated 15N-ammonium (15N-NH4+), 15N-nitrate (15N-NO3-), 15N-glycine or baseline assemblages not exposed to 15N label. We grew biofilms exposed to 15NH4+, 15NO3−, 15N-glycine, or no 15N addition using nutrient-diffusing substrates (NDS). We grew biofilms exposed to 15NH4+, 15NO3−, 15N-glycine, or no 15N addition using nutrient-diffusing substrates (NDS; Tank et al. 2007). To construct NDS, we filled 30-mL plastic cups (Polycon, Madan Plastics) with agar amended with either no additions (baseline), 0.5 M 15N as 15NH4+ (15NH4Cl, 98 atom percent, Aldrich), 0.5 M 15N as 15NO3− ( K15NO3-, 98 atom percent, Aldrich) or 0.5 M 15N as 15N-glycine (15N-glycine; 98 atom percent, Aldrich). The agar was capped with a porous glass disc (crucible cover manufactured by LECO) which served as a platform for biofilm growth and exposed the growing biofilms to nutrients which diffused out of the agar. We placed NDS in the Middle Provo River at our study site located immediately below Jordanelle Reservoir for 19 days during summer 2016. At the end of the river deployment, we collected biofilm-colonized discs from the NDS and stored them at -20°C until DNA analyses.

DNA-SIP analyses were conducted by first extracting genomic DNA from each biofilm-colonized disc using a PowerSoil DNA Isolation Kit (MoBio, Carlsbad, California, USA. We next separated the DNA in each sample by density using ultracentrifugation (58,000 rpm, 20°C, at least 72 hours). We collected 28 density fractions from the resulting gradient with a fraction recovery system and pooled the low density fractions containing unlabeled DNA and high density fractions containing 15N labeled DNA in each sample. We performed target metagenomics of the 16S rRNA gene using Illumina Hi-Seq. We then analyzed sequences using Mothur (Schloss et al. 2009) and identified operational taxonomic units (the bacterial equivalent of species) using Megablast at a minimum coverage of 99% and minimum pairwise identity of 97%/ We aligned phylogenetic identities aligned against the SILVA database (Glockner et al. 2017).

To examine ambient nutrient concentrations at our study site, we collected grab samples of river water for total nitrogen (TN) and total phosphorus (TP) analyses and collected samples of river water filtered through pre-combusted Whatman GF/F filters for total dissolved nitrogen, total dissolved phosphorus, nitrite + nitrate (hereafter NO3−), ammonium (NH4+), and soluble reactive phosphorus (SRP) analyses. We estimated the concentration of dissolved organic nitrogen (DON) by subtracting the concentration of NH4+ and NO3− from the concentration of total dissolved nitrogen. The CSV file “N15_ambient_nutrients” reports water column concentrations of total nitrogen (TN), total phosphorus (TP), total dissolved nitrogen, total dissolved phosphorus, ammonium (NH4-N), nitrate (NO3-N + NO2-N), soluble reactive phosphorus (SRP-P) and dissolved organic nitrogen (DON) (nutrient concentrations are mean of 3 replicates).

We have included three CSV files: \"N15_SIP_design\" describes the treatment and fractions associated with each sample, \"N15_OTU\" contains the observed OTU counts by samples, and \"N15_tax\" contains the taxonomic classification OTUs. We have also included a zipped folder (\"N15_SIP_fastq\") containing the raw bacterial fastq files. The Word document “N15_SIP_analytical_methods” describes the analytical methods used to measure nutrient concentrations.

References:
Glöckner, F. O., P. Yilmaz, C. Quast, J. Gerken, A. Beccati, A. Ciuprina, G. Bruns, P. Yarza, J. Peplies, R. Westram, and W. Ludwig. 2017. 25 years of serving the community with ribosomal RNA gene reference databases and tools. Journal of Biotechnology.

Schloss, P. D., S. L. Westcott, T. Ryabin, J. R. Hall, M. Hartmann, E. B. Hollister, R. A. Lesniewski, B. B. Oakley, D. H. Parks, C. J. Robinson, J. W. Sahl, B. Stres, G. G. Thallinger, D. J. Van Horn, and C. F. Weber. 2009. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology 75:7537–7541.