ABSTRACT:

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.

ABSTRACT:

This resource contains the results of a nutrient uptake incubation experiment conducted at a mountain and urban site on the Logan River in northern Utah. The experiments were performed with biofilms grown for 14-15 days on nutrient diffusing substrates (NDS), 1-oz plastic cups filled with agar and capped with a fritted glass disc. We performed the nutrient uptake experiment by incubating biofilm-colonized discs in clear plastic jars filled with stream water spiked with nitrogen (N) and phosphorus (P) at a series of concentrations. The CSV file "nutrient uptake treatments" lists the treatments used in the nutrient uptake experiment at each site. Biofilms were incubated in situ for 2 hours at midday. Samples for dissolved nutrient analysis were collected from the nutrient treatment solutions used to fill the jars and from each jar at the end of the incubation. The dissolved oxygen concentration in each jar was measured at the start and end of the incubation. We calculated nutrient uptake rates as the rate of loss in water nutrients and net primary production as the change in dissolved oxygen concentration. We measured biofilm biomass as chlorophyll a. The CSV file “nutrient uptake results” contains summary statistics (mean, standard deviation, count) of nutrient uptake rates, net primary production rates, and chlorophyll a concentrations in nutrient uptake treatments at each site. The Word document “nutrient uptake analytical methods” contains the analytical methods used to measure nutrient concentrations.

We also examined biofilm nutrient limitation at each site using NDS. We constructed nutrient limitation NDS by filling 1-oz plastic cups with agar amended with either no nutrients (control), 0.5 M NH4-N (N), 0.5 M PO4-P (P), or 0.5 M NH4-N + 0.5 M PO4-P (N+P). NDS were then capped with a fritted glass disc and placed in the stream at each site during the same period that NDS for the nutrient uptake experiment were deployed. Biofilm biomass was measured as chlorophyll a and ash-free dry mass. The CSV file “nutrient limitation results” contains summary statistics of chlorophyll a concentration and ash-free dry mass in each nutrient treatment at each site. The Word document “nutrient uptake analytical methods” contains the analytical methods used to measure chlorophyll and ash-free dry mass.

ABSTRACT:

This resource contains the results of an experiment to test the effects of nutrients and pharmaceuticals on stream biofilms at montane and urban sites in the Logan River, Red Butte Creek, and Middle Provo River watersheds located in northern Utah. We constructed contaminant exposure substrates (CES) by filling 1-oz plastic cups with agar amended with individual and combined additions of nutrients (nitrogen, phosphorus, iron) and pharmaceuticals (caffeine, diphenhydramine). The CSV file “treatments_CES” lists the contaminant treatments included in the CES experiment. We capped the agar with an inorganic (fritted glass disc) or organic (cellulose sponge) substrate to select for biofilm assemblages dominated by autotrophic and heterotrophic microbes, respectively, and then deployed CES in the river at each site for 18 - 26 days.

At the end of the deployment period, we used biofilms grown on CES to perform a series of in-stream incubations. We measured respiration and productivity using a modified light-dark bottle incubation method and nitrogen fixation using an acetylene reduction assay. We measured biofilm biomass (chlorophyll a, ash-free dry mass) and calculated Autotrophic Index values (calculated as chlorophyll a concentration divided by ash-free dry mass). The CSV file “biomass_function_CES” contains summary statistics (mean, standard deviation, count) of respiration rates, gross primary production rates, nitrogen fixation rates, chlorophyll a concentrations, ash-free dry mass, and Autotrophic Index values of biofilms on each contaminant treatment and substrate type at our study sites. The Word document “methods_CES” describes the analytical methods used to measure chlorophyll and ash-free dry mass.

To examine microbial community composition of biofilms grown on CES, were used target metagenomics of the 16S rRNA and 18S rRNA genes to identify bacterial and eukaryotic taxa, respectively. The Word document "methods_CES" describes our sequence analysis methods. The folders "bacteria_fastq" and "eukaryotes_fastq" contain the bacterial and eukaryotic fastq files, respectively and the CSV files "bacteria_design_CES" and "eukaryotes_design_CES" describe the contaminant treatment and study location for each sample. The CSV files “bacteria_tax_CES” and “eukaryotes_tax_CES” contain taxonomic classification information for bacterial and eukaryotic OTUs, respectively. The CSV files “bacteria_otu_CES” and “eukaryotes_otu_CES” list the number of sequences of each bacterial and eukaryotic OTU, respectively, in contaminant treatments, with both datasets rarefied to the smallest sample size (bacteria = 27,704 sequences; eukaryotes = 1,523 sequences). The CSV file "bacteria_abun_photo_core_CES" lists bacterial OTUs that were abundant (≥0.1% relative abundance) and rare (<0.1 relative abundance) and potential photoautotrophs and potential heterotrophs. The file also lists bacterial OTUs that were identified as core taxa in each contaminant treatment by land-use combination, where core taxa were defined as OTUs present in at least 75% of samples in a specific grouping.

We characterized light availability, water temperature, and nutrient concentrations at each study site. The Word document "methods_CES" describes our methods for measuring these site characteristics and the analytical methods used to measure nutrient concentrations. The CSV file “site_characteristics_CES” contains percent canopy openness, transmitted incoming PAR, transmitted solar shortwave radiation, degree days, total nitrogen, total phosphorus, ammonium, nitrate, soluble reactive phosphorus, total dissolved iron, and total ferrous iron concentrations at each site.

We examined water column pharmaceutical concentrations at one site on each river using Polar Organic Contaminant Integrative Samplers (POCIS). POCIS were deployed for 20-26 days during summer and fall 2015. The masses of 19 pharmaceuticals which had sorbed onto the POCIS were measured using high performance liquid chromatography combined with tandem mass spectrometry. The CSV file “POCIS_CES” reports concentrations of pharmaceuticals that accumulated in each POCIS (expressed as ng/POCIS) and time-weighted average concentrations of pharmaceuticals (expressed as ng/L), calculated using the resulting pharmaceutical masses and uptake rates reported in the literature. Average daily discharge was calculated using time series discharge data collected by the iUTAH project.