Stream biofilm 13C-hemicellulose DNA stable isotope probing experiment
|Authors:||Elizabeth Ogata Sandra Udy Michelle Baker Zachary Aanderud|
|Storage:||The size of this resource is 19.1 KB|
|Created:||Aug 06, 2016 at 4:43 p.m.|
|Last updated:|| Jul 05, 2018 at 7:11 p.m.
|Citation:||See how to cite this resource|
This resource contains the results of a 13C-hemicellulose DNA stable isotope probing (DNA-SIP) experiment that tested how nutrients and light exposure influence hemicellulose decomposition and hemicellulose-degrading bacterial populations. We conducted our experiment with stream biofilms grown on nutrient diffusing substrates (NDS) in the Middle Provo River (Utah), at a site below Jordanelle Reservoir (June 1, 2016 - June 20, 2016). To construct the nutrient diffusing substrates, we filled 1-oz plastic cups with unamended agar and then capped the agar with a fritted glass disc, which served as a platform for biofilm colonization. To assess potential nutrient limitation of the stream biofilms grown for our hemicellulose DNA-SIP experiment, we also deployed NDS containing agar amended with either no nutrients (control), nitrogen (N; 0.5 M NH4-N), phosphorus (P; 0.5 M PO4-P), or N and P (N+P) and measured biomass (chlorophyll a, ash-free dry mass) and calculated Autotrophic Index values. The CSV file “hemicellulose DNA-SIP nutrient limitation” contains summary statistics (mean, standard deviation, count) of chlorophyll, ash-free dry mass, and Autotrophic Index values on each nutrient treatment. The Word document “hemicellulose DNA-SIP analytical methods” describes the analytical methods used to measure chlorophyll and ash-free dry mass. To characterize conditions at the site, we collected water column samples for total and dissolved nutrient analyses and calculated degree days from time series water temperature data collected as part of the NSF-funded iUTAH project (Award number 1208732). The CSV file “hemicellulose DNA-SIP site characteristics” reports degree days and water column concentrations of total nitrogen (TN), total phosphorus (TP), ammonium (NH4-N), nitrate (NO3-N + NO2-N), and soluble reactive phosphorus (SRP-P) (nutrient concentrations are mean of 3 replicates). The Word document “hemicellulose DNA-SIP analytical methods” describes the analytical methods used to measure nutrient concentrations.
Biofilms grown on unamended NDS were used to perform the hemicellulose DNA-SIP experiment. Biofilm-colonized discs were placed in clear glass jars containing filter-sterilized river water amended with 13C-hemicellulose (approximately 540 µmol C L-1). We tested eight combinations of nutrient (control, N, P, N+P) and light exposure (light, dark) treatments. Nutrient treatments were applied by adding N (2.5 mg NH4-N L-1) and/or P (0.36 mg PO4-P L-1) to the appropriate jars. To apply the light exposure treatments, we wrapped dark treatment jars in aluminum foil and left the light treatment jars unwrapped. We incubated jars for 10 days on a shaker table (50 rpm) in a growth chamber held at a temperature of 12°C set to a 15-hour photoperiod, which was illuminated using cool white fluorescent bulbs (4200 K color temperature, Sylvania Supersaver, Osram Sylvania Products Inc.). The average photosynthetically active radiation, measured with a LI-COR LI-190 quantum sensor, was 27.3 µE m-2 sec-1. We collected biofilms and water samples from each treatment on day 0 and day 10. Biofilms were frozen for DNA-SIP analyses. Water samples were collected for dissolved organic carbon (DOC) concentration and dissolved organic matter (DOM) fluorescence characterization analyses. We used DOC and DOM data to calculate Fluorescence Index (FI), Freshness Index (BIX), Humification Index (HIX) and specific ultraviolet absorbance (SUVA254; calculated by dividing the absorbance at 254 nm by the DOC concentration). The CSV file “hemicellulose DNA-SIP DOC and DOM” contains summary statistics of DOC and DOM data in each hemicellulose incubation treatment. The Word document “hemicellulose DNA-SIP analytical methods” describes the analytical methods used to measure DOC and DOM.
DNA-SIP analyses were conducted by first extracting genomic DNA from each biofilm-colonized disc. 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 13C labeled DNA in each sample. We then composited the low and high density fractions from all samples within each hemicellulose incubation treatment. We performed target metagenomics of the 16S rRNA gene. The Word document “hemicellulose DNA-SIP analytical methods” describes the DNA-SIP community composition analysis methods. The CSV file “hemicellulose DNA-SIP shared OTU” lists the number of sequences for each OTU in the low and high density fractions of each hemicellulose incubation treatment. The CSV file “hemicellulose DNA-SIP taxonomy” contains OTU classification information.
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Resource Level Coverage
|hemicellulose DNA-SIP nutrient limitation variables||chlorophyll a concentration (mg/m2), ash-free dry mass (g/m2), Autotrophic Index (calculated by dividing ash-free dry mass (mg/m2) by chlorophyll a (mg/m2))|
|hemicellulose DNA-SIP site characteristics variables||total nitrogen concentration (mg N/L), total phosphorus concentration (mg P/L), ammonium concentration (mg NH4-N/L), nitrate concentration (mg NO3-N + NO2-N/L), soluble reactive phosphorus concentration (mg SRP-P/L), degree days|
|hemicellulose DNA-SIP DOC and DOM variables||dissolved organic carbon concentration (DOC; mg/L), Freshness Index (BIX), Humification Index (HIX), Fluorescence Index (FI), Specific ultraviolet absorbance (SUVA254; calculated by dividing the absorbance at 254 nm by the DOC concentration)|
This resource was created using funding from the following sources:
|Agency Name||Award Title||Award Number|
|National Science Foundation||iUTAH-innovative Urban Transitions and Aridregion Hydro-sustainability||1208732|
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