Stream biofilm 13C-hemicellulose DNA stable isotope probing experiment

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 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 folder "hemi_SIP_fastq" contains the bacterial fastq files and the CSV file "hemi_SIP_design" describes the treatment, day, and fractions associated with each sample. The CSV file “hemi_SIP_OTU” lists the number of sequences for each OTU in the low and high density fractions of each hemicellulose incubation treatment, with all samples rarefied to the smallest sample size (2,743 sequences). The CSV file “hemi_SIP_tax” contains OTU classification information.

本资源收录了一项13C-半纤维素DNA稳定同位素探针（DNA-SIP）实验的结果，该实验旨在探究营养物质与光照如何影响半纤维素分解及半纤维素降解细菌群落。实验于犹他州中部普罗沃河（Middle Provo River）的营养扩散基材（NDS）上培养的溪流生物膜中开展，实验地点位于乔丹内尔水库下方（2016年6月1日至6月20日）。为构建营养扩散基材，我们使用未经修改的琼脂填充1盎司塑料杯，并在琼脂表面覆盖一层多孔玻璃碟，作为生物膜定殖的平台。为评估我们半纤维素DNA-SIP实验中培养的溪流生物膜的营养限制潜力，我们还部署了含有琼脂的NDS，其中添加了无营养物质（对照组）、氮（N；0.5 M NH4-N）、磷（P；0.5 M PO4-P）或氮和磷（N+P）并测量了生物量（叶绿素a，无灰分干重）并计算了自养指数值。CSV文件“半纤维素DNA-SIP营养限制”包含了每种营养处理下叶绿素、无灰分干重和自养指数值的汇总统计（均值、标准差、计数）。Word文档“半纤维素DNA-SIP分析方法”描述了测量叶绿素和无灰分干重的分析方法。为表征现场条件，我们收集了水柱样品进行总营养和溶解营养分析，并从NSF资助的iUTAH项目（资助编号1208732）收集的水温时间序列数据中计算了积温。CSV文件“半纤维素DNA-SIP现场特征”报告了积温和总氮（TN）、总磷（TP）、铵（NH4-N）、硝酸盐（NO3-N + NO2-N）和可溶性活性磷（SRP-P）的水柱浓度（营养浓度均为3个重复的平均值）。Word文档“半纤维素DNA-SIP分析方法”描述了测量营养浓度的分析方法。

未经修改的NDS上生长的生物膜被用于进行半纤维素DNA-SIP实验。生物膜定殖的碟片被放置在含有经过滤灭菌的河流水并添加13C-半纤维素的透明玻璃罐中（约540 µmol C L-1）。我们测试了八种营养（对照组、N、P、N+P）和光照暴露（光照、黑暗）处理的组合。通过向适当的罐中添加N（2.5 mg NH4-N L-1）和/或P（0.36 mg PO4-P L-1）来应用营养处理。为了应用光照暴露处理，我们将黑暗处理罐用铝箔包裹，而将光照处理罐保持未包裹。在生长室内（温度设定为12°C，光照周期为15小时，使用色温为4200 K的冷白荧光灯泡照明）的摇床桌上（50 rpm）孵化罐体10天。使用LI-COR LI-190量子传感器测得的平均光合有效辐射为27.3 µE m-2 sec-1。在第0天和第10天收集了每个处理的生物膜和水样。生物膜被冷冻以进行DNA-SIP分析。水样被收集以进行溶解有机碳（DOC）浓度和溶解有机物（DOM）荧光表征分析。使用DOC和DOM数据计算荧光指数（FI）、新鲜度指数（BIX）、腐殖化指数（HIX）和特定紫外吸收（SUVA254；通过将254 nm处的吸光度除以DOC浓度计算）。CSV文件“半纤维素DNA-SIP DOC和DOM”包含了每个半纤维素孵化处理中DOC和DOM数据的汇总统计。Word文档“半纤维素DNA-SIP分析方法”描述了测量DOC和DOM的分析方法。

通过首先从每个生物膜定殖碟片中提取基因组DNA，进行DNA-SIP分析。然后，使用超速离心（58,000 rpm，20°C，至少72小时）根据密度将每个样品中的DNA分离。使用分级回收系统从得到的梯度中收集了28个密度分数，并将每个样品中包含未标记DNA的低密度分数和包含13C标记DNA的高密度分数合并。我们进行了针对16S rRNA基因的目标宏基因组学分析。Word文档“半纤维素DNA-SIP分析方法”描述了DNA-SIP群落组成分析方法。文件夹“hemi_SIP_fastq”包含细菌fastq文件，CSV文件“hemi_SIP_design”描述了与每个样本相关的处理、天数和分数。CSV文件“hemi_SIP_OTU”列出了每个OTU在低密度和高密度分数中的序列数量，所有样本都被稀疏化到最小的样本大小（2,743个序列）。CSV文件“hemi_SIP_tax”包含OTU分类信息。