AIMS_GP_approach3_CHLA

This study quantifies chlorophyll-a (as a proxy for biomass of microbial photoautotrophs) on rock surfaces (µg/cm2) and in surface waters (µg/L) collected from non-perennial stream sites located within the South Fork of King’s Creek at Konza Prairie Biological Station. At the USGS gage located on the mainstem (06879560; est. 1979), Kings Creek is a 5th order intermittent stream draining 1059-ha of tallgrass prairie in the Kansas Flint Hills.
Sample collection followed a synoptic survey design to support the sampling goals of the Aquatic Intermittency effects on Microbiomes in Streams (AIMS) Project. During June 2021, a field team co-collected datasets characterizing stream microbiota, biogeochemistry, and hydrology across 50 locations within a sub-drainage of the South Fork of Kings’ Creek. The 50 sites were selected to balance multiple competing priorities: (i) strategically targeting existing monitoring infrastructure with long-term data (n=14); (ii) including sites near several known springs and tributary junctions (n=9); and (iii) including a range of drainage area and topographic wetness index (TWI) values (n=27), both of which have been correlated with flow permanence elsewhere. For a detailed description of the site selection process, please see (Swenson et al., 2024).
Samples were collected during the period of June 4th to June 8th, 2021, following the AIMS Microbial Field Sampling protocol (Zeglin and Busch, 2024). Briefly, representative samples of (i) surface water and (ii) epilithon (rock surface biofilms) were collected from each site (where present) using aseptic technique. All equipment was either pre-sterilized (e.g., Nalgene bottles, syringes, filter holders and plastic containers) or was cleaned with 95% ethanol between sites and subsequently flame sterilized (forceps for handling filters) or rinsed in stream water (wire brushes for scraping rocks). Foil packets containing GFF filters (Whatman 1825025) were pre-ashed and pre-labeled for each sample. At each site, the stream was divided into three areas (compartments) of equal width across the wetted width of the channel, or inferred channel width at dry sites (n=5). For water, a 360 mL composite sample was collected (120 mL from each of the three compartments), and a known volume of the composite sample was filtered (up to 60 mL) by pushing it through a GFF filter via a syringe and filter holder. The volume filtered was recorded and the filter was saved in its pre-labeled foil packet for chlorophyll-a analysis. For epilithon, three rocks (one from each compartment) were randomly collected, and their top surfaces were scraped using wire brushes, with area scrapped quantified by using a 25-cm2 template to limit scrub area on each rock, or for rocks that did not fit the template, by measuring and estimating rocks surface areas manually. Scraped biofilms were rinsed using a maximum of 50 mL of deionized water, and the rinsed materials (slurry) were collected and mixed in pre-sterilized plastic containers. At each site, up to 10-mL of epilithic biofilm slurry was filtered onto a GFF filter, the volume filtered was recorded and the filter was saved in its pre-labeled foil packet for chlorophyll-a analysis. In the field, all filters were temporarily stored in plastic bags on ice or under ice packs, and at the end of each field day were stored in a freezer (-20ºC).
Frozen filters were later shipped on dry ice from the Zeglin lab at Kansas State University (Manhattan, KS) to the Kuehn lab at the University of Southern Mississippi (Hattiesburg, MS). In the lab, chlorophyll-a was extracted from GFF filters in 5-10 ml of 90% ethanol at 80ºC for 5 minutes, steeped overnight at 4ºC (in darkness), and immediately quantified the following day by fluorescence using a Shimadzu 10ADvp series high performance liquid chromatography (HPLC) equipped with a Shimadzu RF10Axl fluorescence detector (excitation 430nm, emission 670 nm) (Meyns et al. 1994). Chlorophyll-a content quantified and calculated in terms of rock surface area sampled (µg/cm2) or concentration in surface waters (µg/L).
References:
Meyns, S., Illi, R., & Ribi, B. (1994). Comparison of chlorophyll-a analysis by HPLC and spectrophotometry: where do the differences come from? Archiv für Hydrobiologie, 129-139.
Swenson, L. J., Zipper, S., Peterson, D. M., Jones, C. N., Burgin, A. J., Seybold, E., ... & Hatley, C. (2024). Changes in Water Age During Dry‐Down of a Non‐Perennial Stream. Water Resources Research, 60(1), e2023WR034623. https://doi.org/10.1029/2023WR034623
Zeglin, L., M. Busch (2024). AIMS SOP - Microbial Field Sampling, HydroShare, http://www.hydroshare.org/resource/4b071711215341118330c22f18b5d20d

本研究定量测定了康扎草原生物站（Konza Prairie Biological Station）国王溪（King’s Creek）南支流的间歇性河流点位采集的岩石表面（单位：µg/cm²）及地表水（单位：µg/L）中的叶绿素a（chlorophyll-a，作为微生物光合自养生物（microbial photoautotrophs）生物量的替代指标）。设于国王溪干流的美国地质调查局（USGS）水文测站（gage，编号06879560，1979年启用）监测显示，国王溪为五级河流（5th order）间歇性河流，汇水面积达1059公顷，流域覆盖堪萨斯州弗林特丘陵（Kansas Flint Hills）的高草草原（tallgrass prairie）。

样本采集采用同步调查设计（synoptic survey design），以支撑“溪流微生物组的水生间歇性影响（AIMS）项目（Aquatic Intermittency effects on Microbiomes in Streams (AIMS) Project）”的采样目标。2021年6月，野外团队在国王溪南支流的子汇水区（sub-drainage）内的50个点位同步采集了溪流微生物组、生物地球化学及水文相关数据集。这50个点位的选取兼顾了多项优先级目标：（i）战略性覆盖具备长期监测数据的现有监测基础设施（共14个）；（ii）纳入多个已知泉点及支流交汇处附近的点位（共9个）；（iii）涵盖一系列汇水面积与地形湿度指数（TWI, topographic wetness index）值的点位（共27个）——这两类指标在其他研究中均已被证实与径流持久性（flow permanence）相关。点位选取的详细说明参见Swenson等人（2024）的研究。

样本采集于2021年6月4日至6月8日期间进行，遵循《AIMS微生物野外采样标准操作流程》（Zeglin与Busch，2024）。简要而言，研究人员采用无菌操作技术（aseptic technique），从每个点位（若存在对应样本）采集两类代表性样本：（i）地表水；（ii）石表生物膜（epilithon）。所有采样设备要么经过预灭菌处理（如耐洁（Nalgene）瓶、注射器、滤器支架（filter holder）及塑料容器），要么在点位间使用95%乙醇（95% ethanol）清洁后，经火焰灭菌（flame sterilized，处理抓取滤膜的镊子）或用溪流河水冲洗（处理刮取岩石的钢丝刷（wire brushes））。为每个样本预先灼烧灭菌并标记好含玻璃纤维滤膜（GFF filters, Whatman 1825025）的铝箔袋。

在每个点位，研究人员将河道按湿宽（wetted width，干涸点位则按推断的河道宽度）均分为三个等宽区域。对于地表水样本，采集360mL混合样（composite sample，三个区域各取120mL），随后取已知体积的混合样（最多60mL），通过注射器与滤器支架将其压滤至玻璃纤维滤膜上，记录过滤体积后，将滤膜置于预先标记好的铝箔袋中，用于叶绿素a分析。对于石表生物膜样本，随机采集三块岩石（每个区域各一块），使用钢丝刷刮取其顶面生物膜：刮取面积通过25cm²的模板限定，若岩石尺寸无法适配模板，则通过手动测量估算其表面积。刮取的生物膜用至多50mL去离子水（deionized water）冲洗收集，将冲洗得到的浆液（slurry）混合至预灭菌的塑料容器中。每个点位取至多10mL石表生物膜浆液，过滤至玻璃纤维滤膜上，记录过滤体积后，将滤膜置于预先标记好的铝箔袋中用于叶绿素a分析。野外采样期间，所有滤膜暂存于带冰或冰袋的塑料袋中，每日野外工作结束后置于-20℃冰柜保存。

后续，冻存的滤膜通过干冰（dry ice）从堪萨斯州立大学（Kansas State University）泽格林实验室（Zeglin lab，堪萨斯州曼哈顿市）运送至南密西西比大学（University of Southern Mississippi）库恩实验室（Kuehn lab，密西西比州哈蒂斯堡市）。实验室分析阶段，研究人员将玻璃纤维滤膜中的叶绿素a用5~10mL 90%乙醇于80℃萃取5分钟，随后于4℃黑暗条件下浸泡过夜，次日即刻使用配备岛津RF10Axl荧光检测器（fluorescence detector，激发波长430nm，发射波长670nm）的岛津10ADvp系列高效液相色谱（HPLC, high performance liquid chromatography）通过荧光法定量（Meyns等人，1994）。最终叶绿素a含量按采样岩石表面积（单位：µg/cm²）或地表水浓度（单位：µg/L）进行计算与报告。

参考文献：
1. Meyns, S., Illi, R., & Ribi, B. (1994). 高效液相色谱与分光光度法测定叶绿素a的对比：差异来源探析. 《Archiv für Hydrobiologie》, 129-139.
2. Swenson, L. J., Zipper, S., Peterson, D. M., Jones, C. N., Burgin, A. J., Seybold, E., et al. (2024). 非季节性河流干涸过程中的水龄变化. 《Water Resources Research》, 60(1), e2023WR034623. https://doi.org/10.1029/2023WR034623
3. Zeglin, L., M. Busch (2024). AIMS标准操作流程——微生物野外采样, HydroShare, http://www.hydroshare.org/resource/4b071711215341118330c22f18b5d20d