Coastal water with coastal microbial concentrate (COAST): Microbial cell and micro-aggregate counts with parallel beta-galactosidase activity measurements

A mesocosm experiment was carried out in and October of 2015 (Mesocosm#2) using coastal water collected near Galveston Bay, Texas. Four mesocosm treatments were prepared in triplicate for this study: (1) Control (seawater only); (2) WAF (oil + seawater); (3) Chemically-enhanced WAF (CE-WAF; oil + Corexit + seawater); (4) Diluted CE-WAF (1:10 dilution). Initial mesocosm volumes were 79L. Prior to experiment initiation, phytoplankton were collected from coastal Galveston, TX waters and added to each tank to stimulate interactions between phytoplankton, oil, and Corexit. Starting at time-point zero and every 12 hours thereafter, water samples were collected from each mesocosm in clean, opaque, Nalgene bottles. For cell and aggregate counts, 10mL of water was collected from each mesocosm and fixed with 2% formalin. Fixed samples were stained with DAPI (45uM final concentration), filtered onto 25mm, 0.2um black polycarbonate filters, and counted directly via fluorescent microscopy. For beta-galactosidase measurements, parallel samples of 10um-filtered and unfiltered water were amended with 4-methylumbelliferyl beta-D-galactopyranoside. Florescence was quantified using a Tecan Spark 10M multimode microplate reader.

本研究于2015年10月开展中型实验生态系（mesocosm）实验，实验编号为中型实验生态系#2（mesocosm#2），所用海水采自得克萨斯州加尔维斯顿湾附近沿岸海域。本研究设置4组中型实验生态系处理组，每组均设置3个生物学重复：(1) 对照组（仅天然海水）；(2) 水包油分散液组（WAF，oil + seawater，即原油+海水）；(3) 化学强化型水包油分散液组（CE-WAF，Chemically-enhanced WAF，即原油+Corexit+海水）；(4) 稀释型化学强化水包油分散液组（按1:10比例稀释的CE-WAF）。每组中型实验生态系的初始体系体积为79L。实验启动前，研究人员从得克萨斯州加尔维斯顿沿岸海域采集浮游植物（phytoplankton），接种至每个试验槽中，以促进浮游植物、原油与Corexit之间的相互作用。自实验零时起，此后每间隔12小时，使用洁净的不透明Nalgene瓶从每个中型实验生态系中采集水样。针对细胞与聚集体计数检测：从每个中型实验生态系中采集10mL水样，采用2%福尔马林固定；固定后的样品以终浓度45μM的DAPI染色，随后过滤至直径25mm、孔径0.2μm的黑色聚碳酸酯滤膜上，通过荧光显微镜直接计数。针对β-半乳糖苷酶（beta-galactosidase）活性检测：取平行水样分别经10μm滤膜过滤与未过滤处理，向其中加入4-甲基伞形酮-β-D-半乳糖吡喃糖苷（4-methylumbelliferyl beta-D-galactopyranoside）作为底物；采用Tecan Spark 10M多功能酶标仪定量检测荧光信号。