粘土/蓝藻 Floccules 沉降速率的测定
Summary
在受控实验室环境中, 在自然环境中观察到的海洋领域内粘土和细菌细胞的相互作用和沉淀是最好的。在这里, 我们描述了一个详细的协议, 其中概述了一个新的方法来测量粘土和蓝藻 floccules 沉降率。
Abstract
在很大程度上讨论了沉积细粒、富含有机沉积物的机制。具体而言, 研究了粘土颗粒与反应性、浮游蓝藻细胞相互作用对沉积记录的影响。这种相互作用是页岩沉积模型的潜在主要贡献者。在实验室设置中, 这些材料的絮凝和沉淀速率可以在受控环境中进行检查和测量。在这里, 我们详细介绍了一个测量蓝藻/粘土混合物沉降速率的协议。通过对两个样品实验的描述, 证明了这种方法: 第一次使用高岭土 (一种脱水的高岭土) 和细长7002 (一种海洋球形蓝藻), 第二次使用高岭土和Synechocystis 6803 (淡水球形蓝藻)。蓝藻文化是混合在一个专门设计的坦克设备的不同数量的粘土, 优化, 以允许连续, 实时视频和摄影记录。抽样程序是详细的, 以及收集后的协议, 以精确测量叶绿素a , 其中蓝藻细胞的浓度在悬浮可以确定。通过实验复制, 构造出显示沉降速率的剖面。
Introduction
利用目前的环境条件和过程来推断过去的沉积机制, 长期以来一直是沉积学的基础。虽然现代沉积类似物, 如黑海, 已被用来了解沉积的有机丰富, 细粒度矿床, 实验室的实验有可能进一步揭示页岩矿床的起源。黑色页岩成因的一个研究领域是原始地层的沉积速率和机理。传统上, 据推测, 在沉积速率、初级生产力和有机物呼吸率等环境中形成的黑色页岩, 在1、2 的沉淀物中促进有机物的保存. ,3。然而, 蓝藻和粘土絮凝的作用在很大程度上仍然不值得考虑。这种絮凝机理可以迅速沉积出富含有机物的细粒沉积物, 并且不需要低氧。考虑到这一前提, 该协议有两个目标: 1) 测量蓝藻/粘土 floccules 的沉降速率, 2) 实时可视化沉积过程。这种方法, 除了地球化学分析, 已被用来证明蓝藻/粘土絮凝实际上可能是一个重要的机制, 页岩形成1。该方法最初拟用于模拟页岩沉积时, 适用于生物和环境修复等其他学科, 其中粘土输入对细菌代谢和种群的影响需要测量。
已进行了大量的研究, 以观察蓝藻和粘土的絮凝, 以减轻有害藻类绽放2,3,4,5,6,7,8,9,10,11,12. 然而, 在测量细胞浓度的同时, 这些研究并没有应用蓝藻/粘土絮凝来模拟岩石记录的沉积。因此, 这些研究缺乏视觉成分, 这在模拟过去的沉积过程时可能是至关重要的。此外, 大多数研究都利用细胞计数 (例如, Pan等.11), 这可能是费力的。我们的方法, 最近在测量蓝藻絮凝的进展, 确定蓝藻细胞浓度的变化, 通过测量叶绿素a (智利a) 在离散时间间隔。与视觉数据进行配对是一种新的方法, 可用于推断沉积条件。所生成的图像也可用于计算杜等 al工作后的沉降速率。13. 视觉和数字数据的结合加强了结果的可靠性。此外, 我们还概述了附加的协议, 允许沉积的死生物量和粘土也被观察到。在考虑过去的沉积环境时, 这一点很重要, 那里的生物量可能发生了共生。在絮凝过程中死生物量的差异 (例如, 絮凝速率的降低) 可能会产生沉积的影响。
Protocol
1. 准备蓝藻文化 利用固体培养基制备接种培养基 获取无菌蓝藻细胞从美国类型的文化收集或巴斯德文化收集。例如, 单细胞, 海洋细长7002 是从巴斯德文化收集, 它将被称为细长以后。 在含有固体介质的板上保持细长细胞 (A + 液体介质14和1.5% 琼脂15)。这些将被提到以后作为接种文化。 在30–50µmol…
Representative Results
当暴露在粘土, 蓝藻细胞被带出悬浮22。在这里给出的代表性结果表明了这一点。为了确定粘土对蓝藻种群的影响, 并观察沉降速率, 进行了两次试验,细长和Synechocystis暴露在50克/升高岭土 (表 5–6, 图 2–3)。蓝藻文化按步骤1所述增长。随后, 在设置了坦克 (步骤 2) 后, 稀释后的细长文化倒?…
Discussion
蓝藻细胞-粘土相互作用的絮凝反应在生态学和工程2、3、4、5、6、7 等领域引起了很大的兴趣. ,8,9,10,11,12, 然而, 这?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
提交人感谢加拿大自然科学和工程研究理事会提供的资金 (05448、165831和 213411)。
Materials
| cyanobacteria (in this study: Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803) | Pasteur Culture Collection | PCC 7002 or PCC 6803 | used to inoculate the plates |
| agar | Thermo Scientific | CM0003 | used to fill two petri dishes |
| Petri plates (standard bacteriology, 100 x 15 mm) | Sarstedt | 82.1473.001 | 2 required |
| 1 L heat resistant Erlenmeyer flask | Pyrex | 4980-125 | 1 required |
| 250 mL heat resistant Erlenmeyer flask | Pyrex | 4980-250 | 1 required |
| Nichrome inoculating loop with handle | Fisher Scientific | 14-956-103 | 1 required |
| tinfoil | Reynolds Wrap Aluminum Foil | 89079-067 | 50 cm required; used to cover foam stopper and neck of erlenmeyer flasks |
| growth media (e.g. A+) | 1050 mL required; produced using composition described in tables 1-4 | ||
| Bunsen Burner | Fisher Scientific | S95941 | 1 required |
| plastic tubing | Fisher Scientific | S504591 | 1 m required; used to create the bubbling apparatus |
| sponge stopper | Jaece Industries Inc | 14-127-40E | 1 required; hole made in center for pipette; used for constructin the bubbling apparatus |
| acrylic sheet | Home Depot | Optix clear acrylic sheet model # MC-102S | 1 required; used to construct acrylic tank (20 x 30 x 5.1 cm) |
| clear waterproof silicone adhesive | Home Depot | Loctite clear silicone model # 908570 | 1 required; used to construct acrylic tank (20 x 30 x 5.1 cm) |
| camera or video recorder | Panasonic | HC-V770 HD camcorder | 1 required |
| tripod | Magnus | VT-300 | 1 required |
| black cloth | primomart | EAN 0726670162199; Part number 680254blacknappedfr | 1 required; duvetyne light block-out cloth; approximatly 152 x 213 cm to cover tank experiment |
| heat resistant serological pipet | corning incorporated C708510 | 13-671-101G | 1 required; used to create the bubbling apparatus |
| sample vials | Dynalon | S30467 | at least 12 (will vary with time interval chosen) |
| heat resistant glass pipette | Fisher Scientific | Corning Incorporated C708510, 13-671-101G | 1 required; used to create the bubbling apparatus; Polystyrene serological pipet would also work, but should be connected to the tubing and stopper after the rest of the apparatus is autoclaved. |
| microcentrifuge | Eppendorf | 22 62 120-3 | 1 required;Comparable products may be used if capable of centrifuging 1.5 -2 mL microfuge tubes at 13,000 x g |
| vortex machine (Vortex-Genie 2) | Scientific Industries, Inc | SI-0236 | 1 required |
| 100% methanol | Fisher Scientific | A412-500 SDS | at least 12 mL (1mL per sample) required; Caution: Flammable, toxic. Wear gloves and safety glasses. Do not use or store near ignition source. Alternate sources may be used. |
| cuvettes (1.6 mL, polystyrene) | Sarstedt | 67.742 | at least 12 required |
| spectrophotometer | Fisher Scientific | 222-271600 | 1 required; Pharmacia Biotech Novaspec ll could also be used. |
| light bulbs | Home Depot | model # 451807; internet #205477895; store SKU #1001061538 | 6-8 bulbs required to provide light for the tank experiments |
| pipette (Pipetman Classic P1000 | Gilson | F123602 | used to collect samples |
| 37 % Hydrochloric acid | Sigma-Aldrich | 258148 | Caution: Corrosive and toxic. Wear lab coat, safety glasses and acid-resistant gloves while using. Prepared to 4 N before use by dilution into deionized water in a chemical fumehood. |
| Foam stopper (small) | Canlab | T 1385 | |
| Foam stopper (large) | Canlab | T 1387 | Requires some intact stoppers and some with a single hole through the centre |
| 30 °C incubator/growth room with continuous illumination | 1 required | ||
| 70 % Ethanol | Fisher Scientific | BP8201500 | 30 mL required;Caution: Toxic and flammable. Wear lab coat and safety glasses |
| hydrophobic air filter (Midisart 2000, 0.2 µm) | Sartorius | 17805 | 1 required |
| clay (e.g. kaolin) | Fisher Scientific | MFCD00062311 | at least 50 g required |
| microfuge tubes (2 mL, polypropylene) | Sarstedt | 72.695.500 | Comparable products may be used. At least 12 (will vary with time interval chosen) |
| 1000 µL pipet tips | Sarstedt | 70.762 | 1 required |
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Cite This Article
Playter, T., Konhauser, K., Owttrim, G. W., Whitford, D. S., Warchola, T., Hodgson, C., Mloszewska, A. M., Sutherland, B., Zonneveld, J., Pemberton, S. G., Gingras, M. K. Determination of the Settling Rate of Clay/Cyanobacterial Floccules. J. Vis. Exp. (136), e57176, doi:10.3791/57176 (2018).