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)。

1. 准备蓝藻文化
<ol><li>
利用固体培养基制备接种培养基
	<ol>
<li>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。</li>
		<li>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。</li>
		<li>在30–50µmol 光子 m-2 s-1,15,18的连续光线下孵化出32至2°c 的板块。</li>
		<li>重复步骤 1.1–1.3, 并将 BG-1116中的 + 介质替换为淡水蓝藻物种的步骤 1.1.2, 如Synechocystis sp 680316、17, 以后称为Synechocystis.有关第1-4 和 BG-11 介质的组成, 请参阅表。</li>
	</ol>
</li>
	<li>
准备液体培养: 每个坦克实验需要一个400毫升蓝藻文化。
	<ol>
<li>收集一个250毫升和两个1升耐热锥形瓶。</li>
		<li>用4米盐酸 (hcl) 溶液 (每瓶30毫升的 hcl 溶液) 冲洗每个烧瓶, 接着是蒸馏水。 注意: 盐酸具有腐蚀性和毒性。在使用4米 HCl 溶液时, 请确保佩戴了实验室大衣、安全护目镜和耐酸手套。在实验室水槽中执行这一步骤, 并按照当地规定处理4米 HCl 溶液</li>
		<li>用50毫升液体介质 (A + 或 BG-11) 填充250毫升烧瓶。用400毫升液体介质 (a + 或 BG-11) 和其他 1 l 烧瓶填充一个1升烧瓶, 其中有600毫升的介质 (a + 或 BG-11)。</li>
		<li>用透气的泡沫塞子封住每个烧瓶, 用锡纸盖上泡沫塞子和烧瓶颈。</li>
		<li>标签和高压釜的烧瓶在121°c 和100帕25分钟. 允许烧瓶冷却到室温。</li>
		<li>用70% 酒精消毒表面, 制备层流罩。</li>
		<li>收集电线接种回路, 本生燃烧器, 冷却50毫升液体培养基瓶 (从步骤 1.2.5) 和接种文化 (从步骤 1.1)。把这些物品放在消毒的气流罩里。</li>
		<li>使用本生燃烧器的火焰, 简单地消毒电线接种回路, 让它降温。</li>
		<li>沿接种培养的表面拖动冷却循环以收集蓝藻细胞。</li>
		<li>从250毫升瓶中取下泡沫塞子和箔帽 (不接触泡沫塞子), 通过火焰短暂传递锥形瓶的边缘。</li>
		<li>倾斜的烧瓶, 使媒体靠近烧瓶的颈部, 并插入接种环涂上细胞的瓶子。一旦循环被淹没在介质中, 搅动接种循环以移除细胞。</li>
		<li>使用本生燃烧器火焰, 除去接种回路, 重新消毒烧瓶的唇部。</li>
		<li>更换泡沫塞子和箔帽到锥形瓶 (不接触泡沫塞子)。</li>
		<li>在本生燃烧器火焰中短暂消毒接种回路。</li>
		<li>准备一个生长室 (称为生长室), 温度保持在30°c15,18和光强度是30–50µmol 光子 m-2 s-1,15,18.湿度不受主动控制。</li>
		<li>允许接种的50毫升培养在 150 rpm 的生长室进行孵化, 保持温度为30摄氏度。在搅拌过程中保持瓶子的嘴, 以控制蒸发损耗, 防止污染。</li>
		<li>允许蓝藻文化生长96小时。一个成功的文化应该出现明亮的绿色和有一个光学密度 (OD) 在750毫微米0.4–0.6。</li>
		<li>一旦文化已经充分增长, 将50毫升的文化和 1 L 烧瓶包含400毫升液体介质 (从步骤 1.2.5) 在层流罩。确保流动罩在步骤1.2.6 后灭菌。</li>
		<li>在层流罩内对两个烧瓶重复步骤1.2.10。</li>
		<li>将50毫升的文化倒入1升瓶中的400毫升培养基中, 用本生燃烧器火焰重新杀菌1升烧瓶的唇。</li>
		<li>在泡沫塞子和箔帽的地方, 附上一个无菌鼓泡装置, 由泡沫塞子、吸管、塑料管、内衬过滤器和箔盖到烧瓶口。</li>
		<li>搅动 1 L 烧瓶在 150 rpm 在30°c 与涌泉设备附有空气泵, 通过解决方案15,18在300毫升/分钟循环湿空气混合物。</li>
		<li>允许培养基在生长室内生长30摄氏度, 120–168 h。一个成功的文化应该出现明亮的绿色和有 OD750 毫微米0.4–0.6。</li>
		<li>重复步骤 1.2, 产生控制文化, 这将不会与粘土混合。</li>
		<li>使用死生物量的实验需要额外的步骤。蒸压釜1升瓶养殖60分钟, 在121摄氏度, 并允许它冷却到室温。</li>
	</ol>
</li>
</ol>2. 实验设置
<ol><li>将一个长方形的丙烯酸罐 (长度为20厘米, 宽5.1 厘米, 高度为30厘米) 放在荧光灯库的前面 (九 T8 灯泡, 至少2600流明;图 1)19。</li>
	<li>在灯和油箱之间放一层半透明的白色塑料片, 以分散光线, 透过水箱照射。这个设置是由萨瑟兰等。19
</li>
	<li>将丙烯酸罐标在高度为10厘米处, 从底座垂直测量。所有测量都应在水柱的高度进行, 以确保所有取样的一致性。</li>
	<li>将相机放在三脚架上, 在坦克前面1米处记录每个实验。视频摄像机是推荐图像可以从视频文件中提取, 并通过使用数学建模软件, 视频文件可以用来模型沉降动力学19。</li>
	<li>将黑色布放在轻型银行、坦克和照相机上, 以屏蔽外部光源的实验 (图 1)。</li>
</ol>3. 絮凝实验协议
<ol><li>收集400毫升的文化 (步骤 1.2), 一个大的毕业圆筒, 和600毫升的额外生长介质在 1 L 烧瓶 (步骤 1.2.5)。</li>
	<li>稀释400毫升培养 (第4-6 毫克/毫升的初始细胞浓度) 与额外的生长培养基 (A + 或 BG-11) 到最终体积的1升使用的毕业气缸。将此解决方案添加到亚克力罐中。</li>
	<li>准备2毫升离心管标签和放置在一个方便的位置附近的丙烯酸罐。测试50克粘土。</li>
	<li>开始视频录制。在多次实验的情况下, 一个带有实验标识符的符号可以暂时在摄像机前举行。</li>
	<li>使用吸管, 采取1毫升的样品, 并将其放置在标签离心管。使用此示例通过测量 OD750 nm值来确定初始蓝藻单元格计数。把所有样品都用三份, 以确保准确的结果。</li>
	<li>用搅拌棒将粘土 (50 克) 快速倒入水箱中, 10–15 s。</li>
	<li>启动定时器, 并采取最初的1毫升样品为智利的决心使用 P1000 吸管 (样品时间0)。</li>
	<li>在适当的时间间隔 (例如, 1 分钟, 2 分钟, 3 分钟, 4 分钟, 5 分钟, 10 分钟, 15 分钟, 30 分钟, 60 分钟, 180 分钟, 240 分钟) 进行额外的取样。</li>
	<li>实验完成后, 保存视频文件, 关闭摄像机, 并处理所有的样本以确定 (步骤 4). </li>
	<li>蒸压釜剩余的粘土/蓝藻溶液。根据蓝藻种类、当地法规和实验室策略, 使用适当的方法处理解决方案。用肥皂和水清洗水箱, 用蒸馏水冲洗, 晾干。</li>
	<li>准备一个没有粘土添加的控制实验。在同一时间点取样品。这些数据可以与其他实验数据进行比较, 以确认由于粘土絮凝而导致沉降速率提高, 而不是自然沉降的结果。</li>
	<li>如果在实验中使用死生物量, 则使用相同的实验过程。然而, 解决方案的处置程序不需要热处理。</li>
</ol>4. 样品处理和评估
<ol><li>使用从 Owttrim16修改的过程, 确定每个细胞样品中的叶绿素浓度。收集后, 在室温下使用离心, 在 1.3万 x g上将每个样品中的细胞颗粒3分钟。</li>
	<li>使用 P1000 吸管去除多余的介质, 并添加1毫升100% 甲醇 (20 °c)。将样品全速旋转1分钟, 并用重悬细胞颗粒。 注意: 甲醇是有毒和易燃的。戴上手套和安全眼镜, 保持甲醇远离点火源。</li>
	<li>将样品在-20 摄氏度孵育24小时, 以利于提取叶绿素a。</li>
	<li>孵化后, 颗粒的细胞碎片, 如4.1 所述, 转移绿色上清到一个试管使用吸管, 并把试管到分光光度计。允许样品在分光光度计中休息1分钟, 以确保样品中剩余的粘土沉淀并不会干扰测量。</li>
	<li>通过测量 665 nm 和 652 nm 的吸光度, 以及在 750 nm 上的 OD, 确定 spectrophotometrically 浓度。智利的浓度是由公式智利a = 16.29 x (665 od750)-8.54 x (652 od750) 20。浓度被用作细胞浓度的代表。此外, 一个转换因子 7.4 x 1010细胞/l = 10 克/升21可以用来计算在克的一些蓝藻物种的细胞浓度。</li>
	<li>绘制计算值与时间的数值。</li>
</ol>

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</ol>

作者没有什么可透露的。

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?...

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。

<table><tbody><tr><td>cyanobacteria (in this study: &lt;em&gt;Synechococcus&lt;/em&gt; sp. PCC 7002 and &lt;em&gt;Synechocystis&lt;/em&gt; sp. PCC 6803)</td><td>Pasteur Culture Collection</td><td>PCC 7002 or PCC 6803</td><td>used to inoculate the plates</td></tr><tr><td>agar</td><td>Thermo Scientific</td><td>CM0003</td><td>used to fill two petri dishes</td></tr><tr><td>Petri plates (standard bacteriology, 100 x 15 mm)</td><td>Sarstedt</td><td>82.1473.001</td><td>2 required</td></tr><tr><td>1 L heat resistant Erlenmeyer flask</td><td>Pyrex</td><td>4980-125</td><td>1 required</td></tr><tr><td>250 mL heat resistant Erlenmeyer flask</td><td>Pyrex</td><td>4980-250</td><td>1 required</td></tr><tr><td>Nichrome inoculating loop with handle</td><td>Fisher Scientific</td><td>14-956-103</td><td>1 required</td></tr><tr><td>tinfoil</td><td>Reynolds Wrap Aluminum Foil</td><td>89079-067</td><td>50 cm required; used to cover foam stopper and neck of erlenmeyer flasks</td></tr><tr><td>growth media (e.g. A+)</td><td></td><td></td><td>1050 mL required; produced using composition described in tables 1-4</td></tr><tr><td>Bunsen Burner</td><td>Fisher Scientific</td><td>S95941</td><td>1 required</td></tr><tr><td>plastic tubing</td><td>Fisher Scientific</td><td>S504591</td><td>1 m required; used to create the bubbling apparatus</td></tr><tr><td>sponge stopper</td><td>Jaece Industries Inc</td><td>14-127-40E</td><td>1 required; hole made in center for pipette; used for constructin the bubbling apparatus</td></tr><tr><td>acrylic sheet&amp;nbsp;</td><td>Home Depot</td><td>Optix clear acrylic sheet model # MC-102S</td><td>1 required; used to construct acrylic tank (20 x 30 x 5.1 cm)</td></tr><tr><td>clear waterproof silicone adhesive</td><td>Home Depot</td><td>Loctite clear silicone model # 908570</td><td>1 required; used to construct acrylic tank (20 x 30 x 5.1 cm)</td></tr><tr><td>camera or video recorder</td><td>Panasonic</td><td>HC-V770 HD camcorder</td><td>1 required</td></tr><tr><td>tripod</td><td>Magnus</td><td>VT-300</td><td>1 required</td></tr><tr><td>black cloth</td><td>primomart</td><td>&amp;nbsp;EAN 0726670162199; Part number 680254blacknappedfr</td><td>1 required; duvetyne light block-out cloth; approximatly 152 x 213 cm to cover tank experiment</td></tr><tr><td>heat resistant serological pipet</td><td>corning incorporated C708510</td><td>13-671-101G</td><td>1 required; used to create the bubbling apparatus</td></tr><tr><td>sample vials&amp;nbsp;</td><td>Dynalon</td><td>S30467</td><td>at least 12 (will vary with time interval chosen)</td></tr><tr><td>heat resistant glass pipette</td><td>Fisher Scientific</td><td>Corning Incorporated C708510, 13-671-101G</td><td>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.</td></tr><tr><td>microcentrifuge</td><td>Eppendorf</td><td>22 62 120-3</td><td>&amp;nbsp;1 required;Comparable products may be used if capable of centrifuging 1.5 -2 mL microfuge tubes at 13,000 x g</td></tr><tr><td>vortex machine (Vortex-Genie 2)</td><td>Scientific Industries, Inc</td><td>SI-0236</td><td>1 required</td></tr><tr><td>100% methanol</td><td>Fisher Scientific</td><td>A412-500 SDS</td><td>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.</td></tr><tr><td>cuvettes (1.6&amp;nbsp; mL, polystyrene)</td><td>Sarstedt</td><td>67.742</td><td>at least 12 required</td></tr><tr><td>spectrophotometer</td><td>Fisher Scientific</td><td>222-271600</td><td>1 required; Pharmacia Biotech Novaspec ll could also be used.</td></tr><tr><td>light bulbs</td><td>Home Depot</td><td>model # 451807; internet #205477895; store SKU #1001061538</td><td>6-8 bulbs required to provide light for the tank experiments</td></tr><tr><td>pipette (Pipetman Classic P1000</td><td>Gilson</td><td>F123602</td><td>used to collect samples</td></tr><tr><td>37 % Hydrochloric acid</td><td>Sigma-Aldrich</td><td>258148</td><td>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.</td></tr><tr><td>Foam stopper (small)</td><td>Canlab</td><td>T 1385</td><td></td></tr><tr><td>Foam stopper (large)</td><td>Canlab</td><td>T 1387</td><td>Requires some intact stoppers and some with a single hole through the centre</td></tr><tr><td>30 &amp;deg;C incubator/growth room with continuous illumination</td><td></td><td></td><td>1 required</td></tr><tr><td>70 % Ethanol</td><td>Fisher Scientific</td><td>BP8201500</td><td>30 mL&amp;nbsp; required;Caution: Toxic and flammable. Wear lab coat and safety glasses</td></tr><tr><td>hydrophobic air filter (Midisart 2000, 0.2 &amp;micro;m)</td><td>Sartorius</td><td>17805</td><td>1 required</td></tr><tr><td>clay (e.g. kaolin)</td><td>Fisher Scientific</td><td>MFCD00062311</td><td>at least 50 g required</td></tr><tr><td>microfuge tubes (2 mL, polypropylene)</td><td>Sarstedt</td><td>72.695.500</td><td>Comparable products may be used. At least 12 (will vary with time interval chosen)</td></tr><tr><td>1000 &amp;micro;L pipet tips</td><td>Sarstedt</td><td>70.762</td><td>1 required</td></tr></tbody></table>

determination of the settling rate of clay/cyanobacterial floccules

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。

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?...

Watch this Scientific Journal Video about Determination of the Settling Rate of Clay/Cyanobacterial Floccules at JoVE.com

Determination of the Settling Rate of Clay/Cyanobacterial Floccules

粘土/蓝藻 Floccules 沉降速率的测定

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。

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction ...

determination-of-the-settling-rate-of-claycyanobacterial-floccules

Research

JoVE Journal

Environment

6.9K 次观看.  University of Alberta. 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。

视频: Determination of the Settling Rate of Clay/Cyanobacterial Floccules

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

一个铁（II）的实验室模拟的前寒武纪富海洋上升流系统探索光合细菌生长

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

科研

环境科学

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

测定蓝细菌中的糖原含量

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

生物化学

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

流式细胞仪对水生生物膜的表征

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Combined Size and Density Fractionation of Soils for Investigations of Organo-Mineral Interactions

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF relies on sequential density separation and sedimentation steps to isolate (1) the free light fraction (uncomplexed organic matter), (2) the occluded light fraction (uncomplexed organic matter trapped in soil aggregates) and (3) a variable number of heavy fractions (soil minerals and their associated organic matter) differing in composition. Provided that the parameters of the CSDF (dispersion energy, density cut-offs, sedimentation time) are properly selected, the method yields heavy fractions of relatively homogeneous mineral composition. Each of these fractions is expected to have a different complexing ability towards organic matter, rendering this a useful method to isolate and study the nature of organo-mineral interactions. Combining density and particle size separation brings an improved resolution compared to simple size or density fractionation methods, allowing the separation of heavy components according to both mineralogy and size (related to surface area) criteria. As is the case for all physical fractionation methods, it may be considered as less disruptive or aggressive than chemically-based extraction methods. However, CSDF is a time-consuming method and furthermore, the quantity of material obtained in some fractions can be limiting for subsequent analysis. Following CSDF, the fractions may be analyzed for mineralogical composition, soil organic carbon concentration and organic matter chemistry. The method provides quantitative information about organic carbon distribution within a soil sample and brings light to the sorptive capacity of the different, naturally-occurring mineral phases, thus providing mechanistic information about the preferential nature of organo-mineral interactions in soils (i.e., which minerals, what type of organic matter).

Combined size and density fractionation (CSDF) is a method to physically separate soil into fractions differing in texture (particle size) and mineralogy (density). The purpose is to isolate fractions with different reactivities towards soil organic matter (SOM), in order to better understand organo-mineral interactions and SOM dynamics.

土壤联合粒径与密度的分离研究

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF ...

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO3&#8722; for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO3&#8722; transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the 15N-labeled ammonium (15NH4+) accumulation rate in 15NO3&#8722; added incubation. The determination of the 14NH4+ and 15NH4+ concentrations described in this paper is comprised of the following steps. First, the NH4+ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO3&#8722; via persulfate oxidation. Third, the NO3&#8722; is converted to N2O via an N2O reductase deficient denitrifier. Finally, the converted N2O is analyzed using a previously developed quadrupole gas chromatography&#8211;mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

A series of methods to determine the potential DNRA rate based on 14NH4+/15NH4+ analyses is provided in detail. NH4+ is converted into N2O via several steps and analyzed using quadrupole gas chromatography&#8211;mass spectrometry.

基于14NH 4 + /4+ 15NH 4= 通过顺序转换为 N2O 的分析，测量异化硝酸盐降低至铵的潜在率

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has ...

A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances

Ecotoxicity data is a requirement for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH). The algal toxicity test is frequently used in regulatory risk assessment of chemicals. In order to achieve high reliability and reproducibility the development of standardized guidelines is vital. For algal toxicity testing, the guidelines require stable and uniform conditions of parameters such as pH, temperature, carbon dioxide levels and light intensity. Nanomaterials and other so-called difficult substances can interfere with light causing a large variation in results obtained hampering their regulatory acceptance. To address these challenges, we have developed LEVITATT (LED Vertical Illumination Table for Algal Toxicity Tests). The setup utilizes LED illumination from below allowing for a homogenous light distribution and temperature control while also minimizing intra-sample shading. The setup optimizes the sample volume for biomass quantification and does at the same time ensure a sufficient influx of CO2 to support exponential growth of the algae. Additionally, the material of the test containers can be tailored to minimize adsorption and volatilization. When testing colored substances or particle suspensions, the use of LED lights also allows for increasing the light intensity without additional heat generation. The compact design and minimal equipment requirements increase the possibilities for implementation of the LEVITATT in a wide range of laboratories. While compliant with standardized ISO and OECD guidelines for algal toxicity testing, LEVITATT also showed a lower inter-sample variability for two reference substances (3,5-Dicholorophenol and K2Cr2O7) and three nanomaterials (ZnO, CeO2, and BaSO4) compared to Erlenmeyer flasks and microtiter plates.

We demonstrate algal toxicity testing for difficult substances (e.g., colored substances or nanomaterials) using a setup illuminated vertically with an LED.

纳米材料和其他难物质藻类毒性试验的小型装置

Ecotoxicity data is a requirement for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH). The algal ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

生物工程

Early Detection of Cyanobacterial Blooms and Associated Cyanotoxins using Fast Detection Strategy

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of cyanobacteria and related cyanotoxins in water samples and in an organic matrix, such as bivalve extracts. FDS combines remote/proximal sensing techniques with analytical/bioinformatics analyses. Sampling spots are chosen through multi-disciplinary, multi-scale, and multi-parametric monitoring in a three-dimensional physical space, including remote sensing. Microscopic observation and taxonomic analysis of the samples are performed in the laboratory setting, which allows for the identification of cyanobacterial species. Samples are then extracted with organic solvents and processed with LC-MS/MS. Data obtained by MS/MS are analyzed using a bioinformatic approach using the online platform Global Natural Products Social (GNPS) to create a network of molecules. These networks are analyzed to detect and identify toxins, comparing data of the fragmentation spectra obtained by mass spectrometry with the GNPS library. This allows for the detection of known toxins and unknown analogues that appear related in the same molecular network.

A fast-multidisciplinarystrategy for early detection of cyanobacterial blooms and associated cyanotoxins is described here. It allows for the detection of cyanobacteria and related cyanotoxins in water samples and in organic matrices, such as bivalve samples, in 24 h.

使用快速检测策略早期检测蓝藻花和相关氰毒素

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of ...

化学

<meta charset="utf8"></head><body>与真菌伴侣共培养蔗糖分泌蓝细菌的方案

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being explored as a method for solar-driven bioproduction. One approach involves the use of CO2-fixing cyanobacteria that secrete organic carbon to support the metabolism of a co-cultivated heterotroph, which in turn transforms the carbon into higher-value goods or services. In this protocol, a technical description to assist the experimentalist in the establishment of a co-culture combining a sucrose-secreting cyanobacterial strain with a fungal partner(s), as represented by model yeast species, is provided. The protocol describes the key prerequisites for co-culture establishment: Defining the media composition, monitoring the growth characteristics of individual partners, and the analysis of mixed cultures with multiple species combined in the same growth vessel. Basic laboratory techniques for co-culture monitoring, including microscopy, cell counter, and single-cell flow cytometry, are summarized, and examples of nonproprietary software to use for data analysis of raw flow cytometry standard (FCS) files in line with FAIR (Findable, Accessible, Interoperable, Reusable) principles are provided. Finally, commentary on the bottlenecks and pitfalls frequently encountered when attempting to establish a co-culture with sugar-secreting cyanobacteria and a novel heterotrophic partner is included. This protocol provides a resource for researchers attempting to establish a new pair of co-cultured microbes that includes a cyanobacterium and a heterotrophic microbe.

<meta charset="utf8"></head><body>异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

This protocol provides a comprehensive guideline for the setup and quantitative monitoring of co-cultures, including photoautotrophic sugar-secreting cyanobacteria and heterotrophic yeasts.

异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being ...

粘土/蓝藻 Floccules 沉降速率的测定

摘要

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异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量