这项工作的一部分是由财政研究所的创新促进了科学和技术战略基础研究（IWT-SBO）项目阳光和BIOSOLAR细胞的支持。埃里克更大胆和BackKim阮都承认他们对珠跳动过程的优化贡献。

1。样品制备有样品制备列为步骤1.1和1.2两个备用协议。这两种方法都同样适合并得到相似的结果，但是，如果在有限的藻培养物体积量是可用的，方法1.1推荐。 注：对于任一协议，根据该协议，整个准备两个附加珠打浆机管但不添加藻它们被用作空白。以这种方式，在从提取自所用的材料组分造成的气相色谱峰可以被识别和量化。 1.1。样品制备协议选项1：建议在藻类培养的数量有限的可利用<ol><li>确定的藻类干重的浓度（g / L）的培养液，例如所描述的布鲁尔等人 1。 </li><li>转移量的培养液含有5-10毫克藻干重在玻璃离心管中。计算量的使用步骤1.1.1确定的生物量浓度转移的确切数额。 </li><li>离心5分钟，1,200×g的。 </li><li>弃去上清液的一部分，留下大约为0.25毫升管。 </li><li>通过重新悬浮于剩余的上清液中的藻类温和上下吹打颗粒与完整细胞沉淀转移到用200μl移液管的珠打浆机管。 </li><li>冲洗离心管和移液管玻璃与±0.15毫升milliQ水中和液体传送到同一珠打浆机管。 </li><li>离心机珠打浆机管在最大转速，以确保没有气泡留在管的底部一分钟。它可以存储封闭珠打浆机管在-80℃下</li><li>冻干含有样品的珠打浆机管。它可以存储在密闭珠打浆机管在-80℃下</li></ol> 1.2。样品制备协议选项2 <ol><li>离心机的藻类培养液中的不确定量和弃上清。这是没有必要的，以确定生物量浓度或测量所使用的音量。 </li><li>测量或用存在于培养基中的主盐的浓度计算的培养基的渗透压。 </li><li>通过重悬细胞沉淀在相同体积洗涤细胞沉淀，如在步骤1.2.1使用的甲酸铵溶液，这近似于培养基的渗透压摩尔浓度。一个equiosmolar甲酸铵溶液可以防止细胞的洗涤过程中的细胞裂解。 </li></ol>注意：洗涤细胞是必要的，以消除存在于培养基中的盐。因为在他们的培养基中的盐浓度高的，这是用于在海洋微藻脂肪酸分析尤其重要。如果盐仍然存在，这会导致细胞干重将被在T后确定的数额高估他的协议。甲酸铵用于洗涤细胞，因为该解决方案将冻干过程中完全蒸发，不留任何残余物。 <ol start="4"><li>离心机藻液弃上清。 </li><li>冻干细胞沉淀。 </li><li>称取5〜10毫克冻干粉微藻成珠打浆机管。记录准确重量。 </li></ol> 2。细胞破碎和脂质提取注意：本节介绍了一个广泛的细胞分裂过程。可能的话，一些细胞破碎步骤是多余的，那么广泛破碎细胞可能会产生相同的结果对于一些微藻物种或生物量从某些培养条件而得。然而，这将需要验证每个特定情况。因此，建议广泛破坏协议被推荐为适合微藻的所有材料的通用方法。 <ol><li>权衡tripentadecanoin的确切数额（TRiacylglycerol含3 C15：0脂肪酸），并将其添加到一个精确已知量的4:5（体积/体积）氯仿：甲醇;以这种方式tripentadecanoin的浓度精确已知。目的是为50毫克/升的浓度Tripentadecanoin用作脂肪酸的定量的内标。 </li><li>加入1 ml氯仿：甲醇4点05（体积/体积）含tripentadecanoin每个打浆管（在试剂和设备表中指定的）。检查是否有剩余的beadbeater管帽内无珠，这将导致泄漏的管子。使用正位移移液管准确加入溶剂。 </li><li>珠打珠打浆机管每次8倍，在2500转60秒，用120秒内每跳动之间。 </li><li>从珠打浆机管将溶液转移到一个干净的耐热15毫升玻璃离心管铁氟龙插入螺丝帽。确保所有的珠子从珠打浆机管转移为好。 </li><li>到了h时珠打浆机管中，加入1 ml氯仿：甲醇4点05（V / V）含tripentadecanoin成珠打浆机管，靠近帽和混合，并将该溶液从步骤2.4转移到同一玻璃管。洗管3次（总共加入4ml氯仿：甲醇4点05（V / V）含tripentadecanoin用于每个样品 - 1毫升在步骤2.2中加入3毫升3洗涤步骤）。 </li><li>涡流的玻璃管，持续5秒声处理及在超声浴中10分钟。 </li><li>添加将2.5毫升的MilliQ水，含有50mM 2 - 氨基-2 - 羟甲基丙烷-1,3 - 二醇（TRIS）和1M NaCl的，​​这是用一个HCl溶液中设置pH至7。这将导致氯仿和甲醇之间的相分离：水。 1M NaCl的用于增强脂质朝向氯仿相中的平衡。 </li><li>涡旋5秒，然后超声处理10分钟。 </li><li>离心5分钟，在1200×g的。 </li><li>使用玻璃巴斯德pipett整个氯仿相（底部相）转移到一个干净的玻璃管Ë。确保不转让任何相间或顶部阶段。 </li><li>加入1 ml氯仿旧管（：水溶液含有甲醇）重新提取样品。 </li><li>涡旋5秒，然后超声处理10分钟。 </li><li>离心5分钟，在1200×g的。 </li><li>使用玻璃巴斯德吸管和游泳池与步骤2.10第一氯仿馏分收集氯仿相（下相）。 </li><li>如果困难是经验丰富的收集整个氯仿部分在上一步中，重复步骤2.11-2.14。否则执行步骤2.16。 </li><li>从管中蒸发氯仿在氮气流中。之后可以存储这个步骤的样品在-20℃的氮气气氛下进行。 </li></ol> 3。酯交换反应脂肪酸甲酯<ol><li>加入3毫升甲醇中含有5％（体积/体积）硫酸，以含有干燥萃取脂质（管原本含有的氯仿级分）的管子和盖紧管。 </li><li>涡持续5秒。 </li><li>在一个块中的加热器或水浴中孵育样品3小时，在70℃。周期性地（每隔±30分钟）确保样品不沸腾（由于一个不正确地闭合盖子）和涡流管。在该反应中脂肪酸被甲基化到其脂肪酸甲基酯（脂肪酸甲酯）。 </li><li>凉爽的样品至室温，并添加3毫升的MilliQ水和3毫升正己烷。 </li><li>涡旋5秒和15分钟拌上试管旋转器。 </li><li>离心5分钟，在1200×g的。 </li><li>收集2毫升己烷（顶部）相，并用玻璃巴氏吸管把新鲜的玻璃管。 </li><li>加入2ml的MilliQ水收集到的己烷相，以洗。 </li><li>涡旋5秒，离心5分钟，1,200×g的。在此步骤之后，可以存储该样品在-20℃的氮气气氛下（不是必需的相分离）下。 </li></ol> 4。脂肪酸甲酯的U定量唱气相色谱法<ol><li>填GC小瓶用玻璃巴氏吸管的己烷相（上层相）。 </li><li>将瓶盖上的GC小瓶。确保瓶盖完全密封，且不能转动，以防止水分蒸发从药瓶。 </li><li>笔芯GC的清洗溶剂（正己烷），清空废液瓶，并把样品瓶的自动进样器。运行前在GC上，第一次运行2空白GC上的只含有正己烷的实际样品。 </li><li>上运行的GC-FID对样品用Nukol柱（30×0.53毫米×1.0微米）。用250℃的入口温度，并使用氦作为载气，81.7千帕的压力和0.1:1的分流比，总流速：20毫升/分钟。在270℃以40毫升/分钟的H 2的流量为400毫升/分钟的空气流率和He流量为9.3毫升/分钟速率FID检测器温度。设定注射体积为1微升/样品。前，后洗注射器用正己烷。初始烘箱温度设定为90℃0.5分钟，然后升至机智小时20°C /分钟，直到200℃的烘箱内温度达到。然后烘箱的温度保持在200℃下进行39分钟。总运行时间为45分钟。 </li></ol>

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作者什么都没有透露。

所描述的方法可以用于确定内容以及存在于微藻生物质的总脂肪酸的组成。从所有脂质类，包括存储（TAG）以及膜脂质（磷脂和糖脂）衍生的脂肪酸，被检测到。所有脂肪酸的链长的饱和的和度存在于微藻可以被检测和分辨。该方法是基于机械破碎细胞，溶剂为基础的脂质提取，脂肪酸对脂肪酸甲酯的酯交换，并用气相色谱与火焰离子化检测器（GC-FID）组合脂肪酸甲酯的定量。该方法需要少量的?...

脂肪酸是一体微藻生物质的主要成分，通常弥补之间的细胞干重1-3 5-50％。它们主要存在于甘油脂的形式。这些甘油脂又主要包括磷脂，糖脂和甘油三酯（TAG）的。尤其是存在于TAG的脂肪酸是具有商业价值的，因为它们可以被用作用于生产运输燃料，散装化学品，营养品（ω-3脂肪酸）和粮食商品3-6的资源。微藻可以在海的水基种植介质成长，可以具有高得多的单位面积生产率比陆生植物，并在该不适合于农业的位置可以培养在光生物反应器，甚至可能是近海。由于这些原因，微藻通常被认为是一个很有前途的替代陆地植物生产生物柴油等大宗产品3-6。可能没有农业的升和或淡水（当在封闭的光生物反应器或栽培时，海洋微藻的使用）是需要他们的生产。因此，从微藻生物燃料衍生的被认为是第三代生物燃料。 脂肪酸（干重％），脂质类组合物，以及所述脂肪酸长度和饱和度的总细胞含量的微藻物种之间是高度可变的。此外，这些特性随培养条件如营养物可用性，温度，pH值和光强1,2。例如，当暴露在氮饥饿，微藻可积累大量的TAG。在最佳生长条件TAG典型构成的干重计少于2％，但是当暴露在氮饥饿TAG含量可提高到微藻干重的1到40％。 微藻主要生产脂肪酸与16和18的链长个碳原子，但是有些种类可以使长度最多为24个碳原子的脂肪酸。既饱和以及高度不饱和脂肪酸是由微藻生产。后者包括脂肪酸与营养益处（ω-3脂肪酸），如C20：5（二十碳五烯酸，EPA）和C22：6（二十二碳六烯酸; DHA）的量没有蔬菜的替代品存在1,2,4,7。脂肪酸链的长度和饱和度的（分布）也决定了藻类衍生的生物燃料和食用油4,8的性能和质量。 开发的微藻衍生的脂肪酸的商业应用中，都需要的脂肪酸含量和组成可靠的定量分析方法。 由于还指出Ryckebosch 等。9，在微藻脂肪酸分析，从其它底物（ 如植物油，食品，动物组织等）是区别于导致1）微藻是单细胞的刚性细胞壁所包围，复杂的脂质提取; 2）微藻含有各种脂质类和脂质类分布是高度可变的7。这些不同的类脂质有各种各样的化学结构和如极性性质。另外，不是酰基脂质其它脂质类生产; 3）微藻含有多种脂肪酸，范围从在长度12-24个碳原子，同时含有饱和以及高度不饱和脂肪酸。因此，开发了以分析脂肪酸底物比微藻其它方法，可能不适合于分析脂肪酸的微藻。 如由Ryckebosch 等 9评论，常用的脂质提取程序之间的主要区别是在所使用的溶剂系统。由于种类繁多目前在微藻的脂质类的，在每个极性不同，提取的脂的数量将与使用10-12溶剂不同而不同。这导致脂质含量和组成在文学9,10介绍的不一致。根据所用的溶剂系统中，基于溶剂萃取而不破碎细胞的方法通过，例如，珠击或超声处理，可能无法提取，因为一些微藻物种9,13的刚性结构的所有脂质。在不完全的脂质提取的情况下，不同的类脂质的提取效率可以变化14。这也可对测得的脂肪酸组合物的效果，因为脂肪酸的组成是可变的脂质类7之间。 我们的方法是基于机械破碎细胞，溶剂为基础的脂质提取，脂肪酸对脂肪酸甲基酯（脂肪酸甲酯）酯交换反应，并且量化和识别使用气相色谱与火焰电离组合脂肪酸甲酯组成的序列化检测器（GC-FID）。在三酰基甘油（tripentadecanoin）形式的内标是在分析过程之前加入。提取和不完整的酯交换过程中可能发生的损失可以被用于纠正。该方法可用于确定内容以及存在于微藻生物质的脂肪酸的组合物。存在于不同的酰基脂质类，包括存储（TAG）以及膜脂（糖脂，磷脂），被检测，识别和准确和再现地量化由该方法仅使用少量样品（5毫克）的所有脂肪酸。此方法不提供有关不同类脂质的相对丰度信息。然而，该方法可以扩展到从对方1分离脂质类。不同的类脂质的浓度和脂肪酸组成可以被单独确定。 在文献中其他几个方法描述分析脂质微藻。一些方法集中在总亲脂性成分15，而其他方法集中在总脂肪酸9,16。这些替代品包括重量法测定总提取脂质，直接转酯化脂肪酸的利用色谱法结合定量和染色细胞与亲脂性荧光染料。 常用的替代品的使用色谱脂肪酸的量化是用重量法测定17,18脂质定量。的测定重量法优点是缺乏像气相色谱仪的先进和昂贵的设备要求;缓解设定的，因为标准化的分析设备（ 如索氏）的可用性起来的程序，以及一个重量法测定是耗时少比色谱法为基础的方法。使用基于色谱方法对超视距的主要优点呃手的是，在这种方法中仅脂肪酸计量。在一个重量法测定含脂类，如色素或类固醇的非脂肪酸，也包​​括在确定。这些含非脂肪酸的脂质可以弥补脂类总量的很大比例（&gt; 50％）。如果一个人只关心脂肪酸含量（例如，用于生物柴油的应用程序），它会在一个重量法测定是用来被高估。另外，在重量分析确定用来衡量所提取的脂类分析天平的准确度判断为需要将所使用的样本大小。这个量通常比当色谱使用所需的量多。最后，使用色谱法重量分析测定的另一个优点是，色谱提供了有关的脂肪酸组合物的信息。 另一种方法给我们提出的方法是直接酯交换16,19,20。在该方法中脂质提取和脂肪酸的酯交换反应，以脂肪酸甲酯相结合，在一个步骤。这种方法比单独的提取和酯交换步骤更快，但结合这些步骤限制了可用于萃取的溶剂。这可能提取效率产生负面影响。一个单独的脂质提取和酯基转移步骤的另一个优点是，它允许对这些步骤1之间的额外的脂质类分离。这是不可能的，当直接酯交换使用。 其他常用的方法来确定在微藻的脂质含量，包括染色与亲脂性荧光染料，如尼罗河生物质能红色或肾上腺素和测量荧光信号21,22。这些方法的优点在于，它们比其它方法少费力。这些方法的缺点是，所述荧光响应是，由于各种原因，可变SPECI之间ES，栽培条件下，脂质类和分析程序。作为一个例子，一些这些变化是由在染料的吸收差异由微藻引起的。因此使用另一种定量分析方法的校准是必要的，优选为所有不同的培养条件和生长阶段进行。最后，这个方法不提供有关的脂肪酸组成的信息，并且是不准确的和可重现的比基于层析方法。 该方法是基于由拉默斯等 23和Santos 等 24记载的方法，也被通过各种其他作者1,25-27应用。还有其他方法可用，基于相同的原理，并可能提供类似的结果9,28。

<table border="1">
 <tbody>
 <tr>
 <td>Reagent and equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>tripentadecanoin (C15:0 TAG)</td>
 <td>Sigma Aldrich</td>
 <td>T4257</td>
 <td>CAS Number 7370-46-9</td>
 </tr>
 <tr>
 <td>TAG or FAME standards of all fatty acids expected in sample</td>
 <td>Sigma Aldrich </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>TAG or FAME standards of all fatty acids expected in sample</td>
 <td>Lipidox</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>TAG or FAME standards of all fatty acids expected in sample</td>
 <td>Larodan</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Beadbeater</td>
 <td>Bertin Technologies</td>
 <td>Precellys 24</td>
 <td></td>
 </tr>
 <tr>
 <td>beadbeater tubes</td>
 <td>MP Biomedicals</td>
 <td>Lysing matrix E 
 116914500</td>
 <td></td>
 </tr>
 <tr>
 <td>GC-FID</td>
 <td>Hewlett-Packer</td>
 <td>HP6871</td>
 <td></td>
 </tr>
 <tr>
 <td>GC column</td>
 <td>Supelco</td>
 <td>Nukol 25357</td>
 <td></td>
 </tr>
 <tr>
 <td>Positive displacement pipette 100-1000&mu;l</td>
 <td>Mettler Toledo</td>
 <td>MR-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Positive displacement pipet tips C-1000</td>
 <td>Mettler Toledo</td>
 <td>C-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetvuller, Pi-Pump 2 ml</td>
 <td>VWR</td>
 <td>612-1925</td>
 <td></td>
 </tr>
 <tr>
 <td>glass tubes</td>
 <td>VWR</td>
 <td>SCERE5100160011G1</td>
 <td></td>
 </tr>
 <tr>
 <td>TUBE 16 X 100 MM BOROSILICATE 5.1 1 * 1.000</td>
 <td>VWR</td>
 <td>SCERE5100160011G1</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon coated screw-caps</td>
 <td>VWR</td>
 <td>SCERKSSR15415BY10</td>
 <td></td>
 </tr>
 <tr>
 <td>STUART SCIENTIFIC SB2 test tube rotator</td>
 <td>VWR</td>
 <td>445-2101</td>
 <td></td>
 </tr>
 <tr>
 <td>Heated Evaporator/Concentrator</td>
 <td>Cole-Parmer</td>
 <td>YO-28690-25</td>
 <td></td>
 </tr>
 </tbody>
</table>

analysis of fatty acid content and composition in microalgae

的方法，以确定存在于微藻总脂肪酸含量和组成进行说明。脂肪酸是微藻生物质的主要成分。这些脂肪酸可以存在于不同的酰基脂质类。尤其是存在于三酰基甘油（TAG）的脂肪酸是具有商业价值的，因为它们可以用于生产运输燃料，散装化学品，营养品（ω-3脂肪酸）和粮食商品。开发商业应用，都需要脂肪酸含量及组成可靠的定量分析方法。微藻是单细胞通过刚性的细胞壁所包围。 A脂肪酸分析方法应提供足够的细胞破碎解放所有酰基脂质和使用的提取方法应该能够提取所有酰基脂质类。 该方法这里给出存在于微藻所有脂肪酸可以准确和可重复的iDENtified并使用独立于它们的链长，不饱和度，或脂质类它们的一部分少量样品（5毫克）进行定量。 这种方法不提供有关不同脂质类的相对丰度的信息，但可以延伸到彼此分离的脂质类。 该方法是基于机械破碎细胞，溶剂为基础的脂质提取，脂肪酸对脂肪酸甲基酯（脂肪酸甲酯）酯交换反应，并定量分析和鉴定的气相色谱（GC-FID），脂肪酸甲酯的序列。一个TAG内标（tripentadecanoin）加入该分析方法之前，以纠正提取和不完整的酯交换反应过程中的损失。

通过这个过程得到的一个典型的色谱图示于图1。脂肪酸甲酯是由大小和饱和度分开使用的GC色谱柱和协议。较短链长的脂肪酸和多饱和脂肪酸（较少双键）具有更短的保留时间。所使用的GC柱和协议不打算分离脂肪酸异构体（相同的链长度和饱和度的程度，但双键位置不同），但是这可以通过使用一个不同的气相色谱柱和协议来实现。 脂肪酸浓度和组合物可以使?...

Watch this Scientific Journal Video about Analysis of Fatty Acid Content and Composition in Microalgae at JoVE.com

Analysis of Fatty Acid Content and Composition in Microalgae

一种用于脂肪酸含量和组成在微藻基于机械破碎细胞，溶剂为基础的脂质提取，酯交换，以及量化和鉴定用气相色谱法的脂肪酸的测定方法进行说明。一个tripentadecanoin内标是用来弥补提取和不完整的酯交换过程中可能发生的损失。

脂肪酸组分和含量微藻分析

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

analysis-of-fatty-acid-content-and-composition-in-microalgae

Research

JoVE Journal

Environment

59.4K 次观看.  Wageningen University and Research Center. 一种用于脂肪酸含量和组成在微藻基于机械破碎细胞，溶剂为基础的脂质提取，酯交换，以及量化和鉴定用气相色谱法的脂肪酸的测定方法进行说明。一个tripentadecanoin内标是用来弥补提取和不完整的酯交换过程中可能发生的损失。 

视频: Analysis of Fatty Acid Content and Composition in Microalgae

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

优化烟气设置通过电脑模拟来促进微藻生长的光生物反应器中

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

科研

环境科学

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

重金属和微藻的产其他无机污染物的量化

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

内毒素和DNA含量的空气采样滤波器分析

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

提取及土壤微生物磷脂脂肪酸分析

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Composition and Properties of Aquafaba: Water Recovered from Commercially Canned Chickpeas

Chickpea and other pulses are commonly sold as canned products packed in a thick solution or a brine. This solution has recently been shown to produce stable foams and emulsions, and can act as a thickener. Recently interest in this product has been enhanced through the internet where it is proposed that this solution, now called aquafaba by a growing community, can be used a replacement for egg and milk protein. As aquafaba is both new and being developed by an internet based community little is known of its composition or properties. Aquafaba was recovered from 10 commercial canned chickpea products and correlations among aquafaba composition, density, viscosity and foaming properties were investigated. Proton NMR was used to characterize aquafaba composition before and after ultrafiltration through membranes with different molecular weight cut offs (MWCOs of 3, 10, or 50 kDa). A protocol for electrophoresis, and peptide mass fingerprinting is also presented. Those methods provided valuable information regarding components responsible for aquafaba functional properties. This information will allow the development of practices to produce standard commercial aquafaba products and may help consumers select products of superior or consistent utility.

Aquafaba is a viscous juice from canned chickpea that, when stirred vigorously, produces a relatively stable white froth or foam. The primary research goal is to identify the components of aquafaba that contribute viscosifying/thickening properties using nuclear magnetic resonance (NMR), ultrafiltration, electrophoresis, and peptide mass fingerprinting.

Aquafaba 的成分和性质: 从商用罐装鹰嘴中回收的水

Chickpea and other pulses are commonly sold as canned products packed in a thick solution or a brine. This solution has recently been shown to produce ...

The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus area. Biomass capacitance plays an integral role in the avoidance of hydraulic impairment to transpiration. However, high temporal resolution measurements of dynamic changes in the hydraulic capacitance of large trees are rare. Here, we present procedures for the calibration and use of capacitance sensors, typically used to monitor soil water content, to measure the volumetric water content in trees in the field. Frequency domain reflectometry-style observations are sensitive to the density of the media being studied. Therefore, it is necessary to perform species-specific calibrations to convert from the sensor-reported values of dielectric permittivity to volumetric water content. Calibration is performed on a harvested branch or stem cut into segments that are dried or re-hydrated to produce a full range of water contents used to generate a best-fit regression with sensor observations. Sensors are inserted into calibration segments or installed in trees after pre-drilling holes to a tolerance fit using a fabricated template to ensure proper drill alignment. Special care is taken to ensure that sensor tines make good contact with the surrounding media, while allowing them to be inserted without excessive force. Volumetric water content dynamics observed via the presented methodology align with sap flow measurements recorded using thermal dissipation techniques and environmental forcing data. Biomass water content data can be used to observe the onset of water stress, drought response and recovery, and has the potential to be applied to the calibration and evaluation of new plant-level hydrodynamics models, as well as to the partitioning of remotely sensed moisture products into above- and belowground components.

The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees.

电容式传感器监测林木茎含水量的标定及应用

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...

Combining Eye-tracking Data with an Analysis of Video Content from Free-viewing a Video of a Walk in an Urban Park Environment

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. Eye-tracking informatics are known to connect to a range of feelings, health conditions, mental states and actions. But because vision is the result of constant eye-movements, teasing out what is important from what is noise is complex and data intensive. Furthermore, a significant challenge is controlling for what people look at compared to what is presented to them.
The following presents a methodology for combining and analyzing eye-tracking on a video of a natural and complex scene with a machine learning technique for analyzing the content of the video. In the protocol we focus on analyzing data from filmed videos, how a video can be best used to record participants&#39; eye-tracking data, and importantly how the content of the video can be analyzed and combined with the eye-tracking data. We present a brief summary of the results and a discussion of the potential of the method for further studies in complex environments.

The objective of the protocol is to detail how to collect video data for use in the laboratory; how to record eye-tracking data of participants looking at the data and how to efficiently analyze the content of the videos that they were looking at using a machine learning technique.

将眼动追踪数据与从自由观看城市公园环境中的步行视频中获得的视频内容进行分析

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

不同环境条件下生物气溶胶的组成及分布分析

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

气泡柱光生物反应器中绿色微藻的培养及中性脂质的测定

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...

Manufacturing Simple and Inexpensive Soil Surface Temperature and Gravimetric Water Content Sensors

Quantifying temperature and moisture at the soil surface is essential for understanding how soil surface biota respond to changes in the environment. However, at the soil surface these variables are highly dynamic and standard sensors do not explicitly measure temperature or moisture in the upper few millimeters of the soil profile. This paper describes methods for manufacturing simple, inexpensive sensors that simultaneously measure the temperature and moisture of the upper 5 mm of the soil surface. In addition to sensor construction, steps for quality control, as well as for calibration for various substrates, are explained. The sensors incorporate a Type E thermocouple to measure temperature and assess soil moisture by measuring the resistance between two gold-plated metal probes at the end of the sensor at a depth of 5 mm. The methods presented here can be altered to customize probes for different depths or substrates. These sensors have been effective in a variety of environments and have endured months of heavy rains in tropical forests as well as intense solar radiation in deserts of the southwestern U.S. Results demonstrate the effectiveness of these sensors for evaluating warming, drying, and freezing of the soil surface in a global change experiment.

Accurately measuring temperature and water content of the upper 5 mm of the soil surface can improve our understanding of environmental controls on biological, chemical, and physical processes. Here we describe a protocol for manufacturing, calibrating, and conducting measurements with soil surface temperature and moisture sensors.

制造简单、廉价的土壤表面温度和重力含水量传感器

Quantifying temperature and moisture at the soil surface is essential for understanding how soil surface biota respond to changes in the environment. ...