Name: experimental protocol for biodiesel production with isolation of alkenones as coproducts from commercial isochrysis algal biomass
Uploaded: 2016-06-24T15:00:00.000+00:00
Duration: 9 min 10 s
Description: Watch this Scientific Journal Video about Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass at JoVE.com

这项工作是由美国国家科学基金会（CHE-1151492）的支持，西北可再生能源高级联盟（奖学金给J.威尔逊珀耳帖），并通过由WHOI的朋友的私人捐赠。我们感谢凯文R. ​​Steidley和金Ascherl（USDA / ARS / NCAUR）优秀技术援助。

1.微藻生物质和准备注:海洋微藻等鞭金藻。在本研究中所用的"T-异"可以购买（参见材料的列表）。 等鞭金藻多公斤的量能与大海的刺激性气味扑鼻购买含有大约80％的水分和20％的生物质冷冻湿贴（ISO -粘贴），并且是深绿色/近黑色。 等鞭金藻还可以可以用类似的气味购买作为干（95％干）的黄棕色粉末（ 异 -powder）。 <ol><li>在使用剪刀的塑料包装的角2英寸的孔-以干燥的球等鞭金糊，通过切割1打开1千克包。 </li><li>这种球等通过孔粘贴到150mm的×75毫米结晶皿以创建一薄层（约20 MM）挤约300个克</li><li>离开浆料风干室温直到它变得干燥和片状（通常，48 -96小时）。 注意:实际干燥时间可以变化，并且取决于温度。然而，没有差异注意到产量或产品质量与甚至更长的干燥时间（长达两周）。干燥过程也可以由更均匀和/或通过将结晶皿上的温暖板加速（30 - 40℃）。 </li><li>用刮刀刮去从结晶皿干物质量和收集到的纤维素提取套管（长123毫米，43毫米ID）。记录干球等的生物质的重量。 </li></ol> 2.干生物质等鞭金藻的提取索氏<ol><li>加载等鞭金藻含纤维素提取套管（一般为50 - 60克干生物质）转化为索氏提取设备。 </li><li>填用己烷（400毫升）索氏烧瓶，打开冷凝水和热源上，并允许索氏循环为24 - 48小时（直至溶剂的颜色从深绿色去一淡黄色）。 </li><li>关火，并允许该装置冷却到室温，然后断开索格利特萃取器的烧瓶中。 </li><li>使用旋转蒸发器除去己烷并记录己烷可提取的物质（"己烷藻油"（H-AO））的重量。 </li></ol> 3.海藻油和脂肪酸和中性脂肪的分离皂化<ol><li>来自步骤2.4再溶解的H-AO在相同的圆底烧瓶以上，用甲醇:二氯甲烷（2:1，体积= 10×质量藻油）。 </li><li>添加搅拌棒，并附加一个回流冷凝器（卷板:500毫米的长度）。 </li><li>添加H 2 O（体积= 2.67×质量藻油）和KOH（50％重量/重量的藻油）并在搅拌至60℃3小时加热的内容。 </li><li>在冷却到室温后，用旋转蒸发器除去有机溶剂（甲醇和二氯甲烷）。 </li><li>通过倒入一个1传送剩余的含水混合物-L分液漏斗中。添加己烷（相当于在卷到水溶液中），摇动分液漏斗，并允许这些层分离。 </li><li>漏下层水层到三角烧瓶中，倾出上面的有机相到一个单独的锥形。 </li><li>重复步骤3.5和3.6，直到有机层是无色（通常为1-2次）。 </li><li>集中在旋转蒸发合并的有机萃取至中性脂质分离为浅绿色固体（熔点≈60 - 70℃）。 </li><li>酸化用HCl将水相（6M，直到pH〜2的pH试纸指示）。 </li><li>提取从酸化的水相用1升分液漏斗中与步骤3.5和3.6中所述的游离脂肪酸（游离脂肪酸）用己烷（等量的水相）。 </li><li>移除在旋转蒸发器的己烷，得到游离脂肪酸，为深绿色近黑色油状残余物（液体在温度&gt; 30℃）。 </li></ol> 4。酸催化游离脂肪酸酯化和绿色生物柴油生产<ol><li>转移使用甲醇的游离脂肪酸:氯仿（1:1，海藻油的6×体积），以第一溶解游离脂肪酸，然后倒入装有搅拌棒的厚壁高压反应烧瓶中。 </li><li>加浓H 2 SO 4（20％w / w的藻油），密封该烧瓶，并加热该混合物至90℃，同时搅拌1小时。 </li><li>在冷却到室温后，倒入分液漏斗中传送混合物。 </li><li>加入H 2 O（藻油的2×体积），摇动分液漏斗，使各相分离。 </li><li>漏底层到预先称重的圆底烧瓶中并浓缩在旋转蒸发器。记录所得的生物柴油的质量。 </li><li>分析用气相色谱用火焰离子化检测器（GC-FID）28（配备有DB-88（88％cyanopropy气相色谱脂肪酸谱升）methylarylpolysiloxane]柱（30米×0.25毫米内径x 0.20微米膜厚度）。 注意:普通的脂肪酸甲酯通过与商业上获得真实样品的保留时间比较，验证。此外，气相色谱 - 质谱（GC-MS;耦合到质量选择检测器的气相色谱仪）的温度程序和柱的相同条件下进行的分析组件，例如C18:4的量没有可信标准可与施加的结果的GC定量。 </li></ol> 5.生物柴油脱色<ol><li>加热深绿色的生物柴油至60℃在装有搅拌棒的圆底烧瓶中。 </li><li>添加蒙脱土ķ10（MK10）粉末（10-20％w / w的生物柴油的），搅拌1小时。 </li><li>除去从热的圆底，并使溶液冷却至室温。 </li><li>制备由一个圆底烧瓶中并过滤乐趣的过滤装置含道纤维素滤纸（灰分0.007％）。 </li><li>倾使用己烷最小量的冲洗的圆底烧瓶中通过过滤器漏斗的冷却脱色生物柴油。 </li><li>断开圆底烧瓶中的过滤漏斗（这现在包含脱色生物柴油的己烷溶液），并用旋转蒸发器除去己烷，得到橙色/红色的生物柴油。 </li><li>在4℃储存的样品，在此期间不溶性物质的一些沉降（〜10％w / w的）都会发生。 </li><li>通过如在步骤5.4和5.5中所述以产生用于分析一个透明均匀的生物柴油倾析或过滤去除不溶性物质。 </li></ol> 6.从中性脂分离饱和酮的分离纯化<ol><li>溶解在二氯甲烷中最小量的中性脂质（来自步骤3.8）（约50毫升10克中性脂质），并添加用吸管层析柱顶部的溶液（O。ð60毫米，ID 55毫米，长度18"）含有硅胶（230-400目，100克）。 </li><li>洗脱通过硅胶上用压力，用二氯甲烷（约150ml）中作为溶剂的溶液（〜5磅），并收集在250毫升洗脱液圆底烧瓶中。 </li><li>用旋转蒸发器除去二氯甲烷，得到一橙色固体。 </li><li>通过添加约100毫升煮沸己烷，然后用沸腾的己烷的增量额外量的直到溶液是均匀的（总体积〜150毫升）中再结晶用己烷固体。然后，慢慢冷却该溶液至室温，促进结晶。 </li><li>如用冷（0℃）的己烷的少量冲洗烧瓶，在步骤5.4中所述收集使用的过滤装置的结晶烯酮。 </li></ol>

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球等仅仅是一个选择号码工业养殖藻类收获贝类饲料的主要成分，因此，代表所需生物燃料生产规模中的一个。利用藻类，并在这项研究中采用的标准方法，未能使该协议提交给其他组作进一步调查普及。关键步骤包括风干藻（相对于冻干33），溶剂萃取，皂化，与酯化反应。通过这些操作中的一个可以检查从各球等可用30脂质和其它副产品的产量。可以预期，这些可以如不同菌株和栽培方法，31的结果不同，并且也可以由该产品的性质和任何附加的处理由供应商利用（ 例如 ，干燥或冷冻）受到影响。正如我们在这里展示，协议开发编可以成功地应用于不同类型的球等的产品，范围从湿贴到干燥研磨后的粉末。生物柴油的产率然而从粉状生物质下部（7％w / w的干生物量与12％w / w的从干燥的膏），这与较低量海藻油的对应式（h-AO）萃取。这可能表明一个替代提取协议32比索氏装置其它可能更适合用于干粉等鞭金藻的产品。在此研究中使用的球等粉末被通告为含有23-25％的脂类，其类似于我们通过实验从干燥球等鞭金膏得到。33,34,26 尽管不同的颜色开始干生物质中， 异贴昊 ​​和异 -powder昊基本上没有区别，无论是深绿/近黑色固体用约50℃的熔点。有趣的是，游离脂肪酸的中性唇的比例两个正己烷提取物中的ID是不同的。中性脂类的皂化和分离后，获得60％（W / W）从异 -粘贴豪游离脂肪酸和40％（重量/重量）的中性脂质。在异粉末生产的豪平均46％（W / W）的游离脂肪酸和54％（W / W）中性脂质。结果表明，无论是起始粉末的生物质可含有更高量的相对于FA衍生物中性脂质的比等鞭金糊，或粉末状等鞭金藻的索氏提取为中性脂质略有选择性。 不仅是从两个商业等鞭得到的产物的产率生物质不同，而且所产生的生物柴油的脂肪酸谱。这是重要的，作为生物柴油的燃料性能是直接依赖于自然和35个人脂肪酸甲酯的内容要被商业化，所有的生物柴油必须符合在文献ASTM描述的标准D6751或EN 14214，在美国和欧洲分别。规范包括十六烷值和氧化稳定性的润滑性和运动粘度范围和最小值。其他重要的建议中的浊点（CP）或冷过滤堵塞点（CFPP）的形式与冷流性质。我们先前已报道的结果从异糊制备生物柴油的综合燃料的测试。36由于从本研究中的异 -powder产生生物柴油的FAME轮廓类似于先前测试的那些，可以预测某种燃料特性是相似的为生物柴油燃料。例如，多不饱和脂肪酸（PuFAMEs，多于两个双键）占两个FAME混合物的约40％（35.2％和39.9％， 表2）。这将导致氧化稳定性差，有利冷流35有，但是，在这两个biodie的FAME轮廓略有不同SEL燃料。从粉末等鞭产生的生物柴油含有14更高量:0（19.4毫克/克与16.4毫克/克），18:3（13.5毫克/克与8.5毫克/克），以及22:6（11.0毫克/克与6.9毫克/克）脂肪酸甲酯，还较少量18:4（10.4毫克/克与19.8毫克/克）。的包含在ASTM标准的各种燃料性质这些差异的影响的程度还有待调查。 从商用等鞭金藻得到的初始生物柴油是在彩色同样深绿其可以通过叶绿素的存在来解释。36叶绿素及其衍生物已报道对植物油和其相应的生物柴油燃料的稳定性有负面影响。基于Issariyakul和达赖的方法，与生产生物柴油脱色连接greenseed菜籽油36,29，29搅拌我们的绿色生物柴油超过10％（W / W）MK10在60℃1小时，导致一个戏剧性减少在通过目测的颜料含量（ 参考 图 2）。从脱色工艺质量回收率平均为90％。 从异贴豪和ISO-粉豪中性脂肪纯化的烯酮产量是可比的40％和46％w / w的分别（ 表1）。由于中性脂质代表的材料中所含的异粉昊比例较高（54％w / w的对40％W / W），从异粉浩烯酮的产量超过了异贴昊 ​​烯酮产量约10％（25％w / w的比16％W / W）。然而，考虑到异粉末浩本身的产量都比异贴昊 ​​（15％对20％W / W），无论从干生物质等鞭金藻的烯酮总收率都比较相似（0.2×0.4×更低0.4 = 3.2％w / w的从干燥等鞭金膏和0.15点¯x0.54点¯x0.46 = 3.7从球等粉末％）。

最近已出现的兴趣很大回潮在从藻类生物燃料，特别是用于生产液体燃料，如生物柴油1和其他源自生物质的油。2拟议的益处包括某些食品避免与燃料争议3和据说较高的生产率和CO 2减排能力比传统农作物。4这是继能源的水生物种计划（ASP）的近20年美国农业部于1978年开始从藻类调查运输燃料的目的。正如希恩的报告中提出，5项目于1996年结束，主要是因为预计成本，不是用原油的竞争在那个时候（$ 18.46每桶（159升））。而石油的成本自那时以来（2014年87.39 $每桶）6，连接到文艺复兴时期的藻类生物燃料的研究，一些^ h显着增加AVE认为，尽管如此，藻类生物燃料将被证明过于昂贵。7作为一种策略，以抵消生物燃料的生产成本，增值副产品的概念已经评论家7,8和支持者9,10和功能之间成为为关键原因之一追求能源的美国能源部（DOE）"国家生物燃料藻类技术路线图"藻类生物燃料。11 在这里，我们描述了两个独立的燃料流从商业等鞭金藻合拍的方法。我们已经在部分集中在球等 ，因为它已经工业化生产，收获的海水养殖的目的，并且还因为球等鞭金只是藻类的几个物种，除了传统的脂质（ 即脂肪酸）生物合成一类独特的之一称为多不饱和长链烯酮化合物。12烯酮结构的特征在于非常升<html>翁烃链（36-40个碳原子），二至四个非亚甲基间隔反 -double键，和一个甲基或乙基酮（ 图1）。烯酮不饱和度的藻类生长温度敏感，13,14，使得二不饱和C37甲基烯酮（即所谓的"不饱和度指数"）的比例可以被用来作为过去海洋表面温度的代理15日 - 20饱和酮是认为驻留在细胞质脂质体和可以比甘油三酯（代码）更加丰富。21,22在氮或磷的限制，最多在固定相中细胞的碳10〜20％被积累作为烯酮。23,24从一个进化的角度看，烯酮可能已经赞成对TAG的，因为它们的反式 -双键几何形状提供能量储存器的一个更稳定的形式。21 189fig1.jpg"/&gt; 图1的多不饱和长链烯酮的结构共同烯酮甲基37:3从球等例示长烃链长分离（36 - 40个碳）， 反式非亚甲基间隔的双键，并在一个为甲基或乙基酮终止。命名是类似的脂肪酸，其中＃＃是指碳原子数:双键数目<a href="https://www.jove.com/files/ftp_upload/54189/54189fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 我们认为烯酮代表从一个共同的藻类有前途的可再生的碳原料与工业种植的历史。25生物柴油直接从球等的总脂质提取物产生包含由烯酮和污染的显著量（10-15％w / w的）这些高熔化合物会导致较差的低温流动性的燃料。然而，采用t他这里描述皂化/提取技术，烯酮可以去除并回收，从而提高了生物柴油的质量的同时，产生第二产物流。最近我们证明烯酮的转化为液体燃料通过交叉复分解以-2-丁烯（butenolysis）。26 butenolysis反应采用市售的钌复分解引发剂，在低温下迅速发生，且干净提供喷气燃料的一可预测的混合物范围内的碳氢化合物。此反应是在并行自脂肪酸进行用生物柴油的合成，表示朝向为商业上可行的球等生物燃料生产了"生物炼制"的方法27的第一步骤。

<table><tbody><tr><td>&lt;em&gt;Isochrysis&lt;/em&gt;</td><td>Reed Mariculture</td><td>Iso, Raw, Unprocessed, 20%dw</td><td>Live culture paste, 20% w/w biomass</td></tr><tr><td>&lt;em&gt;Isochrysis&lt;/em&gt;</td><td>Necton, S.A</td><td>AADISS004</td><td>PhytoBloom Prof Isochrysis (Freeze-dried)</td></tr><tr><td>Hexanes</td><td>Fisher Chemical</td><td>H291-4</td><td>ACS Certified</td></tr><tr><td>Methanol</td><td>Fisher Chemical</td><td>A452-4</td><td>HPLC Grade</td></tr><tr><td>Dichloromethane</td><td>Fisher Chemical</td><td>D37-4</td><td>Certified/Stabilized</td></tr><tr><td>Soxhlet Apparatus</td><td>Sigma Aldrich</td><td>64826</td><td></td></tr><tr><td>Extraction Thimble</td><td>Sigma Aldrich</td><td>64842</td><td></td></tr><tr><td>B&amp;uuml;chner Funnel</td><td>Chemglass</td><td>CG-1406-25</td><td></td></tr><tr><td>High Pressure Reaction Vessel</td><td>Chemglass</td><td>CG-1880-12</td><td></td></tr><tr><td>Whatman Filter Paper</td><td>GE Life Sciences</td><td>1442-042</td><td>Grade 42, Ash 0.007%, circle, 42.5 mm</td></tr><tr><td>Biodiesel (B100)</td><td>Bellingham Shell</td><td></td><td>The biodiesel (B100) in &lt;strong&gt;Figure 3&lt;/strong&gt; was purchased at a local filling station: Bellingham Shell, Bellingham, WA 98226</td></tr><tr><td>&lt;em&gt;Isochrysis&lt;/em&gt;</td><td>Aquacave</td><td></td><td>In addition to Reed and Necton, &lt;em&gt;Isochrysis&lt;/em&gt; can also be purchased from Aquacave (Gurnee, IL) at: www.aquacave.com (accessed September 30, 2015).</td></tr><tr><td>&lt;em&gt;Isochrysis&lt;/em&gt;</td><td>Brine Shrimp Direct</td><td></td><td>&lt;em&gt;Isochrysis&lt;/em&gt; can also be purchased from Brine Shrimp Direct (Ogden, UT) at: www.brineshrimpdirect.com (accessed September 30, 2015).</td></tr></tbody></table>

experimental protocol for biodiesel production with isolation of alkenones as coproducts from commercial isochrysis algal biomass

从可再生能源和环境可持续发展的源替代品来替代石油燃料的需求日益重要。生物质衍生的生物燃料已经在这方面取得了相当大的关注，从粮食作物如玉米乙醇或生物柴油的大豆但是第一代生物燃料普遍失宠。因此，存在在从国内和优良的非食用源生产液体燃料的方法的发展的极大兴趣。在这里，我们描述了从生产海洋微藻等鞭纯化生物柴油的详细过程。此外，一个唯一的称作多不饱和长链烯酮脂质的套件平行地分离出具有潜在价值的副产品，以抵消生物柴油生产成本。 等鞭金藻的多千克数量从两个商业来源，一个作为湿糊（80％水），该第一处理之前干燥，OT购她干磨粉（95％干）。脂质用己烷萃取在Soxhlet装置以产生含有传统脂肪的藻油（"己烷藻油"）（ 即，甘油三酯，46-60％w / w的）和烯酮（16-25％W / W）。在海藻油的甘油三酯的皂化允许从含有烯酮中性脂质所得游离脂肪酸（游离脂肪酸）的分离。然后游离脂肪酸转化通过酸催化酯化而烯酮被分离并通过结晶从中性脂质纯化生物柴油（ 即 ，脂肪酸甲酯，脂肪酸甲酯）。我们表明，从商用等鞭生物质的生物柴油具有相似但不相同的FAME型材，其特征在于，升高的多不饱和脂肪酸含量（约40％W / W）。从等鞭湿膏开始（w / w的对7％w / w的12％），其可以被追踪，以降低己烷藻OI的量时的生物柴油的产率始终高于从粉状产品等鞭金藻获得的L-。

<html>处理之前，所述等鞭金膏（ 异膏）中的第一次干燥。这是方便地通过加入异 -粘贴到一个大的结晶皿并允许材料风干室温完成在较大的规模。在干燥过程中，一些汇集水的形式（一般红色），其可以通过倾析或移液以加速干燥过程中除去。后约48 - 96小时后，将现在干球等可以刮出的结晶皿中，并作为与海藻般气味（ 图2）一黑/绿片状材料获得。干燥的生物质的产率一般为20％w / w的糊状物如做广告。与此相反，粉末状等鞭产物（ 异粉末）为黄棕色，精细研磨，其直接使用而无需进一步处理的干燥粉末（95％干）（ 图2）。 古尔2"SRC ="/文件/ ftp_upload / 54189 / 54189fig2.jpg"/&gt; 图2.商业球等 。 等鞭金膏（80％湿） 的比较 ，在室温下沿结晶皿的底部扩散和左风干处理前48-96小时。所得的干燥等鞭金就得到深色片状材料（右）是在外观上比商业干粉等鞭金 （左）不同。 <a href="https://www.jove.com/files/ftp_upload/54189/54189fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 索氏用己烷无论是干异 -粘贴或者异型粉末提取了，去除己烷后，海藻油（H-AO）是在外观为暗绿色/近黑色固体（熔点相似。〜50 -60℃）。从贴（" 异贴豪起动时H-AO的产量"）是典型的20％w / w的干球等生物量，与我们以前的结果，26而由商业粉状等鞭金藻的索氏提取（H-AO的产率相一致的" 异粉末浩"）分别为15％重量/ W（ 表1）。 <table fo:keep-together.within-page="1"><tbody><tr><td> 产品（G） </td><td> ISO -粘贴 </td><td> ISO -powder-1 </td><td> 异 -powder-2 </td></tr><tr><td>干重</td><td>三十</td><td> 20 </td><td> 20 </td></tr><tr><td>己烷藻油</td><td> 5.86 </td><td> 2.87 </td><td> 3.11 </td></tr><tr><td>游离脂肪酸</td><td> 3.52 </td><td> 1.34 </td><td> 1.38 </td></tr><tr><td>中性脂质</td><td> 2.34 </td><td> 1.38 </td><td> 1.61 </td></tr><tr><td>烯酮</td><td> 0.94 </td><td> 0.63 </td><td> 0.74 </td></tr><tr><td colspan="4">注:ISO-粉1和ISO-粉2代表和已经在并行处理等鞭金藻粉的两个样品的结果。对于这些产品在ISO粘贴的产量其他报告见参考文献26，32，和33。 </td></tr></tbody></table> 从商业等鞭生物量 表1。 产品产量。 在H-AO酰基甘油在加入在甲醇/ CH 2 Cl 2中KOH水溶液被转化成相应的水溶性羧酸盐（ 即，皂类）。中性脂质包括烯酮然后通过用己烷选择性分区此含水混合物萃取。除去中性脂质后，将皂reacidification然后产生相应的游离脂肪酸（游离脂肪酸），可以从该水溶液被类似提取ueous相己烷。从任一异粘贴昊或ISO -powder豪结合的游离脂肪酸和中性脂肪的总质量回收率始终接近定量。然而，产品的比（ 即，中性脂质+游离脂肪酸）是不同的。从异糊状郝我们得到60％（W / W）游离脂肪酸和40％（重量/重量）中性脂质（ 表1）。相反地， 异粉末浩证明如表1中详述的中性脂质（平均= 54％的中性脂质+ 46％游离脂肪酸）富集。 用H 2 SO 4和甲醇的游离脂肪酸的酯化然后生成脂肪酸甲酯（脂肪酸甲酯， 即 ，生物柴油），如大于90％的产率（ 图3）暗绿色邻近黑色油状液体。通过加热超过蒙脱土K10 29脱色（MK10）粘土然后给了黄/橙色的产品，在外观上与其他商业BI相似odiesel燃料（见材料的列表）（ 图3）。从脱色等鞭金藻的生物柴油燃料的FAME分析结果示于表2中 。 <img alt="图3" src="/files/ftp_upload/54189/54189fig3.jpg" /> 图3 等鞭金藻的比较和大豆生物柴油燃料。格林等鞭生物柴油（中）是通过提取和纯化的游离脂肪酸酯化。脱色生产产品（右）具有类似属性的商业生物柴油（左）。 <a href="https://www.jove.com/files/ftp_upload/54189/54189fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 成名 </td><td> ISO -粘贴 </td><td> ISO -powder </td></tr><tr><td> 14:00 </td><td> 16.4 </td&gt; <td> 19.4 </td></tr><tr><td> 14:01 </td><td> - </td><td> 0.3 </td></tr><tr><td> 15:00 </td><td>跟踪</td><td> 0.3 </td></tr><tr><td> 16:00 </td><td> 10.1 </td><td> 8.8 </td></tr><tr><td> 16:1Δ9 </td><td> 7.6 </td><td> 5.5 </td></tr><tr><td> 16时02分</td><td> ND </td><td> 0.3 </td></tr><tr><td> 16:03 </td><td> ND </td><td> 0.5 </td></tr><tr><td> 18:00 </td><td>跟踪</td><td> 0.2 </td></tr><tr><td> 18:图1B </td><td> 12.1 </td><td> 14.3 </td></tr><tr><td> 18时02分</td><td> 8.1 </td><td> 7.1 </td></tr><tr><td> 18: 的3C </td><td> 8.5 </td><td> 13.5 </td></tr><tr><td> 18时04分</td><td> 19.8 </td><td> 10.4 </td></tr><tr><td> 18:05 </td><td> ND </td><td> 3 </td></tr><tr><td> 20:05 </td><td> ND </td><td> - </td></tr><tr><td> 22时05分</td><td> ND </td><td> 2 </td></tr><tr><td> 22:06 </td><td> 6.9 </TD&gt; <td> 11 </td></tr><tr><td> Σð </td><td> 89.8 </td><td> 96.2 </td></tr><tr><td colspan="3">注: 脂肪酸的命名是#carbons:＃ 顺 -double债券B合并18:1Δ9+ 18:1。Δ11C组Δ6,9,12和Δ9,12,15异构体D中的剩余材料大致是50 :其他50脂肪酸甲酯和非FAME组件（总计〜95％FAME）。 ND =未检出。 </td></tr></tbody></table> 表2. 生物柴油的FAME组成商业异 -past和异 -powder等鞭金藻生物量生产。 中性脂质在40％w / w的从ISO -粘贴昊和54％来自异粉昊（ 表1），得到绿色固体混合物（平均）。通过使用硅胶过滤溶解的中性脂质DCM，得到除去溶剂后，为红/橙色固体，可以用己烷进行重结晶，得到分析纯的烯酮，其为白色固体。这个过程导致了16％（W / W）从异贴豪烯酮的分离收率和25％的收益率从异 -powder昊（ 表1）。

Watch this Scientific Journal Video about Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass at JoVE.com

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

详细方法提出了生产生物柴油的同烯酮从商品等鞭金藻有价值副产品的共隔离沿。

实验协议生产生物柴油与饱和酮的分离从商业余积<em&gt;等鞭金藻</em&gt;藻类生物量

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

experimental-protocol-for-biodiesel-production-with-isolation

Research

JoVE Journal

Environment

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

20.6K 次观看.  Western Washington University. 详细方法提出了生产生物柴油的同烯酮从商品等鞭金藻有价值副产品的共隔离沿。 

视频: Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

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 ...

科研

环境科学

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

一种简单快速的协议用荧光测量中性脂肪在藻细胞

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

化学

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 ...

Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ammonia removal, nitrogen removal and algal growth are compared over an 8-week period in paired sets of small (100 L) and large (1,000 L) reactors designed for algal remediation of landfill wastewater. Contents of the small and large scale reactors were mixed before the beginning of each weekly testing interval to maintain equivalent initial conditions across the two scales. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted to better equalize conditions occurring at both scales. During the short 8-week representative time period, starting ammonia and total nitrogen concentrations ranged from 3.1-14 mg NH3-N/L, and 8.1-20.1 mg N/L, respectively. The performance of the treatment system was evaluated based on its ability to remove ammonia and total nitrogen and to produce algal biomass. Mean &#177; standard deviation of ammonia removal, total nitrogen removal and biomass growth rates were 0.95&#177;0.3 mg NH3-N/L/day, 0.89&#177;0.3 mg N/L/day, and 0.02&#177;0.03 g biomass/L/day, respectively. All vessels showed a positive relationship between the initial ammonia concentration and ammonia removal rate (R2=0.76). Comparison of process efficiencies and production values measured in reactors of different scale may be useful in determining if lab-scale experimental data is appropriate for prediction of commercial-scale production values.

An experimental methodology is presented to compare the performance of small (100 L) and large (1,000 L) scale reactors designed for algae remediation of landfill wastewater. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted based on application.

规模在光合反应器系统对污水的整治藻类比较

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. 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 ...

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

将发电厂的碳捕获与半自动开放式管道池相结合，用于微藻养殖

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas ...

用于从猪废沼气中获取生物甲烷的微藻培养

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

作者聚焦:扩展微藻生物技术以提高生物甲烷产量 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

在半工业化高速率藻类池塘中使用微藻细菌系统进行沼气净化

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

从藻类水热液化使用二维气相色谱时间飞行质谱法水提取部分的定性鉴定

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

实验协议生产生物柴油与饱和酮的分离从商业余积

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此视频中的章节

脂肪酸组分和含量微藻分析

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