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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Technol. 86, 1059-1070 (2005).</li><li>Valenzuela-Espinoza, E., Mill&#225;n-N&#250;&#241;ez, R., Protein N&#250;&#241;ez-Cebrero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein,+carbohydrate,+lipid+and+chlorophyll+a+content+in+Isochrysis+aff.+galbana+(clone+T-Iso)+cultured+with+a+low+cost+alternative+to+the+f/2+medium.">Protein, carbohydrate, lipid and chlorophyll a content in Isochrysis aff. galbana (clone T-Iso) cultured with a low cost alternative to the f/2 medium.</a> Aquacult. Eng. 25, 207-216 (2002).</li><li>Kulkarni, M. G., Dalai, A. K., Bakshi, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+green+seed+canola+oil+for+biodiesel+production.">Utilization of green seed canola oil for biodiesel production.</a> J. Chem. Technol. Biotechnol. 81, 1886-1893 (2006).</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 border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Isochrysis</td>
			<td style="width:107px;">Reed Mariculture</td>
			<td style="width:119px;">Iso, Raw, Unprocessed, 20%dw</td>
			<td style="width:167px;">Live culture paste, 20% w/w biomass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isochrysis</td>
			<td style="width:107px;">Necton, S.A</td>
			<td style="width:119px;">AADISS004</td>
			<td>PhytoBloom Prof Isochrysis (Freeze-dried)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hexanes</td>
			<td style="width:107px;">Fisher Chemical</td>
			<td style="width:119px;">H291-4</td>
			<td>ACS Certified</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methanol</td>
			<td style="width:107px;">Fisher Chemical</td>
			<td style="width:119px;">A452-4</td>
			<td>HPLC Grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dichloromethane</td>
			<td style="width:107px;">Fisher Chemical</td>
			<td style="width:119px;">D37-4</td>
			<td>Certified/Stabilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Soxhlet Apparatus</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">64826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Extraction Thimble</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">64842</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B&#252;chner Funnel</td>
			<td style="width:107px;">Chemglass</td>
			<td style="width:119px;">CG-1406-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High Pressure Reaction Vessel</td>
			<td style="width:107px;">Chemglass</td>
			<td style="width:119px;">CG-1880-12</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Whatman Filter Paper</td>
			<td style="width:107px;">GE Life Sciences</td>
			<td>1442-042</td>
			<td>Grade 42, Ash 0.007%, circle, 42.5 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biodiesel (B100)</td>
			<td style="width:107px;">Bellingham Shell</td>
			<td style="width:119px;"></td>
			<td>The biodiesel (B100) in Figure 3 was purchased at a local filling station: Bellingham Shell, Bellingham, WA 98226</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isochrysis</td>
			<td style="width:107px;">&#160;Aquacave</td>
			<td style="width:119px;"></td>
			<td>&#160;In addition to Reed and Necton, Isochrysis can also be purchased from (Aquacave. (Gurnee, IL) at: www.aquacave.com (accessed September 30, 2015).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isochrysis</td>
			<td style="width:107px;">Brine Shrimp Direct</td>
			<td style="width:119px;"></td>
			<td>Isochrysis 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

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

用于操纵植物诱导土壤异质性的实验协议

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

科研

环境科学

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of a variety of lignocellulosic feedstocks. Here we demonstrate the use of these low-cost protic ILs in the deconstruction of lignocellulosic biomass (Ionosolv pretreatment), yielding cellulose and a purified lignin. In the most generic process, the protic ionic liquid is synthesized by accurate combination of aqueous acid and amine base. The water content is adjusted subsequently. For the delignification, the biomass is placed into a vessel with IL solution at elevated temperatures to dissolve the lignin and hemicellulose, leaving a cellulose-rich pulp ready for saccharification (hydrolysis to fermentable sugars). The lignin is later precipitated from the IL by the addition of water and recovered as a solid. The removal of the added water regenerates the ionic liquid, which can be reused multiple times. This protocol is useful to investigate the significant potential of protic ILs for use in commercial biomass pretreatment/lignin fractionation for producing biofuels or renewable chemicals and materials.

The pretreatment of lignocellulosic biomass with protic low-cost ionic liquids is shown, resulting in a delignified cellulose-rich pulp and a purified lignin. The pulp gives rise to high glucose yields after enzymatic saccharification.

木质纤维素生物质预处理低成本离子液体

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of ...

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

用于检测实验方案<em&gt;蓝藻</em&gt;与抗体基因芯片液体和固体样品

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

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, ...

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

的发展<em&gt; Metarhizium anisopliae</em&gt;作为防腐剂:从隔离到现场表现

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

Experimental System of Solar Adsorption Refrigeration with Concentrated Collector

To improve the performance of solar adsorption refrigeration, an experimental system with a solar concentration collector was set up and investigated. The main components of the system were the adsorbent bed, the condenser, the evaporator, the cooling sub-system, and the solar collector. In the first step of the experiment, the vapor-saturated bed was heated by the solar radiation under closed conditions, which caused the bed temperature and pressure to increase. When the bed pressure became high enough, the bed was switched to connect to the condenser, thus water vapor flowed continually from the bed to the condenser to be liquefied. Next, the bed needed to cool down after the desorption. In the solar-shielded condition, achieved by aluminum foil, the circulating water loop was opened to the bed. With the water continually circulating in the bed, the stored heat in the bed was took out and the bed pressure decreased accordingly. When the bed pressure dropped below the saturation pressure at the evaporation temperature, the valve to the evaporator was opened. A mass of water vapor rushed into the bed and was adsorbed by the zeolite material. With the massive vaporization of the water in the evaporator, the refrigeration effect was generated finally. The experimental result has revealed that both the COP (coefficient of the performance of the system) and the SCP (specific cooling power of the system) of the SAPO-34 zeolite was greater than that of the ZSM-5 zeolite, no matter whether the adsorption time was longer or shorter. The system of the SAPO-34 zeolite generated a maximum COP of 0.169.

With solar energy as the driving force, a novel adsorption refrigeration system has been developed and experimentally investigated. Water vapor and zeolite formed the working pair of the adsorption system. This manuscript describes the setup of the experimental rig, the operation procedure, and the important results.

聚光集热器太阳能吸附制冷实验系统

To improve the performance of solar adsorption refrigeration, an experimental system with a solar concentration collector was set up and investigated. The ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using three recently developed experiments in which dust particles are exposed to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. The UV light source has a narrow bandwidth in wavelength centered at 172 nm. The beam electrons with the energy of 120 eV are created with a negatively biased hot filament. When the vacuum chamber is filled with the argon gas, a thermal plasma is created in addition to the electron beam. Insulating dust particles of a few tens of microns in diameter are used in the experiments. Dust particles are recorded to be lofted to a height up to a few centimeters with a launch speed up to 1 m/s. These experiments demonstrate that photo and/or secondary electron emission from a dusty surface changes the charging mechanism of dust particles. According to the recently developed &#34;patched charge model&#34;, the emitted electrons can be re-absorbed inside microcavities between neighboring dust particles below the surface, causing the accumulation of enhanced negative charges on the surrounding dust particles. The repulsive forces between these negatively charged particles may be large enough to mobilize and lift them off the surface. These experiments present the advanced understanding of dust charging and transport on dusty surfaces, and laid a foundation for future investigations of its role in the surface evolution of airless planetary bodies.

Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies.

紫外辐射或等离子体照射表面的粉尘充电和动员实验方法

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

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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