作者要感谢佐治亚大学农业实验站提供的技术和财政支持。此外，作者还要感谢 Samuel Ogundipe、Ronald Pegg 博士和 Joon Hyuk Suh 博士在分析过程样品方面提供的帮助。

本研究中使用的试剂、耗材和设备列于 材料表中。
1. 生物反应器、液-液萃取系统的构建和集成
<ol><li>准备有机相萃取溶液和储液槽。<ol><li>使用磁力搅拌板和搅拌器将 60 g 三辛基氧化膦 （TOPO） 溶解在矿物油中，制备 2 L 有机相萃取溶液。</li>
		<li>将提取溶液添加到 2 L 玻璃储液器（即 Schott 瓶）中。</li>
		<li>将储液槽放在磁力搅拌板上（图 1A）。连续 LLE 操作期间的推荐混合速度为 150-250 rpm。</li>
	</ol></li>
	<li>为提取溶液储液器准备一个三端口盖。<ol><li>将浸管连接到第一个端口，作为流出端口，为正向提取膜 （FEM） 提供提取溶液（图 1B）。</li>
		<li>粘贴第二个端口，作为来自反向提取膜 （BEM） 的提取溶液的返回端口（图 1C）。</li>
		<li>增加第三个端口，向大气开放，以抑制隔膜泵引起的压力波动。</li>
	</ol></li>
	<li>将隔膜泵连接到萃取膜。<ol><li>将配备聚四氟乙烯 （PTFE） 隔膜泵头的 100 rpm 变速泵驱动器放置在储液罐附近（图 1D）。</li>
		<li>使用柔性泵管（例如，尺寸 16、18），将提取溶液储液储液器流出口（图 1A）连接到隔膜泵的入口，然后将隔膜泵的出口连接到 FEM 底部的壳侧入口（图 1B）。</li>
		<li>使用柔性泵管（例如，尺寸 18）将 FEM 顶部的壳侧出口连接到 BEM 底部的壳侧入口（图 1C）。</li>
		<li>使用柔性泵管（例如，尺寸 16、18）将 BEM 顶部的壳侧出口连接到提取溶液储液罐（图 1A）上的返回端口。 注:该系统组件将从 FEM 中的发酵液中提取的 MCFA 转移到 BEM 中的汽提溶液中。</li>
	</ol></li>
	<li>准备水相剥离溶液和储液槽。<ol><li>取 0.5 M 硼酸溶液并使用 NaOH 将其调节至 pH 9，制备 3.25 L 水相剥离溶液。</li>
		<li>将溶液倒入带有磁力搅拌棒的 3.5 L 玻璃储液槽中。</li>
		<li>将储液槽放在磁力搅拌板上（图 1E）。</li>
	</ol></li>
	<li>为剥离溶液储液槽准备一个四端口盖。<ol><li>将浸管连接到第一个端口，作为流出端口，为 BEM 提供汽提溶液。</li>
		<li>将包含 Y 型接头的第二个端口固定到 （1） 作为 BEM 剥离溶液的回流口，以及 （2） 作为从 pH 控制系统添加 NaOH 的夹带流。</li>
		<li>添加第三个端口，向大气开放，以解决由 NaOH 添加和 MCFA 积累引起的体积增加。</li>
		<li>提供第四个端口以容纳 pH 控制器的 pH 探头。</li>
	</ol></li>
	<li>在汽提液储液罐处安装 pH 控制系统。<ol><li>将设定点为 pH 值为 9 的 pH 控制系统与剥离液储液罐集成（图 1F）。使用 5 M NaOH 作为 pH 控制器的碱溶液，以抵消收集的 MCFA。</li>
		<li>将 pH 探针插入剥离液储液槽端口，并将其悬浮在剥离液中。 注:此 pH 控制系统不需要酸溶液。</li>
	</ol></li>
	<li>准备生物反应器连接端口。<ol><li>在生物反应器上指定两个端口，一个流出口和一个回流口，用于连接液-液萃取 （LLE） 系统（图 1G，H）。</li>
		<li>将浸管连接到流出端口。富含 MCFA 的肉汤将从生物反应器的流出口泵入 LLE 系统。 注意:肉汤首先进入中空纤维膜过滤器，然后再进入萃取膜，以去除细胞和其他固体，以防止结垢。</li>
		<li>在回流端口插入三通接头以接收:（1） 从 FEM 回收的 MCFA 耗尽肉汤和 （2） 来自中空纤维膜过滤器的含细胞的截留物。</li>
	</ol></li>
	<li>安装中空纤维膜过滤器。<ol><li>在生物反应器附近放置一个 300 rpm 变速泵驱动器（图 1I）。</li>
		<li>将亲水性空心纤维膜（图 1J）固定在蠕动泵上方的环形支架上。</li>
		<li>在泵驱动器上堆叠两个蠕动泵头，大（例如 17 号）和小（例如 16 号）。 注:较大的泵头连接到中空纤维膜过滤器，以确保渗透流速大于流入 FEM 的流速。如果不是这种情况，渗透液的消耗速度将比产生渗透液的速度快，从而导致真空形成。</li>
		<li>使用柔性泵管（例如 17 号）将生物反应器的流出口连接到大型泵头入口。</li>
		<li>使用柔性泵管（例如 17 号）将大泵头出口连接到过滤器底部管侧入口处的亲水性中空纤维膜过滤器。</li>
		<li>盖上过滤器的顶部壳侧端口，以防止空气流入。 注:富含 MCFA 的肉汤（含有细胞）将通过中空纤维管向上流动并返回生物反应器。透明肉汤（无细胞）将穿过 0.2 μm 亲水性聚醚砜 （PES） 膜并收集在过滤器的壳侧。</li>
	</ol></li>
	<li>连接 FEM.<ol><li>将第一个疏水性中空纤维膜组件 （FEM） 固定在泵上方的环形支架上（图 1B）。</li>
		<li>使用柔性泵管（例如，尺寸 16、18）将壳侧出口处的亲水性中空纤维膜过滤器（图 1J）连接到小泵头入口。</li>
		<li>使用柔性泵管（例如，尺寸 16、18）将小泵头出口连接到模块底部管侧入口处的 FEM。</li>
		<li>使用耦合接头和三通接头将压力表（图 1K）和限流阀（图 1L）连接到 FEM 的管侧出口。</li>
		<li>使用柔性泵管（例如 18 号）将 FEM 的管侧出口连接到生物反应器的回流口。 注:该系统组件将 MCFA 从清发酵液转移到提取液中，然后将清汤返回生物反应器。</li>
	</ol></li>
	<li>连接 BEM。<ol><li>在生物反应器附近放置一个配备蠕动泵头（例如，尺寸 16）的 300 rpm 变速泵驱动器（图 1M）。</li>
		<li>将第二个疏水性中空纤维膜组件固定在蠕动泵上方的环形支架上（图 1C）。</li>
		<li>使用柔性泵管（例如，尺寸 16、18），将汽提液储液罐流出口连接到蠕动泵入口，将泵出口连接到 BEM 底部的管侧入口。</li>
		<li>使用耦合接头和三通接头将压力表和限流阀连接到 BEM 的管侧出口。</li>
		<li>使用柔性泵管（例如，尺寸 16、18）将 BEM 顶部的管侧出口连接到汽提溶液储液罐回流口。 注意:该系统组件将 MCFA 从提取溶液转移到 BEM 中的剥离溶液，然后将剥离溶液返回到其储液罐中。</li>
	</ol></li>
</ol>2. 启动液-液萃取系统的运行
<ol><li>灌注和循环水相线。<ol><li>打开汽提液蠕动泵/BEM（图 1M）并设置泵速以达到 25-250 mL·min-1 之间的恒定流速。边界元法可以容纳相对较大的流速范围。最初从保守的高流速开始，以确保足够的 MCFA 提取。在运行过程中，可以在以后的运行过程中逐渐降低流速，以延长泵设备和管道的使用寿命。 注意:流速不应太低，以允许 MCFA 在生物反应器肉汤中积累（参见代表性结果）。</li>
		<li>缓慢关闭 BEM 壳侧出口处的针阀（图 1C），以建立 ~5 psig 的背压。</li>
		<li>打开生物反应器/FEM 蠕动泵（图 1I）并设置泵速以达到 25-250 mL·min-1 之间的恒定流速。FEM 可以容纳相对较大的流量范围。最初从保守的高流速开始，以确保足够的 MCFA 提取。在运行过程中，可以在以后的运行过程中逐渐降低流速，以延长泵设备和管道的使用寿命。 注意:流速不应太低，以允许 MCFA 在生物反应器肉汤中积累（参见代表性结果部分）。</li>
		<li>缓慢关闭 FEM 壳侧出口处的针阀（图 1B）以建立 ~5 psig 的背压。</li>
		<li>目视检查回流管线，以确保流量恒定，并且管线已灌注。</li>
		<li>验证透明肉汤是否收集在中空纤维膜过滤器的壳侧（图 1J）。 注:填充 FEM 并在中空纤维膜过滤器和 FEM 之间建立稳定的流动需要几个小时。流速不应太低，以允许 MCFA 在生物反应器肉汤中积累（参见代表性结果部分）。</li>
	</ol></li>
	<li>灌注并循环有机相线。<ol><li>打开有机相萃取溶液隔膜泵（图 1D）并设置泵速以达到 5.0-50 mL·min-1 之间的恒定流速。最初从保守的低流速开始，以最大限度地降低有机相萃取中的压力并最大限度地降低交叉风险。如有必要，可以在操作后期逐渐增加流速，以提高提取效率</li>
		<li>等待 FEM 和 BEM 填满。</li>
		<li>目视检查萃取溶液储液罐处的回流口，以确保恒定流量。</li>
		<li>确认没有有机相溶液进入汽提液或发酵液管线。如果发生交叉，则可以看到有机相的小液滴。如果发生这种情况，请降低隔膜泵的速度，并根据需要略微增加 FEM 或 BEM 的背压。不要超过 10 psig。 注:为防止膜交叉，在灌注有机相管路之前，必须在水相管路内建立流速和背压。</li>
	</ol></li>
	<li>从生物反应器中连续提取 MCFA。<ol><li>LLE 系统应完全运行。允许系统在生物反应器运行期间连续运行。</li>
		<li>每天测量生物反应器中的 MCFA 浓度，以确保 MCFA 的充分提取。如果生物反应器中的 MCFA 浓度升高，这通常表明发酵液通过 FEM 的流速不足。这也可能表明由于污染和需要维护而导致膜通量减少（参见步骤 2.6）。 注意:SCFA 和 MCFA 浓度可以根据 Ge 等人 11 描述的方法通过气相色谱法测量。</li>
	</ol></li>
	<li>监测 MCFA 在剥离溶液储液罐中的积累。<ol><li>在批处理周期中每天测量剥离溶液中的 MCFA 浓度。LLE 系统连续将 MFCA 从生物反应器转移到剥离溶液中，随着时间的推移增加 MCFA 浓度。该工艺可以长时间运行以产生高 MCFA 滴度。MCFA 的体积生产率 （mM C·L-1·d-1） 可以使用公式 111 估算。 注:体积生产率 = <img alt="Equation 1" src="/files/ftp_upload/66956/66956eq01.jpg" style="margin: auto;"> （方程 1） 哪里: Cb，n= 第 n 天发酵液中的 MCFA 浓度，mM C Cs，n= 第 n 天剥离液中的 MCFA 浓度，mM C Cs，n-1 = 第 n-1 天剥离液中的 MCFA 浓度，mM C Vs，n= 第 n 天的剥离液体积，L Vb，n= 第 n 天的发酵液体积（生物反应器体积），L HRTn= 生物反应器在第 n、d 天的水力停留时间 Tn= 第 n、d 天 Tn-1= 第 n-1 天，d</li>
		<li>为确保稳定运行，通过测量测量时间点之间 MCFA 浓度的变化并应用方程 211，定期计算 LLE 系统的提取速率 （mM C·d-1）。 注: <img alt="Equation 2" src="/files/ftp_upload/66956/66956eq02.jpg" style="margin: auto;"> （式 2） 哪里: Cs，n= 第 n 天剥离液中的 MCFA 浓度，mM C Cs，n-1 = 第 n-1 天剥离液中的 MCFA 浓度，mM C Vs，n= 第 n 天的剥离液体积，L Tn= 第 n、d 天 Tn-1= 第 n-1 天，d</li>
		<li>为了保持提取溶液的足够转移速率，请在 MCFA 浓度达到 80% 饱和之前用新批次替换剥离溶液。 注:正己酸的最大溶解度为 10.3 g·L-1 在 25 °C 时，正辛酸的 L-1 为 0.67 g·L-1 在 25 °C 下。</li>
	</ol></li>
	<li>更换剥离液。<ol><li>关闭隔膜泵（图 1D）。</li>
		<li>关闭剥离溶液蠕动泵（图 1M）。</li>
		<li>使用软管夹夹住 BEM 的管侧入口和管侧出口。</li>
		<li>关闭 pH 控制系统并取下剥离液储液槽盖，同时保持端口连接连接（如果可能）。</li>
		<li>带走剥离溶液储液器（图 1E）。</li>
		<li>用一批使用 NaOH 调节至 pH 9 的新鲜 0.5 M 硼酸水溶液替换剥离溶液储液器（参见步骤 1.4）。将盖子重新贴在储液槽上。</li>
		<li>从 BEM 的管侧入口和管侧出口拆下软管夹。</li>
		<li>打开剥离溶液蠕动泵（图 1M），然后打开隔膜泵（图 1D）。系统操作现已恢复。</li>
	</ol></li>
	<li>膜维护。<ol><li>每三个月从 LLE 系统中取出一次 FEM 和 BEM 进行清洁。三个月是保守估计的清洁频率。 注意:根据应用，用户可以或多或少地清洁膜。膜性能下降的迹象在代表性结果部分描述。在维护期间，应关闭泵和 pH 控制器。膜制造商应提供清洁说明。</li>
		<li>将液体从 LLE 系统排入单独的容器中，从有机相萃取溶液管线开始，然后是发酵液管线和汽提液管线。</li>
		<li>清洁并重新安装膜后，将液体放回各自的储液槽中。</li>
		<li>使用上述方法重新启动 LLE 系统（参见步骤 2.1-2.2）。</li>
	</ol></li>
</ol>

通过链伸长和液-液萃取实现可持续的 MCFA 生产

<ol><li>Palomo-Briones, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Near-neutral+ph+increased+n-caprylate+production+in+a+microbiome+with+product+inhibition+of+methanogenesis%5BTitle%5D)+AND+%22Chem+Eng+J%22%5BJournal%5D)">Near-neutral ph increased n-caprylate production in a microbiome with product inhibition of methanogenesis.</a> Chem Eng J. 446, 137170(2022).</li>
<li>Wang, J., Yin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+production+of+medium-chain+carboxylates+through+chain+elongation%3A+An+overview%5BTitle%5D)+AND+%22Biotechnol+Adv%22%5BJournal%5D)">Biological production of medium-chain carboxylates through chain elongation: An overview.</a> Biotechnol Adv. 55, 107882(2022).</li>
<li>Watanabe, S., Tsujino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+of+medium-chain+triglycerides+in+foods%5BTitle%5D)+AND+%22Front+Nutr%22%5BJournal%5D)">Applications of medium-chain triglycerides in foods.</a> Front Nutr. 9, 802805(2022).</li>
<li>Meijaard, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+environmental+impacts+of+palm+oil+in+context%5BTitle%5D)+AND+%22Nat+Plants%22%5BJournal%5D)">The environmental impacts of palm oil in context.</a> Nat Plants. 6 (12), 1418-1426 (2020).</li>
<li>Meijaard, E., Abrams, J. F., Juffe-Bignoli, D., Voigt, M., Sheil, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coconut+oil%2C+conservation+and+the+conscientious+consumer%5BTitle%5D)+AND+%22Curr+Bio%22%5BJournal%5D)">Coconut oil, conservation and the conscientious consumer.</a> Curr Bio. 30 (13), R757-R758 (2020).</li>
<li>Angenent, L. T., Magdalena, J. A., Jeon, B. S., Usack, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eco-mimicry+opens+new+doors+for+bioprocess+engineers%5BTitle%5D)+AND+%22Joule%22%5BJournal%5D)">Eco-mimicry opens new doors for bioprocess engineers.</a> Joule. 4 (10), 2074-2077 (2020).</li>
<li>Shahab, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+heterogeneous+microbial+consortium+producing+short-chain+fatty+acids+from+lignocellulose%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A heterogeneous microbial consortium producing short-chain fatty acids from lignocellulose.</a> Science. 369 (6507), 1214(2020).</li>
<li>Magdalena, J. A., Angenent, L. T., Usack, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+measurement%2C+application%2C+and+effect+of+oxygen+in+microbial+fermentations%3A+Focusing+on+methane+and+carboxylate+production%5BTitle%5D)+AND+%22Fermentation%22%5BJournal%5D)">The measurement, application, and effect of oxygen in microbial fermentations: Focusing on methane and carboxylate production.</a> Fermentation. 8 (4), 138(2022).</li>
<li>Agler, M. T., Spirito, C. M., Usack, J. G., Werner, J. J., Angenent, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+highly+specific+and+productive+process+for+n-caproic+acid+production%3A+Applying+lessons+from+methanogenic+microbiomes%5BTitle%5D)+AND+%22Water+Sci+Technol%22%5BJournal%5D)">Development of a highly specific and productive process for n-caproic acid production: Applying lessons from methanogenic microbiomes.</a> Water Sci Technol. 69 (1), 62-68 (2013).</li>
<li>Gildemyn, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Upgrading+syngas+fermentation+effluent+using+Clostridium+kluyveri+in+a+continuous+fermentation%5BTitle%5D)+AND+%22Biotechnol+Biofuels%22%5BJournal%5D)">Upgrading syngas fermentation effluent using Clostridium kluyveri in a continuous fermentation.</a> Biotechnol Biofuels. 10 (1), 83(2017).</li>
<li>Ge, S., Usack, J. G., Spirito, C. M., Angenent, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+n-caproic+acid+production+from+yeast-fermentation+beer+in+an+anaerobic+bioreactor+with+continuous+product+extraction%5BTitle%5D)+AND+%22Env+Sci+Technol%22%5BJournal%5D)">Long-term n-caproic acid production from yeast-fermentation beer in an anaerobic bioreactor with continuous product extraction.</a> Env Sci Technol. 49 (13), 8012-8021 (2015).</li>
<li>Saboe, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+recovery+of+bio-based+carboxylic+acids%5BTitle%5D)+AND+%22Green+Chem%22%5BJournal%5D)">In situ recovery of bio-based carboxylic acids.</a> Green Chem. 20 (8), 1791-1804 (2018).</li>
<li>Kaur, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reactive+extraction+for+in-situ+carboxylate+recovery+from+mixed+culture+fermentation%5BTitle%5D)+AND+%22Biochem+Eng+J%22%5BJournal%5D)">Reactive extraction for in-situ carboxylate recovery from mixed culture fermentation.</a> Biochem Eng J. 160, 107641(2020).</li>
<li>Carvajal-Arroyo, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+and+extraction+of+medium-chain+carboxylic+acids+at+a+semi-pilot+scale%5BTitle%5D)+AND+%22Chem+Eng+J%22%5BJournal%5D)">Production and extraction of medium-chain carboxylic acids at a semi-pilot scale.</a> Chem Eng J. 416, 127886(2021).</li>
<li>Choi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+biphasic+extractive+fermentation+for+hexanoic+acid+production+from+sucrose+by+Megasphaera+elsdenii+NCIMB+702410%5BTitle%5D)+AND+%22Appl+Biochem+Biotechnol%22%5BJournal%5D)">In situ biphasic extractive fermentation for hexanoic acid production from sucrose by Megasphaera elsdenii NCIMB 702410.</a> Appl Biochem Biotechnol. 171 (5), 1094-1107 (2013).</li>
<li>Grootscholten, T., Steinbusch, K., Hamelers, H., Buisman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+medium+chain+fatty+acid+productivity+using+chain+elongation+by+reducing+the+hydraulic+retention+time+in+an+upflow+anaerobic+filter%5BTitle%5D)+AND+%22Bioresour+Technol%22%5BJournal%5D)">Improving medium chain fatty acid productivity using chain elongation by reducing the hydraulic retention time in an upflow anaerobic filter.</a> Bioresour Technol. 136, 735-738 (2013).</li>
<li>Grootscholten, T., Dal Borgo, F. K., Hamelers, H., Buisman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promoting+chain+elongation+in+mixed+culture+acidification+reactors+by+addition+of+ethanol%5BTitle%5D)+AND+%22Biomass+Bioenergy%22%5BJournal%5D)">Promoting chain elongation in mixed culture acidification reactors by addition of ethanol.</a> Biomass Bioenergy. 48, 10-16 (2013).</li>
<li>Cermak, S. C., Evangelista, R. L., Kenar, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distillation+of+natural+fatty+acids+and+their+chemical+derivatives%5BTitle%5D)+AND+%22Distillation-Advances+from+Modeling+to+Applications+Chapter%22%5BJournal%5D)">Distillation of natural fatty acids and their chemical derivatives.</a> Distillation-Advances from Modeling to Applications Chapter. 5, 109-142 (2012).</li>
<li>Agler, M. T., Spirito, C. M., Usack, J. G., Werner, J. J., Angenent, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chain+elongation+with+reactor+microbiomes%3A+Upgrading+dilute+ethanol+to+medium-chain+carboxylates%5BTitle%5D)+AND+%22Energy+Environ+Sci%22%5BJournal%5D)">Chain elongation with reactor microbiomes: Upgrading dilute ethanol to medium-chain carboxylates.</a> Energy Environ Sci. 5 (8), 8189-8192 (2012).</li>
</ol>

没有利益冲突。

生物生产的 MCFA 通常与各种有机化合物（包括 SCFA 和醇类）一起存在于混合物中2。因此，需要选择性分离过程来有效回收和利用它们。这里开发的 LLE 系统从这些混合物中选择性地连续提取 MCFA，同时保留 SCFA 和醇类。此功能使 LLE 系统特别适用于发酵应用，例如微生物链伸长，其中 MCFA、SCFA 和醇构成主要代谢物8。具体来说，LLE 系统允许通过去除 MCFA 来进行链延伸过程，防止产品抑制1，同时将 SCFA 和醇反应物留在发酵液中用于随后的生物转化。通过修改特定的萃取溶液，可以针对其他应用定制 LLE 系统。例如，通过从萃取剂溶液混合物中去除 TOPO，可以使用相同的 LLE 系统连续提取发酵过程中产生的 SCFA。
因此，与其他方法相比，LLE 方法的意义在于为这些生物加工和生物技术应用提供更强大的 MCFA 提取技术。用不混溶液体进行 原 位双相萃取是从发酵液中提取 MCFA 的另一种方法15。但是，这种方法相对效率较低。在水相（即发酵液）和有机相之间形成乳化层，严重限制了传质速率。相层之间最小的界面流体混合也限制了传质。另一个缺点是微生物细胞与有机相直接接触，导致夹带、抑制和细胞死亡15。最后， 原位 双相萃取需要频繁维护以去除和更换有机相。
在生物反应器内应用高稀释率是避免产品抑制的另一种方法16。高稀释率可以通过在生物反应器中保持高反应物浓度来实现高生产率。然而，这种方法是不利的，因为它会导致生物质冲刷、产生大量流出物和高底物损失（即 SCFA 和醇类），从而导致产量低。这些缺点可以通过固定化生物质和污水回收来缓解，但这些干预措施增加了系统的复杂性17。最后，产品流中的 MCFA 浓度被稀释，使 MCFA 效率低下且成本高昂。
一种新的提取方法可能涉及使用单个正向萃取膜连续蒸馏 MCFA，该膜将有机相和水相物理分离，从而保留和保护微生物生物量。MCFA 将被选择性提取到有机相中，然后进行蒸馏。抽余液可以连续循环到提取膜上。然而，连续蒸馏在技术上具有挑战性，尤其是在实验室环境中，并且可能导致化学萃取剂在长期运行期间变质或损失。蒸馏还可能导致有机相和 MCFA 产物的热降解18.
LLE 工艺通过结合几个关键特征和加工步骤，避免了与这些替代方法相关的许多缺点。首先，亲水性中空纤维膜过滤器具有双重目的，即保护生物质细胞（生物催化剂）免受 FEB 中的萃取剂溶液的影响，同时提供富含 MCFA 的透明滤液，从而减少 LLE 系统中的结垢和固体积累。其次，为了防止液体交叉，我们采用了针阀，在每个膜接触器的管侧产生背压。这种预防措施保持了轻微的跨膜压力梯度，防止疏水性有机溶剂在 FEM 和 BEM 中从壳侧意外泄漏到水性管侧。此外，液体流配置为从 FEM 和 BEM 的底部平行流向顶部，以防止气泡滞留，这些气泡可能聚集在膜组件内，从而降低传输效率并导致残留。此外，该方法使用带有耐化学腐蚀的 PTFE 泵头的隔膜泵来泵送含有腐蚀性 MCFA 的萃取剂溶液，从而保护系统免受腐蚀和故障的影响，从而影响萃取过程。最后，pH 控制的碱性汽提液保持 pH 梯度，允许 MCFA 通过 LLE 系统以高速率从生物反应器连续转移到汽提液储液罐，MCFA 在那里去质子化并积累至高滴度，促进下游产品回收。
这种 LLE 方法适用于从实验室规模的生物反应器（高达 6 L 工作体积）中连续提取 MCFA，并已在多项研究中验证了长期操作 1,9,11,19。LLE 方法也可用于更大规模的应用14（即中试规模的生物反应器），但需要按比例缩放的膜和流体处理设备。但是，该方法确实存在一些局限性，主要是在维护和系统复杂性方面。由于该工艺设计为连续运行，因此必须经常维护膜组件和泵，从而导致相当长的停机时间。另一个缺点是剥离溶液需要相对大量的 NaOH 和硼酸。此外，MCFA 具有腐蚀性，会导致某些 LLE 系统组件随着时间的推移而劣化。例如，塑料连接器和膜外壳可能会变脆，需要在运行过程中更换。最后，LLE 系统中的流体处理网络很复杂，涉及许多容易产生泄漏的连接点。然而，这些限制和缺点中的大多数是连续膜分离过程的典型特征，应该预料到。
总体而言，该 LLE 协议为选择性 MCFA 提取提供了一种稳健而有效的方法，这对推进不同领域的研究具有重要意义。该方法在发酵过程中对细胞外代谢产物的原位回收的精密发酵领域有许多相关应用。LLE 可能是传统下游加工 （DSP） 方法的低成本替代方案，例如运行后离心、微过滤和超滤或批量进行的溶剂萃取。事实上，DSP 通常是工业发酵过程中的主要成本驱动因素。使用 LLE 进行连续产品提取还可以实现连续发酵，与传统的分批或补料分批方法相比，可显著提高操作的生产率和运行时间效率。此外，未来的研究可以研究有机溶剂以外的萃取介质，例如深共熔溶剂或离子液体。最后，本协议中描述的 LLE 系统旨在用于实验室环境中的实验目的;因此，仍有相当大的优化研究空间，以降低能源需求、膜面积以及总体提取产量和速率。

中链脂肪酸 （MCFA） 是高价值的合成砌块化学品，由链长从 6 个 （C6） 到 12 个 （C12） 碳组成。MCFA 在食品、动物饲料、制药、化妆品、香料、抗菌剂和化学合成中具有工业应用 1,2,3。目前，大多数 MCFA 来自来自东南亚和南美洲的棕榈油和椰子油 4,5。与棕榈油和椰子油生产相关的严重生态破坏已得到利益相关者和公众的广泛认可。研究人员正在探索生物学方法（例如，精确和开放培养发酵），以使用低价值底物或有机废物作为原料更可持续地生成MCFA6,7。生产 MCFA 的一种可持续方法是使用称为微生物链伸长 （CE） 的工艺升级回收有机废物流。这种二次发酵生物过程类似于厌氧消化，因为它利用了厌氧开放培养微生物组的多功能性，但 CE 系统不是促进甲烷形成，而是故意抑制产甲烷途径。在碳不能被最大程度还原为 CH4 的微生物组中，H2 也不能被消耗氢的古细菌维持在 10-4 个大气压以下，通常将长链羧酸盐分解成乙酸盐的β氧化反应（例如，C6 → C4 → C2）可以逆转（例如，C2 → C4 → C6 等），只要还原化合物（即 电子供体），例如乙醇或乳酸8.在这种新陈代谢中，经过伸长的脂肪酸分子充当电子受体。因此，CE 工艺生成的碳长度为 1 （CH4） 的产品不是厌氧消化中，而是产生碳长度为 6 到 8 的 MCFA。一个庞大且不断增长的市场已准备好接收这些绿色平台化学品。然而，到目前为止，尚未证明 CE 工艺可以以可观的速率产生碳长度超过 8 个碳的 MCFA。
这些 MCFA 的高效提取不仅对于所需产品的回收很重要，而且对于防止产品抑制和推动微生物组产生更多的 MCFA也很重要 1。随着 MCFA 浓度的增加，MCFA 代谢受到抑制，热力学上变得不那么有利。通过连续移除 MCFA，可以保持生产率。此外，由于 SCFA 是链伸长过程的子结构，因此不应将其从发酵液中取出。靶向萃取剂混合物应选择性地从含有 SCFA 的发酵液中提取 MCFA 产品。
在这里，展示了一种稳健而实用的方法，可以使用液液萃取 （LLE） 系统从含 SCFA 的发酵液中连续提取 MCFA，该系统包括疏水性聚丙烯正向中空纤维膜萃取器、选择性有机萃取剂溶液 （三辛基膦氧化物 [TOPO]9,10,11）和反向中空纤维膜提取器。在 LLE 系统上游安装了细胞保护过滤器，以保留生物量并减少膜污染。MCFA 以质子化形式从水性发酵液（通常具有 pH 设定点 <5.8）中正向提取到有机萃取剂溶液（即 3% TOPO （w/v） 矿物油中），然后反向提取到碱性汽提溶液 （pH = 9） 中，在那里它们去质子化并浓缩至高滴度用于下游加工。特定的 pH 设定点是必不可少的，因为它们决定了 LLE 过程每个阶段之间的浓度梯度，确保 MCFA 从发酵液到汽提液的净转移。使用正向和反向萃取膜的 LLE 可实现高萃取率，同时最大限度地减少醇类和 SCFA 的共萃取。有机溶剂佐剂 TOPO 能够形成 MCFA 复合物。这些复合物比水更易溶于有机相，因此具有较高的 MCFA 选择性。LLE 过程还避免了与现有方法相关的许多缺点，这些将在讨论部分讨论。使用这种 LLE 方法的长期实施已在多项研究中得到证明 9,10,11。虽然这种方法特别适用于通过微生物链延伸生产 MCFA 的应用，但它也适用于需要选择性分离具有相似化学性质的化合物的其他应用，因为有机萃取剂系统可以定制。

<table><tbody><tr><td>10 L Media Bottle</td><td>Duran</td><td>218018658</td><td></td></tr><tr><td>3.5 L Media Bottle</td><td>Duran</td><td>218016957</td><td></td></tr><tr><td>Boric acid, 99.5%,&amp;nbsp;</td><td>ThermoScientific (Fisher Scientific)</td><td>327132500</td><td></td></tr><tr><td>Hydrophilic MINIKROS 20CM 0.2UM PES 1MM 1.5TC X 3/4TC</td><td>Repligen</td><td>N02-P20U-10-N</td><td></td></tr><tr><td>L/S Variable-Speed Pump Drive; 100 rpm</td><td>MasterFlex (VWR)</td><td>MFLX07528-10</td><td></td></tr><tr><td>L/S Variable-Speed Pump Drive; 300 rpm</td><td>MasterFlex (VWR)</td><td>MFLX07528-20</td><td></td></tr><tr><td>Light Mineral Oil, NF (4 Liters) (CAS: 8042-47-5)</td><td>Thomas Scientific</td><td>C761Z18</td><td></td></tr><tr><td>Liqui-Cel 2.5x8 X50 membrane CO2, PP Housing Viton O-rings (0.5-3 gpm (0.1-0.7 m3/h)), 1/4-in FNPT connections</td><td>3M</td><td>LC-02508X50-G453</td><td></td></tr><tr><td>Magnetic Stirrer, 20 L Capacity, 110 V</td><td>Cole-Parmer</td><td>EW-04661-29</td><td></td></tr><tr><td>Masterflex L/S Precision Pump Tubing, Tygon, Size 14</td><td>MasterFlex (VWR)</td><td>MFLX06402-14</td><td>Specific tubing size will depend on application.</td></tr><tr><td>Masterflex L/S Precision Pump Tubing, Tygon, Size 16</td><td>MasterFlex (VWR)</td><td>MFLX06402-16</td><td>Specific tubing size will depend on application.</td></tr><tr><td>Masterflex L/S Precision Pump Tubing, Tygon, Size 17</td><td>MasterFlex (VWR)</td><td>MFLX06402-17</td><td>Specific tubing size will depend on application.</td></tr><tr><td>Masterflex L/S Precision Pump Tubing, Tygon, Size 18</td><td>MasterFlex (VWR)</td><td>MFLX06402-18</td><td>Specific tubing size will depend on application.</td></tr><tr><td>MasterFlex L/S Standard Pump Head for Precision Tubing L/S 14, Polycarbonate Housing, CRS Rotor</td><td>MasterFlex (VWR)</td><td>MFLX07014-20</td><td>Specific pump head size will depend on application.</td></tr><tr><td>MasterFlex L/S Standard Pump Head for Precision Tubing L/S 14, Polycarbonate Housing, CRS Rotor</td><td>MasterFlex (VWR)</td><td>MFLX07014-20</td><td>Specific pump head size will depend on application.</td></tr><tr><td>MasterFlex L/S Standard Pump Head for Precision Tubing L/S 16, Polycarbonate Housing, CRS Rotor</td><td>MasterFlex (VWR)</td><td>MFLX07016-20</td><td>Specific pump head size will depend on application.</td></tr><tr><td>MasterFlex L/S Standard Pump Head for Precision Tubing L/S 17, Polycarbonate Housing, CRS Rotor</td><td>MasterFlex (VWR)</td><td>MFLX07017-20</td><td>Specific pump head size will depend on application.</td></tr><tr><td>MasterFlex L/S Standard Pump Head for Precision Tubing L/S 18, Polycarbonate Housing, CRS Rotor</td><td>MasterFlex (VWR)</td><td>MFLX07018-20</td><td>Specific pump head size will depend on application.</td></tr><tr><td>MasterFlex PTFE-diaphragm pump head, 10 to 100 mL/min</td><td>MasterFlex (VWR)</td><td>MFLX07090-62</td><td></td></tr><tr><td>Oakton 220 pH/ORP/Temperature Controller, 1/8 DIN</td><td>Spectrum Laboratory Products</td><td>664-12595-E1</td><td></td></tr><tr><td>Oakton 220 pH/ORP/Temperature Controller, 1/8 DIN</td><td>Spectrum Laboratory Products</td><td>664-12595-E1</td><td></td></tr><tr><td>Oakton Female BNC-to-Stripped Wire Adapter</td><td>Spectrum Laboratory Products</td><td>664-12592-E1</td><td></td></tr><tr><td>pH Probe with BNC Connector</td><td>ThermoScientific</td><td>10010-788</td><td>Any pH probe with a BNC connector will suffice.&amp;nbsp;</td></tr><tr><td>Precision Flow-Adjustment Valve, White Polypropylene, 1/4 NPT Male x Male</td><td>McMaster-Carr</td><td>7792K57</td><td></td></tr><tr><td>ProConnex&amp;nbsp; Fittings Kits - A</td><td>Repligen</td><td>ACPX-KT2-01N</td><td>Compatible with Hydrophilic MINIKROS Filter</td></tr><tr><td>ProConnex Fittings Kits - B</td><td>Repligen</td><td>ACPX-KT1-01N</td><td>Compatible with Hydrophilic MINIKROS Filter</td></tr><tr><td>Sodium Hydroxide Pellets for Analysis</td><td>Sigma Aldrich</td><td>1.06498</td><td></td></tr><tr><td>Stainless-Steel Pressure Gauge 0-60 psi Stainless Steel 1/4&quot; NPT 2.5&quot; Face Dial</td><td>NA</td><td>XJ-219</td><td>Any comparable pressure gauge covering 0-60 psig range will suffice.&amp;nbsp;</td></tr><tr><td>Trioctylphosphine oxide (TOPO)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>346187-100G</td><td></td></tr></tbody></table>

continuous liquid-liquid extraction of medium-chain fatty acids from fermentation broth using hollow-fiber membranes

中链脂肪酸 （MCFA;碳长度:C6-C12） 是高价值的平台化学品，服务于各种工业应用，包括绿色抗菌剂、食品配料、动物饲料添加剂、化妆品、香料、药品和结构脂质。目前，大多数 MCFA 由源自东南亚和南美洲的棕榈油和椰子油制成。传统的棕榈和椰子果实收获方法在这些地区造成了相当大的生态破坏。因此，研究人员正在开发生物学方法（例如，精确和开放培养发酵），以使用低价值底物（例如甲醇、乙醇、乳酸盐）或有机废物作为原料，更可持续地生产 MCFA。微生物链延伸 （CE） 是一种快速成熟的开放培养发酵平台，它以工业相关的速率将短链脂肪酸 （SCFA;碳长度:C1-C5） 转化为这些 MCFA 的一个子集。然而，MCFA 产物的连续 原位 提取是必要的，不仅是为了避免产品抑制，而且是为了促进 MCFA 以纯净和可用的形式回收。使用中空纤维膜和靶向萃取剂混合物的液-液萃取 （LLE） 已被证明是从含有 SCFA 的发酵液中选择性提取 MCFA 产品的可靠方法。在这里，以 CE 作为参考发酵系统，以 3% （w/v） 矿物油中的三辛基氧化膦作为萃取系统，展示了 LLE 在连续去除 MCFA 中的应用。从含 SCFA 的肉汤中选择性地去除从含 SCFA 的肉汤中去除从戊酸 （C5） 到辛酸 （C8） 的脂肪酸，并在半间歇碱性汽提溶液中浓缩至高滴度，用于下游加工。

MCFA 产物在碱性水相剥离溶液中的稳定积累（图 2）和发酵液中相对稳定的 MCFA 浓度（数据未显示）表明积极的 MCFA 提取结果。 图 2 显示了连续 LLE 操作期间剥离溶液的三个半批量循环。一个周期包括两个阶段:批次更换阶段（图 2:第 24 天、第 46 天和第 68 天）和 MCFA 积累阶段（图 2:第 0-24 天、第 25-46 天、第 47-68 天）。对于这种特殊的发酵和 LLE 系统，周期持续时间约为 20-24 天。然而，循环持续时间因应用而异，因为它取决于多种因素，包括生物反应器体积、生物生产率、剥离液体积、中空纤维膜面积和 LLE 系统内的液体再循环速率。在批处理循环中，由于发酵液中存在的各种小有机酸（例如腐植酸、黄腐酸）的低水平共提取，剥离溶液的颜色可能会从透明变为黄棕色（图 3）。MCFA 提取的阴性结果表现为 MCFA 产物在剥离液中的缓慢积累和发酵液中 MCFA 浓度相对于预先设定的基线升高。
在这些发酵过程中，己酸盐的生物生产率通常高于辛酸盐;因此，与辛酸盐相比，己酸酯在剥离溶液中以更快的速度积累是很常见的。此外，短链脂肪酸（如乙酸盐和丁酸盐）以较低的量聚集在剥离溶液中是正常的，如图 2 所示。矿物油中的 TOPO 对 MCFA 的亲和力高于 SCFA，这会导致 MCFA 的选择性去除。Saboe 等人12、Kaur 等人13、Carvajal-Arroyo 等人14 和 Ge 等人11 的研究表明，TOPO 在涉及水溶液的多种应用中对脂肪酸具有高选择性。 图 4 显示了第二个批次周期期间 MCFA 与 SCFA 的分区比率。可以预期 MCFA:SCFA 分区比率在一个批处理周期中的几天内大于 40:1。MCFA:SCFA 分区比率将随着提取过程接近伪稳态而趋于稳定。如果几天后仍未达到 >40 的比率，则表明 TOPO 萃取剂已降解或洗脱。如果发生这种情况，应制备新的提取溶液（参见步骤 1.1）。如果该比率在平台期之后降低，则表明 MCFA 已积累超过其饱和点的 80%。如果发生这种情况，应准备新的剥离溶液（参见步骤 1.4）
MCFA 萃取效率低可能是由于 LLE 系统内的流速不足造成的。 在图 5 中，发酵液和汽提液循环管线中的抽速降低，以说明液体再循环速率降低对 MCFA 提取效率的影响。提取效率定义为剥离溶液中提取的 MCFA 相对于生物反应器产生的总 MCFA 加上 LLE 提取的 MCFA 的百分比。在正常操作期间，可以预期提取效率大于 85%（图 5，第 1-14 天）。当泵速较低时（图 5，第 14 天），提取效率会相应地降低。当泵速恢复足够时，萃取效率可能需要几天时间才能恢复。这可能是由于剥离液（较高）的提取速率差异（低于发酵液）导致提取液中 MCFA 的稳态浓度降低引起的。
其他几个因素也会导致萃取效率降低，包括 （1） 膜污染，（2） 由于堵塞，LLE 系统每个阶段的流体流动受限，（3） 膜接触器中形成气穴，以及 （4） 使汽提溶液中的 MCFA 浓度接近其饱和点。膜污染表现为膜通量相对于初始条件随时间推移而减少。虽然在 FEM 中不太可能形成生物膜，但由于细胞碎片和其他悬浮固体的积累，可能会发生结垢。此外，虽然 BEM 是无菌的，但由于脂肪酸盐随着时间的推移在膜接触器或管道内沉淀，流动可能会受阻。但是，膜接触器的日常维护和清洁（参见步骤 2.6）应防止结垢和盐沉淀问题的发展。由于定位不当，膜式接触器的顶壳侧有时会形成气穴。膜式接触器应从垂直方向略微倾斜，以确保壳侧出口位于最高点，从而允许形成的任何气体从接触器中逸出。LLE 系统中的流体流动配置为从承包商的底部流向顶部，以帮助冲洗气穴。最后，当汽提液中 MCFA 浓度非常高时，BEM 中 MCFA 从提取液到汽提液的转移会减少。可以通过更频繁地更换剥离解决方案来解决此问题。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66956/66956fig01.jpg"> 图 1:液-液萃取系统概述。 显示主要系统组件、各种流体回路和流向的图解。主要系统组件标记如下:（A） 有机相萃取溶液储液器，（B） 正向交换膜，（C） 后向交换膜，（D） 萃取溶液隔膜泵，（E） 水相汽提液储液器，（F） pH 控制系统，（G） 生物反应器流出口，（H） 生物反应器回流端口，（I） 正向交换膜和中空纤维膜过滤器蠕动泵，（J） 中空纤维膜过滤器， （K） 压力表、（L） 针阀和 （M） 汽提液蠕动泵。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66956/66956fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66956/66956fig02.jpg"> 图 2:剥离溶液中的脂肪酸积累。 数据显示了连续液-液萃取操作期间汽提溶液三个批次循环期间的短链脂肪酸和中链脂肪酸浓度。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66956/66956fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66956/66956fig03.jpg"> 图 3:萃取后剥离液的颜色变化。 显示水相剥离溶液在批次循环之前（即批次前）和之后（即批次后）的颜色变化的照片。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66956/66956fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66956/66956fig04.jpg"> 图 4:剥离溶液中的脂肪酸比例。 数据显示了在连续液-液萃取操作期间汽提溶液的批量循环中，中链脂肪酸与短链脂肪酸的比率。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66956/66956fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66956/66956fig05.jpg"> 图 5:膜流速对萃取效率的影响。 数据显示了运行过程中通过前向和后向交换膜的流速不足对中链脂肪酸提取效率的影响。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66956/66956fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

作者聚焦:优化中空纤维膜以连续液-液萃取中链脂肪酸

开发了一种涉及中空纤维膜的液-液萃取 （LLE） 系统，用于从发酵液中连续选择性地提取中链脂肪酸 （MCFA）。LLE 系统从含有短链脂肪酸和醇类的肉汤中实现了高 MCFA 特异性。此外，MCFA 浓缩在剥离溶液中，以促进产品回收。

使用空心纤维膜从发酵液中连续液-液萃取中链脂肪酸

Medium-chain fatty acids (MCFAs; carbon-lengths: C6-C12) are high-value platform chemicals that serve a variety of industrial applications, including ...

continuous-liquid-liquid-extraction-medium-chain-fatty-acids-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Continuous Liquid-Liquid Extraction of Medium-Chain Fatty Acids from Fermentation Broth Using Hollow-Fiber Membranes

1.0K 次观看.  University of Georgia. 我的研究旨在评估中空纤维膜和从发酵液中连续液-液萃取中链脂肪酸的效率。我试图通过优化提取过程和确定该方法在工业用途中的可扩展性来了解这种膜与传统技术有何不同。目前的实验挑战包括从发酵液中提取中链脂肪酸的效率低下，这通常会导致链延伸微生物的抑制。传统技术经常面临批处理限制、高能耗和高溶剂使用量等问题。与传?...

视频: 作者聚焦:优化中空纤维膜以连续液-液萃取中链脂肪酸

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

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

生物工程

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

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. Biomass-derived biofuels have gained considerable attention in this regard, however first generation biofuels from edible crops like corn ethanol or soybean biodiesel have generally fallen out of favor. There is thus great interest in the development of methods for the production of liquid fuels from domestic and superior non-edible sources. Here we describe a detailed procedure for the production of a purified biodiesel from the marine microalgae Isochrysis. Additionally, a unique suite of lipids known as polyunsaturated long-chain alkenones are isolated in parallel as potentially valuable coproducts to offset the cost of biodiesel production. Multi-kilogram quantities of Isochrysis are purchased from two commercial sources, one as a wet paste (80% water) that is first dried prior to processing, and the other a dry milled powder (95% dry). Lipids are extracted with hexanes in a Soxhlet apparatus to produce an algal oil (&#34;hexane algal oil&#34;) containing both traditional fats (i.e., triglycerides, 46-60% w/w) and alkenones (16-25% w/w). Saponification of the triglycerides in the algal oil allows for separation of the resulting free fatty acids (FFAs) from alkenone-containing neutral lipids. FFAs are then converted to biodiesel (i.e., fatty acid methyl esters, FAMEs) by acid-catalyzed esterification while alkenones are isolated and purified from the neutral lipids by crystallization. We demonstrate that biodiesel from both commercial Isochrysis biomasses have similar but not identical FAME profiles, characterized by elevated polyunsaturated fatty acid contents (approximately 40% w/w). Yields of biodiesel were consistently higher when starting from the Isochrysis wet paste (12% w/w vs. 7% w/w), which can be traced to lower amounts of hexane algal oil obtained from the powdered Isochrysis product.

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

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

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. ...

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 ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

用于表征土壤微生物的脂质提取和分析方法在许多样品的实验中

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

生物化学

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

生物学

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

含脂脂的微流体生产-温度敏感脂质体

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

Efficiency of Liquid-liquid Extraction

Source: <a href="https://jove.unicartagenaproxy.elogim.com/author/Kerry+M._Dooley">Kerry M. Dooley</a> and Michael G. Benton, Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA
Liquid-liquid extraction (LLE) is a separation technique used instead of distillation when either: (a) the relative volatilities of the compounds to be separated are very similar; (b) one or more of the mixture components are temperature sensitive even near ambient conditions; (c) the distillation would require a very low pressure or a very high distillate/feed ratio.1The driving force for mass transfer is the difference in solubility of one material (the solute) in two other immiscible or partially miscible streams (the feed and the solvent). The feed and solvent streams are mixed and then separated, allowing the solute to transfer from the feed to the solvent. Normally, this process is repeated in successive stages using counter-current flow. The solute-rich solvent is called the extract as it leaves, and the solute-depleted feed is the raffinate. When there is a reasonable density difference between the feed and solvent streams, extraction can be accomplished using a vertical column, although in other cases a series of mixing and settling tanks may be used.
In this experiment, the operational goal is to extract isopropanol (IPA, ~10 - 15 wt. %, the solute) from a mixture of C8-to-C10 hydrocarbons using pure water as solvent. A York-Scheibel type (vertical mixers and coalescers, one each per physical stage) extraction column is available. Like most extractors, the overall efficiency (number theoretical stages/physical stages) of this column is quite low, especially in comparison to many distillation columns. The low efficiencies arise from both slow mass transfer (two liquid resistances instead of one as in distillation) and often also from maldistribution of the phases. The effect of agitator speed on both the solute recovery in the extract and the overall column efficiency will be evaluated.

Liquid-liquid Extraction and York-Scheibel Column Operation

液-液萃取效率

Source: Kerry M. Dooley and Michael G. Benton, Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA
Liquid-liquid extraction ...