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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-neutral+ph+increased+n-caprylate+production+in+a+microbiome+with+product+inhibition+of+methanogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+production+of+medium-chain+carboxylates+through+chain+elongation:+An+overview.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+medium-chain+triglycerides+in+foods.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+environmental+impacts+of+palm+oil+in+context.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coconut+oil,+conservation+and+the+conscientious+consumer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eco-mimicry+opens+new+doors+for+bioprocess+engineers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heterogeneous+microbial+consortium+producing+short-chain+fatty+acids+from+lignocellulose.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement,+application,+and+effect+of+oxygen+in+microbial+fermentations:+Focusing+on+methane+and+carboxylate+production.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+highly+specific+and+productive+process+for+n-caproic+acid+production:+Applying+lessons+from+methanogenic+microbiomes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upgrading+syngas+fermentation+effluent+using+Clostridium+kluyveri+in+a+continuous+fermentation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+n-caproic+acid+production+from+yeast-fermentation+beer+in+an+anaerobic+bioreactor+with+continuous+product+extraction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+recovery+of+bio-based+carboxylic+acids.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+extraction+for+in-situ+carboxylate+recovery+from+mixed+culture+fermentation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+extraction+of+medium-chain+carboxylic+acids+at+a+semi-pilot+scale.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+biphasic+extractive+fermentation+for+hexanoic+acid+production+from+sucrose+by+Megasphaera+elsdenii+NCIMB+702410.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+medium+chain+fatty+acid+productivity+using+chain+elongation+by+reducing+the+hydraulic+retention+time+in+an+upflow+anaerobic+filter.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoting+chain+elongation+in+mixed+culture+acidification+reactors+by+addition+of+ethanol.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distillation+of+natural+fatty+acids+and+their+chemical+derivatives.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chain+elongation+with+reactor+microbiomes:+Upgrading+dilute+ethanol+to+medium-chain+carboxylates.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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%,&#160;</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.&#160;</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&#160; 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&#34; NPT 2.5&#34; Face Dial</td>
			<td>NA</td>
			<td>XJ-219</td>
			<td>Any comparable pressure gauge covering 0-60 psig range will suffice.&#160;</td>
		</tr>
		<tr>
			<td>Trioctylphosphine oxide (TOPO)&#160;</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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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

Research

JoVE Journal

Bioengineering

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

888 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

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

高通量，自动化的DNA和RNA的提取从临床样品使用TruTip技术常见的液体处理机器人

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

科研

生物工程

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

细胞的浸入式印刷到改性表面采用连续流动Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

电子舌生成连续的识别模式的蛋白质分析

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

中空纤维生物反应器的<em&gt;在体内</em&gt;般的哺乳动物组织培养

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

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 ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

牺牲纤维膜细胞培养生产细胞外基质纤维

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

离子通道掺杂脂质膜组成的生物分子记忆器的组装与表征

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...