The authors would like to acknowledge Aspen Walker at the University of Pennsylvania for her assistance in reactor maintenance and sample collection.

1.反应器组件<ol><li>根据图 1中的示意性安排的反应器组成。 <ol><li>放置反应器（R）上的混合板，一个搅拌棒加入到反应器中。放置再循环罐（RT）的搅拌盘和反应器的旁边，以使罐的流出物（顶部）端口被引向实验室工作台的边缘。 </li><li>将废液容器（W）再循环罐（RT）的流出物（顶部）端口下面。放置进料罐（FT）旁边的再循环罐（RT）。 注：料罐为5 L.总容量</li></ol></li><li>固定反应器（R）抗倾倒用适当大小的支架和夹具。同样地，固定再循环罐（RT）以防止移动。 </li><li>将氯丁橡胶蠕动泵管中循环（泵A）和饲料（泵B）泵头。请参阅材料表附加管道规格。安装泵头在泵驱动用螺钉proviDED与泵的驱动器。 </li><li>泵A的管连接到反应器，并再循环罐的端口。插入泵B的管的端部进料罐和再循环罐。顶部反应器端口连接到循环罐与管道。适用夹具到管道在反应器端口。 注：光合社区可能从灯提供人工照明中受益。 </li></ol> 2.制备储备溶液，进水/进料溶液和藻类孕育剂<ol><li>制备矿物原液。以下添加到1L的量瓶中，用500ml去离子水：200克碳酸氢钠，40个克磷酸二氢钾，4克硫酸镁，4克氯化铁，4克氯化钙，1克氯化铜，1克钴六水氯化，1克氯化镍六水合物，1克硫酸锌七水合物。添加额外的400ml去离子水。涡流有力地促进盐溶解。继迪斯盐的olution，加去离子水使溶液的总体积达到1L。 </li><li>制备氨原液。在1L容量瓶中，溶解38.214克氯化铵在约900毫升去离子水。溶解后，加去离子水使总体积达1000毫升 注：1 ml储备溶液稀释至1升产生一个10毫克的L -1 铵态氮溶液。 </li><li>制备硝酸盐原液。在1L容量瓶中，溶解72.413克硝酸钾在约900毫升去离子水。溶解后，加去离子水使总体积达1000毫升 注：1 ml储备溶液稀释至1升产生一个10毫克的L -1 NO 3 - -N溶液。 </li><li>准备饲料/进水的解决方案。为了使含有20毫克的L原料溶液-1 NH 4 + -N和20毫克的L -1 NO 3 - -N，稀释加入2mlmmonia原液和2ml硝酸原液至1L总体积。在稀释之前，添加0.5 ml的矿物溶液/ L的溶液正在取得。制备5升进水中总启动反应器中。 </li><li>准备藻类接种。 <ol><li>收集的水的大体积（至少10升）从含藻类水体如河流或池塘。允许藻类留下不受干扰24小时的水样沉降。 </li><li>倒出并丢弃清晰（非藻类含有）水在样品的顶部，样品瓶内留下浓缩的藻类悬浮液。组合来自所有样品的藻类悬浮液到一个容器中，并重复所述沉降和倾析的步骤。 </li><li>测量所述浓缩试样中的生物量。 <ol><li>干纸真空过滤器（0.45微米MCE真空过滤器）和铝称量船O / N中已设置为103℃后，在干燥器降温为在室温30分钟测量共同的烘箱mbined过滤器的质量和重量的船。 </li><li>真空过滤器20毫升浓藻液，并返回过滤器和权衡船烤箱烘干O / N。 </li><li>测量过滤器的组合质量和称重舟。计算出浓缩的样品中生物量密度。 注：水样的总体积，调查将需要收集将取决于水源水体。 </li></ol></li></ol></li></ol> 3.播种并启动反应堆<ol><li>加将750ml进料溶液到反应器中。填再循环罐用500ml进料溶液。 </li><li>使用长吸管轻轻添加包含反应器的底部附近1.5克藻类的接种悬浮液。允许接种物沉降到反应器的底部，确保此通过目视观察，进行到下一步骤之前。 </li><li>一旦细胞已经解决，除去管夹，打开泵A以缓慢的流速（10革命ns（最小值）-1 /38毫升分钟-1）。空气截留在管道将被排出到反应器中。 注：加入750毫升反应器将阻止由泵干扰的任何生物量从离开反应器。挤压管，以确保所有的空气被排出。 </li><li>逐渐进料溶液添加到再循环罐作为溶液泵入反应器。继续加入直到反应器和再循环罐是在容量和流出物开始退出经由顶部端口再循环罐。 注：进料溶液的体积要添加到再循环罐将随加入到反应器中的接种物的体积。 </li><li>倒入剩余进料溶液到进料罐中。 </li><li>设置循环泵（泵A）以19转分钟-1，建立72.5毫升分钟的回流量-1。观察藻类开始从反应器底部到放样。使用在反应器的灰度等级，确定藻类双向omass区域高度。确保高度是在进行到下一步之前，恒定。 </li><li>打开混合板在非常低的速度;的1或2的设定是适当的启动。混合棒将有助于进一步放样生物量，但攻击性的混合会导致藻类离开反应器，进入再循环罐，和在流出物离开。设定搅拌速度在建立在反应器（图2A）内的一个明确的藻类边界所需的设置;藻类生物量区应在高度约10-15厘米。 </li><li>观察藻插头和反应器流体之间的边界清楚后开始进料泵。设置泵25转分钟-1，确立为1.5ml min的流速-1。观察反应器中的流体出口的流出物端口由于重力和位移引起的传入流入流。 </li></ol> 4.样品采集和分析<ol><li>开展样品采集活动PRI或在反应器系统上执行维护。每天收集20毫升污水和进水样品。收集流出物试样从循环槽内。直接从进料罐收集流入样品。 </li><li>真空过滤样品以去除悬浮之前存储和分析的固体。 </li><li>存储的进水和出水样品在-20°C，直到进一步的分析。限制冻融循环样品进行的数量。如果需要的话，可将样品分成等分部分，以保持样品的完整性。 </li><li>进行样品分析，硝酸盐，亚硝酸盐和氨使用标准技术17。 注意：利用离子色谱法（IC）的作者，以产生本文中所呈现的结果。请参阅材料表规范。 </li></ol>

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D., Bentlage, B., Gibbons, T. R., Bachvaroff, T. R., Delwiche, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metatranscriptome+profiling+of+a+harmful+algal+bloom.">Metatranscriptome profiling of a harmful algal bloom.</a> Harmful Algae. 37, 75-83 (2014).</li></ol>

The authors have nothing to disclose and declare that they have no competing financial interests.

本节将首先解决可能的操作问题，以及使用不同的微生物群落所需要的协议变化的讨论。这种反应器设计的优点将讨论的，包括以管理控制氧通量的和在反应器内形成的高密度的絮凝物的能力。目前的挑战和调查可能的途径也将被提及​​。 协议的细微差别和变化 HDBRs种植不同类型的文化中的操作需要运营协议的微小变化，这取决于被调查的物种。充分的混合和...

城市废水通常治疗活性污泥法中，以减少悬浮固体（SS），生物需氧量（BOD），有机和无机氮和磷含量5,6。活性污泥处理，二次处理废水的装置，工作需要在一个曝气池填充有传入的废水和再循环的异养微生物的混合液的有机碳的氧化（通常称为活性污泥）5-7。该混合液，然后进入一个相当大的澄清器（沉淀池）其中污泥沉降更容易收集，要么被布置或再循环回到曝气池，而澄清，处理后的废水可以继续三级处理或消毒被释放到前受纳水体5-7。在辅助净化器的处理过的废水和固体（污泥）的高效分离为一个是正确功能所必需tewater处理系统，因为任何活性污泥不断超越澄清会增加废水5-8 BOD和SS。 对于二级处理的废水，其减少或消除需要大澄清罐，包括附装生长（生物膜）的反应器，膜生物反应器（MBR）的，与颗粒污泥的反应器有许多替代生物过程存在。在生物膜反应器中，形成生物膜，其中土壤微生物天然骨料和附上作为层在固体表面上，允许生物量保留和积累，而不需要一个澄清槽。生物膜反应器可分为三种类型：填充床反应器，流化床反应器，和生物转盘。填充床反应器，诸如滴滤器和生物塔，利用一个固定的固体生长表面5,6。流化床反应器（快堆）依赖于微生物的附着于颗粒，如砂，粒状活性炭（GAC），或玻璃珠，其通过高向上流速9,10保持悬浮状态。旋转生物反应器依赖于形成在安装于旋转轴媒体允许生物膜的生物膜被交替地暴露于空气和液体被治疗5,6。膜生物反应器使用的膜过滤单元，无论是在生物反应器（浸没配置）或经由再循环（侧流配置）5,11外部。该膜用于实现生物质和固体颗粒从处理后的液体11,12良好的分离。颗粒污泥反应器是上流式反应器中，速度13微生物极为致密，沉降颗粒的形成发生时，他们暴露在高肤浅的空气向上流动。 作为另一替代活性污泥法，一种新型的上流式反应器系统，现在被称为高密度生物反应器（卧床），为designeD和通过销售和Shieh（2006）建研究COD去除率由活性污泥从在低F / M条件合成废物流是已知会引起的沉降污泥差形成（即，膨胀污泥）1,7,14。所利用的卧床系统改性流化床反应器中，通常由一个上流式反应器和一个外部再循环罐的。流化床反应器一般操作与再循环流的流速足够高以保持生物膜生长基质悬浮但足够低以使生物膜覆盖的衬底被保留。不像流化床反应器中，卧床描述在销售和Shieh（2006）中使用相对低的循环物流的流速其中，随着外部通气，防止了反应器1内形成的生物量区的中断。随后的研究证明了这一点反应堆设计的能力，使用硝化/反硝化细菌3,4成功治疗了一系列的氮通量。在所有的螺栓IES的卧床内的稳定，生物质致密区的形成不再需要外部絮凝/沉淀工艺1-4。 正如我们在这里报告，使用卧床的生长致密培养物也已在光生物反应器（PBR）配置为藻类的培养试验。我们讨论的好处和这种新颖的反应器系统的藻类培养缺点及其潜在用于克服大的障碍与生物质收获（即，良好的固-液分离15,16）相关联的藻类生物燃料的商业化。以下协议描述了安装，启动，来自样品，并保持与藻类感兴趣的微生物群落的卧床所需的步骤。在异养和硝化/反硝化文化的启动和运行方案的变化也将被提及​​。最后，一​​般的优点，缺点，以及这种新型反应器设计未知数将突出显示。

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Aeration stone</td>
			<td style="width:96px;">Alita</td>
			<td style="width:119px;">AS-3015C</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Aerator</td>
			<td style="width:96px;">Top Fin</td>
			<td style="width:119px;">Air-1000</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Ammonium chloride</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">A9434</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Anion analysis column</td>
			<td style="width:96px;">Shodex</td>
			<td style="width:119px;">IC SI-52 4E</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">Beaker (600 mL)</td>
			<td style="width:96px;">Corning Pyrex</td>
			<td style="width:119px;">1000-600</td>
			<td style="width:259px;">Used as mixing vessel (MV). Addition of hose barbs at the bottom and 500 mL levels. Outside diameter of hose barbs 3/8&#34;.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Calcium chloride</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">C5670</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Cation analysis column</td>
			<td style="width:96px;">Shodex</td>
			<td style="width:119px;">IC YS-50</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Cobalt chloride hexahydrate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">C8661</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Copper chloride</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">222011</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Ferric chloride</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">157740</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Filter (vacuum)</td>
			<td style="width:96px;">Fisherbrand</td>
			<td style="width:119px;">09-719-2E</td>
			<td style="width:259px;">0.45 um membrane filter, MCE, 47 mm diameter</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">Graduated cylinder (1000 mL)</td>
			<td style="width:96px;">Corning Pyrex</td>
			<td style="width:119px;">3025-1L</td>
			<td style="width:259px;">Used as reactor vessel (R). Addition of hose barbs at bottom, 500 mL, and 1 L levels. Outside diameter of hose barbs 3/8&#34;.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">HPLC/IC</td>
			<td style="width:96px;">Shimadzu</td>
			<td style="width:119px;">Prominence</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Magnesium sulfate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">M2643</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Masterflex L/S variable speed drive</td>
			<td style="width:96px;">Masterflex</td>
			<td style="width:119px;">07553-50</td>
			<td style="width:259px;">Drive for recycle and feed pumps (2 needed)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Nickel chloride hexahydrate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">N6136</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Potassium nitrate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">P8291</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">(Monobasic) Potassium phosphate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">P5655</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Pump head</td>
			<td style="width:96px;">Masterflex</td>
			<td style="width:119px;">07018-20</td>
			<td style="width:259px;">Recycle pump head</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Pump head</td>
			<td style="width:96px;">Masterflex</td>
			<td style="width:119px;">07013-20</td>
			<td style="width:259px;">Feed pump head</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Pump tubing</td>
			<td style="width:96px;">Masterflex</td>
			<td style="width:119px;">6404-18</td>
			<td style="width:259px;">Recycle pump tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Pump tubing</td>
			<td style="width:96px;">Masterflex</td>
			<td style="width:119px;">6404-13</td>
			<td style="width:259px;">Feed pump tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Sodium bicarbonate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Zinc sulfate heptahydrate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">Z0251</td>
			<td style="width:259px;"></td>
		</tr>
	</tbody>
</table>

a novel bioreactor for high density cultivation of diverse microbial communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

该卧床被用于培养藻类超过进水氨和硝酸盐浓度的几种比例，同时保持在饲料中总含氮量在40毫克-NL -1。进水和出水样品的日常拍摄;生物质密度取样在各条件的开始和结束。在反应器的平均时间为3-5天达到稳态平衡条件改变之后。在宽范围的条件下流入一个独特的生物量区成立，所观察到的以前的研究（ 图2）。在卧床的藻类培养中发现，除去平均总氮品种的18.4％的饲料（N =...

Watch this Scientific Journal Video about A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities at JoVE.com

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

一种新型生物反应器为不同的微生物群落的高密度栽培

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

a-novel-bioreactor-for-high-density-cultivation-diverse-microbial

Research

JoVE Journal

Bioengineering

16.8K 次观看.  Drexel University. A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

视频: A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

科研

生物工程

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

一个台式生物反应器的操作

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

一种新型声带生物反应器的构建与鉴定

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

培养哺乳动物细胞使用一次性使用气动生物反应器系统

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

用于动态生长测试的温度，光照和pH监测的台式藻类光合生物反应器的构建和设置

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

中国仓鼠卵巢细胞培养中高通量自动化微生物反应器工艺优化

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...