我们想感谢罗杰 Raiche 和大卫 McCrory 允许我们进入雪松和咨询的地点, 长期孵化。我们还感谢雪松现场船员在2013-2014 赛季: 四野铃木, 俊一, 格雷格 Wanger, 格雷森, 柏林和马修 Schechter。另外多亏了四野铃木和赫斯·贝克 Kuenen 的深入研究和培养支持。这项工作是通过向日本促进科学学会 (jsp) KAKENHI 赠款编号17H04969 和26810085的年轻科学家 a 和 B 资助的, 以及日本医学研究和发展局 (17gm6010002h0002) 提供的。美国提供的资金由美国全球海军研究办公室 (N62909-17-1-2038) 和黑暗能源生物圈调查中心 (德碧) (OCE0939564) 和美国宇航局天体生物学研究所-生活地下 (泥路) (NNA13AA92A)。这项工作的一部分是作为日本促进科学学会的一部分进行的: Kazuhito 桥本实验室的东京大学 PE15019 的短期博士后奖学金。

1. 环境孵化用双电极系统的构建
<ol><li>
阳极材料的制备和碳毡电极的处理 (图 2)。
	<ol>
<li>根据期望的生物量富集, 将碳毡切割成相等的尺寸。将每个电极浸泡在90% 乙醇中30分钟, 然后用去离子水冲洗至少8次, 每次冲洗后 sonicating 1 分钟。</li>
		<li>在1米 HCl 中清洗两次电极, 每洗一次至少搅拌12小时。</li>
		<li>在温暖的 (37 °c) 的烤箱中干燥电极 6–12 h 或直到没有液体。</li>
		<li>在聚四氟乙烯板 (不粘表面) 上, 用石墨环氧树脂将电极连接到钛丝上。 注: 我们使用钛线, 因为它的高耐受性好氧腐蚀。</li>
		<li>烘烤电极在120°c 为6小时。</li>
		<li>用欧姆计测试钛线和碳毡之间的电阻, 并确认导线和毡电极之间的电阻小于5欧姆。</li>
	</ol>
</li>
	<li>
铂对阴极材料制备碳毡电极的电镀
	<ol>
<li>浸没碳毡电极, 在步骤1.1 中制备, 在2米的 KOH, 在一个玻璃容器中至少12小时。</li>
		<li>电化学清洗时, 将电极作为工作电极 (我们) 放置在三电极反应器中, 同时还可容纳参考 (RE) 和反电极 (CE)。用鳄鱼夹子把我们、恒电位仪和 CE 连接起来。确认所有与欧姆计的连接。 注: 我们使用了银/AgCl (氯化钾饱和) 电极和铂丝作为 RE 和 CE, 分别。</li>
		<li>使电极在 1.0 V 与含2米的电解质溶液中600s 的银/AgCl (使用足够的量淹没整个电极)。从电化学反应器中取出电极 (由玻璃制成)。将电极在去离子水中冲洗至少8次, 每次冲洗后 sonicating 1 分钟。干电极在100°c 至少12小时。</li>
		<li>要准备电镀溶液, 加入100克柠檬酸, 5 克硫酸钠, 2 克的 hexachloroplatinate (IV) 六水合物到1升的2米硫酸。</li>
		<li>在步骤 1.2. 1–1.2 2 中制备的重量清洗和干电极, 然后覆盖在电镀溶液中的电极, 如步骤1.2.3 中所准备。油脂实验在电镀溶液中的电极每三十年代三次。</li>
		<li>电镀电极通过中毒电极电位在-0.2 V 对 Ag/AgCl 为460s 在镀液。在去离子水中漂洗两次电极, 并丢弃铂废料。</li>
		<li>在去离子水中冲洗电极至少3次, 每次冲洗后 sonicating 二十年代。无需超声波冲洗至少三次。</li>
		<li>干电极在100°c 至少12小时的重量电极, 以量化电镀铂的碳毡电极。</li>
	</ol>
</li>
</ol>2. 双电极系统的施工与安装
<ol><li>
对自然环境中每个电极的安装部位进行了调查。
	<ol>
<li>使用溶解氧探针测定氧浓度。</li>
		<li>检查现场的深度配置文件。 注: 所需的阳极环境条件是一致的水合和缺氧。如果需要, 消除氧光合作用的影响, 屏蔽阳极从光。阴极放置的理想条件是持续的水合, 并接近地表水的好氧。如有必要, 附加浮子以保持阴极表面接触。</li>
	</ol>
</li>
	<li>
燃料电池型2电极孵化系统的构建
	<ol>
<li>通过扭曲两条线, 将所需长度的绝缘导线与电极 (一个阳极和一个铂电镀阴极) 的引线连接起来。覆盖与防水蜡的连接和进一步保护使用船舶级热收缩管。</li>
		<li>用已知电阻的电阻器将两根导线与阴极和阳极连接。 注: 对于生物系统, 较低的电阻 (10 到1000Ω) 导致更一致的生物活动。如果需要, 高电阻电阻将防止生物活动, 作为一个负控制。为了防止电阻和引线之间的任何连接受到腐蚀, 我们用热收缩管保护它们。</li>
	</ol>
</li>
	<li>
随着时间的推移, 电压和温度测井的测量。
	<ol>
<li>检查电阻两端之间的电压, 以从燃料电池反应中估算出当前的产量。</li>
		<li>使用数据记录电压表测量与阳极和阴极相关的连接 (请参阅制造商协议), 以此衡量电压差。 注意: 同时进行温度数据记录是可选的, 但是这些信息可以帮助将当前的变化与非生物相关, 而不是生物学上的波动。</li>
	</ol>
</li>
	<li>
数据记录器和电气连接的保护
	<ol>
<li>使用固定和/或塑料袋, 以保护记录器和所有的电气连接从雨中。</li>
		<li>拧紧塑料袋和电缆, 以防止强风。如图 1所示。</li>
	</ol>
</li>
</ol>3. 从自然环境中采集电极样品
<ol><li>为防止阳极试样因氧污染而损坏, 在厌氧条件下采集电极。</li>
	<li>在收集电极样品前至少30分钟, 将试管放在厌氧位置。例如, 将试管和盖子分别放在池塘底部, 使瓶子内无氧。</li>
	<li>用线切割机从电极上切割钛铅, 将电极样品轻轻地收集到试管中, 并将其密封在厌氧水区。为了保持样品新鲜, 样品收集后立即将样品保存在4摄氏度。 注: 或者, 电极可以直接转移到缺氧 (N2清除) 介质。我们用的是雪松培养基 (铃木等。11) 这是从现场测量的水地球化学设计的, 经修订, 为微生物生长提供足够的养分。这一媒体被修改为不同的实验室实验。</li>
</ol>4. 对目前生产和 DNA 分析的实验室确认
<ol><li>
电极上附着的微生物联营集团目前生产能力的电化学确认。
	<ol>
<li>构造一个电化学反应器14,15与取样电极, 铂导线, 和 Ag/AgCl (氯化钾饱和) 电极作为我们, CE, 和重新, 分别在一个厌氧室。用含有可溶性碳水化合物电子捐献者的雪松培养基填充电化学反应器。</li>
		<li>平衡电极电位在 +0.2 V 与 Ag/AgCl 和测量当前生产。</li>
	</ol>
</li>
	<li>
用微生物 dna 试剂盒从电极样品中提取 dna (见材料表)。
	<ol>
<li>用70% 乙醇清洗厌氧手套箱内, 在铝箔上放一盘消毒皿。 注: 厌氧室保持氧气浓度低于 1 ppm, 维持一个氢气氛在周围〜 2–3%, 以清除氧气存在钯催化剂。</li>
		<li>在手套盒中打开电化学反应器, 将样品电极放在盘子上, 然后切到一个尺寸, 以适合在 DNA 套件中使用的管子。继续执行制造商的协议。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

在本文的研究中, 我们展示了一个微生物联合体的富集, 与原位电流的生产联系在一起。目前, 该系统在短期和长期的时间尺度上支持微生物活性的观察模式。构建功能性双电极 (燃料电池型) 系统的关键步骤是识别和利用环境中具有稳定水位和氧浓度的位置。阴极在空气水界面接触氧气, 而阳极则保持在厌氧条件下, 电极电位差促进 EET 细菌的厌氧呼吸。
我们观察了环境?...

一些矿物还原微生物被证明利用固相矿物作为终端电子受体, 通过细胞外电子传输 (EET) 过程, 通过氧化还原酶1将电子传导到细胞的外部。EET 是关键, 不仅对微生物矿物过程, 但也应用能源和环境技术, 如微生物燃料电池2, 电极合成3, 和生物修复4。新的 EET 细菌是高度追求, 并已广泛研究从基本或应用的角度5。然而, 我们对这些细菌的生态或生物地球化学意义的洞察力有限。大多数 EET 的微生物是从水, 沉淀物, 或厌氧的消化器, 利用固体电子受体, 如 MnO2, Fe2O3或准备电极在实验室6,分离出来的,7,8. 然而, 这些方法往往产生类似的联营集团, 可能会错过可能主宰低能量或低生物量系统的更敏感分类群, 从而使这些微生物适应实验室或无菌文化环境的能力降低9.通常对于低生物量环境, 大量的水从一个地点被过滤, 以集中细菌细胞。然而, EET 的细菌往往表现出厌氧代谢, 因此氧气暴露可能进一步抑制或阻止他们的耕种。替代的现场方法, 以集中细胞而不暴露于氧气, 可以促进分离 EET 细菌。在这里, 我们报告的安装细节的现场电化学技术, 以丰富 EET 能力的微生物在很长一段时间内, 而不需要外部电源。
使用我们的 electrocultivation 实验从一个高碱性的春天在加利福尼亚北部, 雪松10, 我们描述这种现场电化学技术。serpentinization 在雪松中的地球化学特征受到地下的影响。弹簧高度还原, 氧气浓度低于在空气水界面下检测的限度, 突出了在这个功能性缺氧环境11通过 EET 微生物能源生产的潜力。然而, 没有证据支持 EET 的微生物从雪松 (在 16S rRNA 或宏基因组分析)。尽管这种环境被认为是电子受体有限的, 但利用不溶性矿物作为终端电子受体的潜力, 包括矿物, 如由 serpentinization 产生的铁霸矿物 (即,磁铁矿), 尚未广泛调查12。因此, 我们部署了我们的电化学系统在营地春天, 在雪松高 pH 值的春天, 以丰富的 EET 微生物 (图1)13。

<table><tbody><tr><td>Carbon felt sheet</td><td>n/a</td><td>n/a</td><td>Used for anode and cathode</td></tr><tr><td>Titanium wire</td><td>The Nilaco Cooporation</td><td>TI-451485</td><td>Used to construct fuel cell system</td></tr><tr><td>Graphite epoxy</td><td>Electrolytica lnc.</td><td>n/a</td><td>Used to connect the&lt;br/&gt; electrodes and Ti wire</td></tr><tr><td>Drying oven</td><td>Yamato</td><td>DY300</td><td>bake the electrode to&lt;br/&gt; solidify conductive graphite epoxy</td></tr><tr><td>Digital multi meter</td><td>Fluke</td><td>616-1454</td><td>to check the ohmic value&lt;br/&gt; of resistance</td></tr><tr><td>Dissolved oxygen probe</td><td>Sper Science</td><td>#&amp;nbsp; 850045</td><td>to check the oxygen&lt;br/&gt; concentration in the environments</td></tr><tr><td>Resistor</td><td>Sodial</td><td></td><td>Used to construct fuel cell&lt;br/&gt; system</td></tr><tr><td>Conducting wire</td><td>Pico</td><td>81141s</td><td>Used to construct fuel cell&lt;br/&gt; system</td></tr><tr><td>Voltmeter and Data logger</td><td>T&amp;amp;D corporation</td><td>VR-71</td><td>Used for data recording</td></tr><tr><td>Hydrogen Hexachloroplatinate(IV) Hexahydrate</td><td>wako</td><td>18497-13-7</td><td>Used for electropolation</td></tr><tr><td>Citric acid</td><td>Wako</td><td>038-06925</td><td>Used for electropolation</td></tr><tr><td>Sulfuric acid</td><td>Wako</td><td>192-04696</td><td>Used for electropolation</td></tr><tr><td>HCl</td><td>Wako</td><td>083-01095</td><td>Used for electrode washing</td></tr><tr><td>Glass cylinder</td><td>N/A</td><td>N/A</td><td>Custom-made, used as the electrochemical reactor</td></tr><tr><td>PTFE cover and base</td><td>N/A</td><td>N/A</td><td>Custom-made, used as a cover and a foundation of the electrochemical reactor</td></tr><tr><td>Buthyl rubber</td><td>N/A</td><td>N/A</td><td>Custom-made, inserted between each component of electrochemical reactor</td></tr><tr><td>Septa</td><td>GL Science</td><td>3007-16101</td><td>Used as an injection port of electrochemical reactor</td></tr><tr><td>Indium tin-doped oxide (ITO) electrode</td><td>GEOMATEC</td><td>No.0001</td><td>Used as a working electrode, 5&amp;Omega;/sq</td></tr><tr><td>Ag/AgCl KCl saturated electrode</td><td>HOKUTO DENKO</td><td>HX-R5</td><td>Used as a reference electrode, &amp;Phi;0.30mm</td></tr><tr><td>Platinum wire</td><td>The Nilaco Cooporation</td><td>PT-351325</td><td>Used as a counter electrode</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Wako</td><td>191-01305</td><td>Used for The Cedars Media (CMS)</td></tr><tr><td>CaCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Wako</td><td>030-00385</td><td>Used for CMS</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Wako</td><td>011-03015</td><td>Used for CMS</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;&amp;bull; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>135-00165</td><td>Used for CMS</td></tr><tr><td>NaOH&amp;nbsp;</td><td>Wako</td><td>198-13765</td><td>Used for CMS</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Wako</td><td>194-03355</td><td>Used for CMS</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Wako</td><td>164-04295</td><td>Used for CMS</td></tr><tr><td>CABS</td><td>SANTA CRUZ</td><td>SC-285279</td><td>Used for CMS</td></tr><tr><td>Incubator</td><td>TOKYO RIKAKIKAI CO. LTD.</td><td>LTI-601SD</td><td>Used for precultivation</td></tr><tr><td>Autoclave machine</td><td>TOMY SEIKO CO. LTD.</td><td>LSX-500</td><td>Used for sterilization of the electrochemical reactor and the medium</td></tr><tr><td>Clean bench</td><td>SANYO</td><td>MCV-91BNF</td><td>Used to prevent the contamination of the electrochemical reactor and the medium with other microbes</td></tr><tr><td>Centrifuge separator</td><td>Eppendorf</td><td>5430R</td><td>Rotational speed upto 6000&amp;times;g is required</td></tr><tr><td>Nitrogen gas generator</td><td>Puequ CO. LTD.</td><td>PNTN-2</td><td>Nitrogen gas cylinder can also be used instead of gas generator</td></tr><tr><td>UV-vis spectrometer</td><td>SHIMADZU</td><td>UV-1800</td><td>Used for optimization of cell density</td></tr><tr><td>Potentiostat</td><td>BioLogic</td><td>VMP3</td><td>Used for biofilm formation and kinetic isotope effect experiments</td></tr><tr><td>Thermal water circulator</td><td>AS ONE</td><td>TR-1A</td><td>Used for maintanance of temperature of electrochemcial reactor</td></tr><tr><td>Faraday cage</td><td>HOKUTO DENKO</td><td>HS-201S</td><td>Used for electrochemical experiments</td></tr><tr><td>Anaerobic Chamber</td><td>COY</td><td>TypeB (Vinyl)</td><td>TO conduct experiments&lt;br/&gt; under anaerobic condition</td></tr><tr><td>Ultraclean DNA Extraction kit</td><td>MoBio</td><td></td><td></td></tr></tbody></table>

self-standing electrochemical set-up to enrich anode-respiring bacteria on-site

厌氧呼吸耦合电子运输到不溶性矿物 (称为胞外电子传输 [EET]) 被认为是关键的微生物能源生产和持久性在许多地下环境, 特别是那些缺乏可溶性终端电子受体。虽然 EET 的微生物已成功地从不同的环境中分离出来, 但 EET 的细菌多样性仍然不太清楚, 特别是在难以取样、低能或极端环境下, 如许多地下生态。在这里, 我们描述了一个现场电化学系统, 以丰富的 EET 能力的细菌使用阳极作为呼吸终端电子受体。这个阳极连接到能够催化非生物氧还原的阴极。将此方法与使用恒电位仪中毒电极电位的 electrocultivation 方法进行比较, 双电极系统不需要外部电源。我们提供了一个例子, 我们的现场浓缩利用在一个碱性池塘在雪松, 一个地面 serpentinization 遗址在加利福尼亚州北部。以前试图培养的矿物质还原菌是不成功的, 这可能是由于该网站的低生物量的性质和/或低相对丰度的金属还原微生物。在实现双电极富集之前, 我们测量了溶解氧浓度的垂直剖面。这使我们能够将碳毡阳极和铂电镀碳毡阴极放在深度, 以支持厌氧和有氧过程, 分别。在现场孵化之后, 我们进一步丰富了实验室中的阳极电极, 并证实了与通常在雪松上观察到的表面附着或生物膜群落相比, 有明显的微生物群落。这种浓缩后来导致了从雪松中分离出的第一 electrogenic 微生物。这种现场微生物富集的方法有可能极大地增强从低生物量或难以取样生境的 EET 细菌的分离。

使用电压数据记录器成功地测量了当前生产的大约3月, 如图 3所示。这次被选择了, 因为它是春天的最长的稳定的潜伏期, 由于强的秋天降雨影响春天。较短的时期可能是充足的, 虽然一个更长的时期可能提供更强的生物量的丰富。在电化学孵化后, 我们确认了双电极系统的连接, 没有发现系统存在腐蚀的迹象。在低电阻 (1000 Ω) 的双电极系统中, 相对于...

Watch this Scientific Journal Video about Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site at JoVE.com

Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site

现场微生物富集或原位栽培技术可以促进分离难培养的微生物群, 尤其是低生物量或地球化学极端环境。在这里, 我们描述了一个电化学设置不使用外部的电源, 以丰富微生物菌株, 有能力的细胞外电子运输 (EET)。

自立电化学建立富集阳极 respiring 菌现场

Anaerobic respiration coupled with electron transport to insoluble minerals (referred to as extracellular electron transport [EET]) is thought to be ...

self-standing-electrochemical-set-up-to-enrich-anode-respiring

Research

JoVE Journal

Environment

7.6K 次观看.  National Institute for Materials Science. 现场微生物富集或原位栽培技术可以促进分离难培养的微生物群, 尤其是低生物量或地球化学极端环境。在这里, 我们描述了一个电化学设置不使用外部的电源, 以丰富微生物菌株, 有能力的细胞外电子运输 (EET)。

视频: Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

科研

环境科学

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

一种技术，屏幕美国榉木为抵抗山毛榉介壳虫（<em&gt;隐球菌fagisuga</em&gt;林德。）

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

Electrochemically and Bioelectrochemically Induced Ammonium Recovery

Streams such as urine and manure can contain high levels of ammonium, which could be recovered for reuse in agriculture or chemistry. The extraction of ammonium from an ammonium-rich stream is demonstrated using an electrochemical and a bioelectrochemical system. Both systems are controlled by a potentiostat to either fix the current (for the electrochemical cell) or fix the potential of the working electrode (for the bioelectrochemical cell). In the bioelectrochemical cell, electroactive bacteria catalyze the anodic reaction, whereas in the electrochemical cell the potentiostat applies a higher voltage to produce a current. The current and consequent restoration of the charge balance across the cell allow the transport of cations, such as ammonium, across a cation exchange membrane from the anolyte to the catholyte. The high pH of the catholyte leads to formation of ammonia, which can be stripped from the medium and captured in an acid solution, thus enabling the recovery of a valuable nutrient. The flux of ammonium across the membrane is characterized at different anolyte ammonium concentrations and currents for both the abiotic and biotic reactor systems. Both systems are compared based on current and removal efficiencies for ammonium, as well as the energy input required to drive ammonium transfer across the cation exchange membrane. Finally, a comparative analysis considering key aspects such as reliability, electrode cost, and rate is made.
This video article and protocol provide the necessary information to conduct electrochemical and bioelectrochemical ammonia recovery experiments. The reactor setup for the two cases is explained, as well as the reactor operation. We elaborate on data analysis for both reactor types and on the advantages and disadvantages of bioelectrochemical and electrochemical systems.

We demonstrate the extraction of ammonium from an ammonium-rich stream using an electrochemical and a bioelectrochemical system. The reactor setup, operation and data analysis are discussed.

电化学和Bioelectrochemically诱导铵恢复

Streams such as urine and manure can contain high levels of ammonium, which could be recovered for reuse in agriculture or chemistry. The extraction of ...

化学

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

生物化学

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

通过活生物膜表征电子传输

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

用于培养环境微生物的原位模拟厌氧方法

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

作者聚焦:通过原位培养揭开未知环境中陆地厌氧微生物的神秘面纱

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

一套用于培养环境获得性厌氧微生物的 原位 信息模拟培养基格式

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

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.

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 ...

生物工程

<meta charset="utf8"></head><body>DHNA 对 植物乳植杆菌 EET 进行计时安培分析

Characterizing Mediated Extracellular Electron Transfer in Lactic Acid Bacteria with a Three-Electrode, Two-Chamber Bioelectrochemical System

Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron acceptor. This electron acceptor can be an electrode and electrons can be delivered indirectly via a redox-active mediator molecule. Here, we present a protocol to study mediated EET in Lactiplantibacillus plantarum, a probiotic lactic acid bacterium widely used in the food industry, using a bioelectrochemical system. We detail how to assemble a three-electrode, two-chambered bioelectrochemical system and provide guidance on characterizing EET in the presence of a soluble mediator using chronoamperometry and cyclic voltammetry techniques. We use representative data from 1,4-dihydroxy-2-naphthoic acid (DHNA)-mediated EET experiments with L. plantarum to demonstrate data analysis and interpretation. The techniques described in this protocol can open new opportunities for electro-fermentation and bioelectrocatalysis. Recent applications of this electrochemical technique with L. plantarum demonstrated an acceleration of metabolic flux towards producing fermentation end-products, which are critical flavor components in food fermentation. As such, this system has the potential to be further developed to alter flavors in food production or produce valuable chemicals.

<meta charset="utf8"></head><body>用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

Here we present a protocol for characterizing mediated extracellular electron transfer (EET) in lactic acid bacteria using a three-electrode, two-chamber bioelectrochemical system. We illustrate this method with Lactiplantibacillus plantarum and the redox mediator 1,4-dihydroxy-2-naphthoic acid and provide a thorough description of the electrochemical techniques used to evaluate mediated EET.

自立电化学建立富集阳极 respiring 菌现场

摘要

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Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

一种技术，屏幕美国榉木为抵抗山毛榉介壳虫（

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电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

通过活生物膜表征电子传输

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式

一种新型生物反应器为不同的微生物群落的高密度栽培

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一套用于培养环境获得性厌氧微生物的 原位 信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的 原位 信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的 原位 信息模拟培养基格式

一种新型生物反应器为不同的微生物群落的高密度栽培

用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式