博士, s.m., L.M.T. 承认海军研究办公室 (奖 #N0001415WX01038 和 N0001415WX00195), 海军研究实验室, 海军研究实验室 Nanosciences 研究所;M.Y.E.-N。得到美国能源部赠款 DE-FG02-13ER16415 的支持。

1. 交叉微电极阵列 (IDA) 的制备
<ol><li>获取在导电基板上图案的商用 IDA 电极, 或者使用标准平版方法合成它们。28 注: IDA 尺寸和/或材料可以根据所需条件进行不同的实验。这里使用的 IDAs 是商业获得的, 包括两个交叉金电极图案上的玻璃基板连接到大电极垫在阵列的两端。电极被暴露, 而连接电极到大接触板的母线被涂上薄薄的绝缘材料。IDAs 在这里使用的是由两套10µm 和2毫米长金微电极带 (源和排泄) 间隔5µm 分开图案在平板玻璃表面。这里使用的 IDAs 有65个电极对 (130 个总交叉带)。交流带连接到阵列两端的电极垫片。</li>
	<li>电线和绝缘 IDA<ol>
<li>用导电银环氧树脂将电线连接到各大电极垫上。<ol>
<li>根据制造商的指示, 制备导电环氧树脂, 用于使用混合棒或吸管尖的特定环氧树脂 (指示可能因制造商而异)。</li>
			<li>在每个金垫上放一根电线, 用实验室胶带固定到位。用混合棒或吸管尖将银环氧树脂盖在电线和衬垫上。</li>
			<li>小心地移动到一个80°c 烤箱1小时的治疗或治疗根据制造商的建议, 为特定导电银环氧树脂使用。</li>
			<li>在环氧治疗后, 使用万用表确保电线端与垫片之间的电连接。导线和衬垫之间的电阻应 < 5 Ω。此外, 使用万用表来验证没有导电环氧树脂连接多个电极垫, 因为这可以缩短阵列。如果导电环氧树脂连接多个引线, 请使用一个抄写员来隔离引线。</li>
		</ol>
</li>
		<li>通过与热、电、防水绝缘材料的连接涂层, 使 epoxied 连接绝缘。阻燃聚氨酯树脂通常是合适的。<ol>
<li>卸下15毫升锥形离心管 (约2.5 毫升标记) 的尖端作为绝缘材料的模具。用21g 针在底部做两个小孔, 使导线伸出来。</li>
			<li>将 IDA 插入模具中, 通过模具底部的孔线。</li>
			<li>准备特定的绝缘材料。按照所得到的特定树脂提供的指示。</li>
			<li>将绝缘子注入模具, 使银环氧树脂完全覆盖 (〜2.5 毫升), 并允许根据制造商的规格干燥。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 电化学反应器的设置、测试和接种
<ol><li>设置电化学电池。<ol>
<li>将 IDA、反电极和参考电极插入电化学电池。 注: 使用的电化学反应器可以有很大的变化, 只要所有的电极都适合内部。考虑到反应堆的实际极限, 参考电极应尽可能接近工作电极。在这里, 我们使用一个单室反应器与参考电极〜2-3 厘米从工作电极。此外, 计数器电极应大于工作电极, 以确保它不限制在系统中。</li>
		<li>如果使用纯文化, 在反应器内用反电极消毒反应堆。在漂白剂中浸泡10分钟和无菌去离子水10分钟, 消毒参考电极. 对 IDA 进行消毒, 在5秒内浸泡在漂白剂中, 然后在插入反应器之前, 将十年代的无菌去离子水注入。</li>
		<li>用不育培养基填充电化学细胞, 适合生物膜生长。对于G. sulfurreducens17,18, 使用含富马酸的淡水介质。对于 biocathode MCL,9使用人工海水介质。9
</li>
		<li>将电极连接到 bipotentiostat。连接一个 ida 电极到工作铅 1, 其他 ida 电极到工作铅 2, 参考电极到参考导线, 和反电极到计数器引线。</li>
	</ol>
</li>
	<li>
IDAs 的电化学测试。 注意: 这个测试的主要目标是确保两个电极是电隔离的。所有的电化学技术都可用于控制 bipotentiostat 的软件中。<ol>
<li>执行控制电导率测试 (在生物膜生长之前), 以确保适当的 IDA 功能使用 bipotentiostat。<ol>
<li>测量每个电极的开路电位为1分钟。 注: 在某些仪器上, 使用恒电流收集程序并将电流设置为 0 mA, 必须实现开路电位。</li>
			<li>测量电极2的开路电位, 同时在 +0.2 伏至 +0.6 v (vs) 的电极上执行循环伏安法, 在 20 mV/秒。 注: 如果需要, 可以使用其他的边界和扫描速率。但是, 要避免产生氢气或氧气产生的电位。</li>
			<li>验证在2电极上测量的开路电位不会在 CV 使用恒电位仪软件时反映电极1的电位。 注: 电极的开路电位5月2日改变, 但应独立于电极1的电位。</li>
			<li>重复步骤2.2.1.1 通过 2.2.1.3, 除了控制电极2的电位和测量电极1的开路电位。</li>
		</ol>
</li>
		<li>使用 bipotentiostat 软件将工作电极的电位设定为相关活性生物膜所期望的生长电位。例如, 使用 +0.5 v (vs)地杆菌能 sulfurreducens 或 +0.31 v (vs) biocathode MCL。</li>
	</ol>
</li>
	<li>生长相关的活性生物膜<ol>
<li>使用标准无菌微生物技术, 从储存培养/富集所需的电化学活性微生物中接种电化学反应器。对于标准测试, 接种1:20 的比率 (接种到反应堆体积) 的 OD600 = 0.5 区域性。</li>
		<li>设置搅拌在反应器到期望的水平 (~ 200 rpm) 和孵化器或水浴到期望的温度根据生物膜的成长条件感兴趣。对于sulfurreducens, 请使用30°c 进行最佳增长。</li>
		<li>根据感兴趣的微生物的特定要求孵化系统, 直到生物膜桥分离两个电极的间隙。对于固定的G. sulfurreducens生物膜, 孵育7-10 天。biocathode MCL, 孵育7天。在每种情况下, 控制温度在30摄氏度。</li>
	</ol>
</li>
</ol>3. 电化学浇注实验
<ol><li>选择将用于确定浇口测量的电流潜在依赖性的实验参数。
	<ol>
<li>确定将应用于 IDA 以获得所进行的电流 (ISD) 与系统的栅极电位 (EG) 曲线的门电位范围。 注: 检查门电位范围应涵盖所有电位与可能的氧化还原活性。如果没有有关系统的信息可用, 应使用广泛的潜在范围 (-0.55 至 +0.6 V vs)。在研究的基础上, 利用试验和误差的方法对系统进行微调。门电位定义为: <img alt="Equation 1" src="/files/ftp_upload/54671/54671eq1.jpg"> 其中 eD是漏极电位, 而 eS是源电极的电位。所使用的门势范围受特定系统的要求和限制所限制。18 注: 由于这些过程可能会破坏生物膜, 因此应避免产生氧和氢演化的源和漏电位。</li>
		<li>确定源漏电压 (VSD), 它将用作通过胶片从源到排水管的电子传输的驱动力。源漏电压定义为: <img alt="Equation 2" src="/files/ftp_upload/54671/54671eq2.jpg"> 注意: 源漏电压应足够小, 以便 Isd与 Vsd线性缩放。17
</li>
		<li>选择一个扫描速率 (v), 在这种情况下, EG随与 ISD无关的时间线性变化, 以便系统可以近似为每个栅极电位的稳定状态。 注意: 扫描速率小于0.002 伏/秒通常用于生物系统。29,30
</li>
		<li>设置 bipotentiostat 软件, 以执行门的测量 (即扫门电位) 超过选定的范围, 在选定的源漏电压, 并根据所需的扫描速率基于上述考虑。 注意: 对于以前使用sulfurreducens的门控测量,17,19 EG =-0.55 v 到 0.6 v (vs), vSD = 0.01 v 或 0.1 v, v = 0.001 v/秒。对于 Biocathode MCL, EG = 0.25 v 到 0.7 v (vs), VSD = 0.002 V, v = 0.0002 V/秒。<ol>
<li>此外, 使用 vSD = 0.000 v (即在每个单独的电极上同时进行的 CV) 在步骤3.1.3 中选择的同一 V 上执行基线测量。</li>
		</ol>
</li>
		<li>使用3.1.4 中的条件进行浇口测量 (具有可溶性电子供体或受体存在) 和非周转 (无可溶性电子供体或受体) 条件。 注意: 非周转条件是有利的, 因为测量不被氧化或减少可溶性化合物的细胞代谢所掩盖, 虽然应该取得类似的结果, 无论使用后的条件减去背景电流 (在3.1.8 中详细说明)。<ol>
<li>使用与电极上细菌生长相同的反应器介质, 达到周转条件。该培养基含有可溶性电子供体, 例如用于G. sulfurreducens、17或受体的醋酸盐, 如 Biocathode MCL 的氧气。</li>
			<li>通过制造与电极上细菌生长相同的反应器介质来达到非周转条件, 除了省略可溶性电子供体或受体。在确保恒电位仪关闭后, 去除介质灌装, 并在没有阳极系统或阴极系统受体的电子供体的情况下加入新鲜介质。另外, 还可以设置一个连续的流动系统, 以便在 nonturnover 条件下用所需的介质慢慢替换介质。 注: 如果氧是可溶电子受体 (对于 biocathode MCL), 喷雾系统的混合物为 ~ 15% CO2和 85% N2 (或将保持介质中 pH 值的气体混合物)。</li>
		</ol>
</li>
		<li>在完成浇口测量后, 使用恒电位仪软件将每个电极的电位设置回增长电位, 以允许系统重新稳定 (使用与2.2.2 相同的值)。</li>
		<li>如果满足上述所有条件 (ISD独立于 v 并且与 vSD线性扩展), 则使用以下等式将 ISD值转换为电导率31 <img alt="Equation 3" src="/files/ftp_upload/54671/54671eq3.jpg"> <img alt="Equation 4" src="/files/ftp_upload/54671/54671eq4.jpg">
</li>
		<li>其中 G 是电导和 S 是一个比例因子, 是系统依赖, 和因素, 如电极大小, 缝隙大小, 和生物膜高度。对于某些系统, S 可以由预定方程确定。31也可以使用建模软件进行数值计算, 如前所述。17
</li>
		<li>减去背景电流以识别所进行电流的形状和大小。将在 vsd = 0.00 v 中生成的电流减去从 vsd = 0.01 v 所生成的电流, 或者从使用 vsd = 0.01 v 的源电流减去漏电流. 任何一种方法都可以删除背景电流, 从而使只有传导电流。</li>
	</ol></li>
	<li>温度依赖性电化学浇注测量
	<ol>
<li>确定感兴趣的温度范围。这个范围是高度依赖于被研究的系统, 然而生理上相关的温度应该使用。 注: 以前的研究使用了10摄氏度到30摄氏度的温度范围来研究温微生物在生理相关条件下的电子传输。17
</li>
		<li>获得循环水浴或孵化器来调节反应堆温度和控制电抗器, 以确保介质的设定点和实际温度相同。<ol>
<li>将温度计或热电偶放在 IDA 所在的控制反应堆中。</li>
		</ol>
</li>
		<li>使用下面描述的两个过程之一的 bipotentiostat, 使 ISD测量 (见 3.1.4) 在营业额和 nonturnover (步骤 3.1.5) 条件下选择的温度范围。<ol>
<li>生成 ISD vs EG曲线, 如上文所述 (见 3.1), 对于每一个温度范围内的兴趣。对于显示氧化还原电导率的材料, 如sulfurreducens和 Biocathode MCL, 从每个温度的峰值电流用于确定 ISD与 T 依赖性。 注意: 此方法用于为每个温度生成完整的 ISD与 EG曲线, 但需要的时间比第二个选项多。</li>
			<li>或者, 设置并按住 IDA 的栅极电位, 利用 bipotentiostat (从 ISD vs EG曲线、步骤3.1.4 获得最大电流) 并记录使用 bipotentiostat 的最大传导电流, 同时温度在使用水浴或孵化器的板载控制所选择的温度范围之间循环。 注: 在这里使用的电极/反应堆几何, iSD和 T 被允许稳定至少20分钟, 平均稳定 ISD用于每个温度。根据具体的系统, 可能需要或多或少的稳定时间。这种方法比第一个快, 对生物膜造成的压力更小。但是, 不会生成完整的浇口曲线。</li>
			<li>将温度从一个集合点循环到另一个位置, 再使用孵化器或水浴的板载控制来确定反应的可逆性, 以确保温度循环不会损害生物膜。</li>
		</ol>
</li>
		<li>使用孵化器或水浴的板载控制, 将温度重置为正常的生长温度, 使系统稳定。 注: 对于氧化还原导体, ISD与 1/T 数据可以配合阿伦尼乌斯速率表达式, 它允许计算激活能量, 如下所示: <img alt="Equation 5" src="/files/ftp_upload/54671/54671eq5.jpg"> <img alt="Equation 6" src="/files/ftp_upload/54671/54671eq6.jpg"> 其中 Ea是相邻氧化还原辅助因子与 k 之间电子传输的活化能, 是玻尔兹曼常数. </li>
	</ol></li>
</ol>

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作者没有什么可透露的。

在 IDA 的安装过程中, 测试源和漏油是否在电化学浇口测量之前没有短路是至关重要的, 因为这将改变 ISD与 EG曲线, 并可能导致错误的结果和解释。选择 vsd和 v (例如, 当前线性依赖于 vsd并且独立于五个) 也是至关重要的。如果不是这种情况, 那么上面描述的方程不能用来计算电导率。
至少必须考虑两个背景电流, 并从进行的电流测量中移除。第?...

胞外电子输运 (EET) 是一种使某些微生物在细胞内代谢过程和不溶性电子受体之间传输电子的过程, 它是由天然矿物质到电极。在某些情况下, EET 使微生物在电极表面形成导电的多细胞厚生物膜, 而与电极不直接接触的细胞仍然可以利用它作为代谢电子受体或捐献者。在诸如微生物合成、污染物传感/去除、远程能源生成和存储等各种应用的电极催化剂等生物膜中, 有相当大的兴趣,7,8,9 、10、11、12、13、14由于微生物所执行的新陈代谢过程多种多样, 微生物生物膜的耐久性比较以酶为基础的 bioelectrodes。15,16此外, EET 通路可能会被用来电控或信号改变自然发生或基因工程微生物代谢过程中涉及, 例如, 在生产所需的产品或检测目标分析或刺激的。电催化生物膜的电导率与其他生物材料不同, 是其催化性能的一个核心方面, 但对于电极环境下的底层 EET 过程却知之甚少,众所周知的是高度争议的。17,18,19,20,21,22,23,24
这里描述的是一个2电极法测量电导率通过生活, 电极生长生物膜使用交叉电极阵列 (IDAs)。IDAs 包括在平板玻璃表面上图案的平行矩形电极, 使每一个其它波段连接在阵列的另一侧, 导致2电极 (源和漏)。对 IDA 的仔细检查 (例如, 参见 #1 的图 6.12b) 揭示了分隔相邻带的缝隙也以这样的方式连接在一起, 形成一个单独的缝隙, 在分离两个电极的阵列之间来回编织。结果是一个长而窄的缝隙, 分离源和排泄电极, 产生非常高的源漏电流, 当导电材料形成, 铸造, 聚合, 或种植 (在这里所考虑的生物膜类型的情况下) 阵列。此外, 由于电容充电和导电材料的氧化状态随栅极电位的变化而改变, 电极的小尺寸导致了小背景电流, 因为电导率所需的材料量使用 IDAs 的测量是如此之小。本文介绍了基于 IDA 的电化学门控技术, 开发了用于表征薄膜导电聚合物,2,3,4,25最近才被应用于生活系统。18另一种用于测量活生物膜电导率的技术使用了大格式的拆分源和排泄电极和源表来设置栅极电位。26,27但是, 对这些方法的关注以前已经详细说明过。18
下面的协议封装了我们对活体地杆菌能 sulfurreducens 和 biocathode MCL 生物膜进行电导率测量的经验。G. sulfurreducens是一种能够使用不溶性材料 (包括电极) 作为唯一代谢电子受体的模型电极还原有机体。此外, 它形成厚的生物膜, 能够传输电子在多个细胞长度, 使它成为一个理想的模型有机体研究阳极远距离细胞外电子转移。我们还包括详细的研究 biocathode MCL, 一个有氧, 自养混合社区生物膜分离的底栖微生物燃料电池的阴极。Biocathode MCL (命名为三主要成分– Marinobacter、 Chromatiaceaea和Labrenzia) 能够将电极氧化为其唯一的电子供体, 并在多个细胞长度上传输电子, 使它是一个有趣的阴极系统来研究。此外, biocathode MCL 有最高的报告电导率的生活系统迄今使用这些方法。在本议定书中列入这些不同的活性生物膜, 是为了强调这项技术适用于通过任何能够与电极电相互作用的生物生物膜来测量电子的传输。

<table><tbody><tr><td>IDAs</td><td>CH Instruments</td><td>012125</td><td>Manufactured by ALS-Japan; sold by CH Instruments</td></tr><tr><td>Wire</td><td>Digikey</td><td>W7-ND</td><td></td></tr><tr><td>Conductive silver epoxy</td><td>Electron microscopy sciences</td><td>12670-EE</td><td></td></tr><tr><td>Insulating material</td><td>3M</td><td>2131-B</td><td>Scotchast flame retardant compound</td></tr><tr><td>15 mL conical centrifuge tube</td><td>VWR</td><td>89004-368</td><td></td></tr><tr><td>21g needle</td><td>VWR</td><td>BD-305165</td><td></td></tr><tr><td>5 mL pipette tips</td><td>VWR</td><td>82018-842</td><td></td></tr><tr><td>5 mL pipettor</td><td>VWR</td><td>89079-976</td><td></td></tr><tr><td>Freshwater medium components</td><td>Sigma Aldrich</td><td>All standard laboratory chemicals</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Ammonium chloride</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Sodium phosphate monobasic</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Sodium bicarbonate</td><td></td><td></td><td></td></tr><tr><td>Artificial seawater medium components</td><td>Sigma Aldrich</td><td>All standard laboratory chemicals</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Sodium chloride</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Magnesium chloride hexahydrate</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Magnesium sulfate heptahydrate</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Potassium chloride</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Sodium bicarbonate</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Calcium chloride dihydrate</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Ammonium chloride</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Potassium phosphate dibasic</td><td></td><td></td><td></td></tr><tr><td>Ag/AgCl reference electrode</td><td>Basi</td><td>MF-2079</td><td></td></tr><tr><td>Graphite rod counter electrode</td><td>Electron microscopy sciences</td><td>70230</td><td></td></tr><tr><td>Recirculating water bath</td><td>Thermo Scientific</td><td>152-5256</td><td></td></tr><tr><td>Bipotentiostat</td><td>Pine Instruments</td><td>WD-20</td><td>http://www.voltammetry.net/pine/aftermath/user</td></tr><tr><td>Stir bars</td><td>VWR</td><td>58947-114</td><td></td></tr><tr><td>&lt;em&gt;G. sulfurreducens&lt;/em&gt; culture</td><td>ATCC</td><td>51573</td><td></td></tr><tr><td>Jacketed reactor</td><td>Pine Instruments</td><td>RRPG085</td><td></td></tr></tbody></table>

characterizing electron transport through living biofilms

在这里, 我们演示了电化学浇口的方法, 用于表征电极生长的微生物生物膜在生理相关条件下的电导率。1这些测量是用在交叉电极 (IDA) 阵列的专门配置中, 在玻璃表面上图案的源和漏电极对水介质中活生物膜进行的。在连接源和排泄的缝隙中生长的生物膜。电位被应用到电极 (eS和 eD) 上, 通过电极之间的生物膜生成源漏电流 (ISD)。电导率对栅极电位的依赖性 (源和漏电位的平均值, EG = [eD + ES]/2) 是通过系统地改变栅极电位和测量产生的源漏来确定的。当前。电导率对栅极电位的依赖性提供了有关在调查的特定生物膜电导率基础上的胞外电子传输过程的机械信息。此处描述的电化学浇口测量方法直接基于 Wrighton2、3和同事和刻录默里4、5、6和同事在1980是研究薄膜导电聚合物。

IDAs 进行了有线、绝缘和测试, 以确保两个电极彼此电隔离 (图 1)。反应器组装, 接种与G. sulfurreducens, 并孵化, 直到生物膜弥合电极之间的差距。可以直观地看到sulfurreducens生物膜覆盖阵列。其他生物膜可能要求研究员做电化学门控测量, 以查看两个电极是否已电连接。显微镜也应用于验证阵列电极之间的连接。电化学浇注实验是为了确定<...

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Characterizing Electron Transport through Living Biofilms

提出了一种在生理相关条件下测量活体微生物生物膜电导率的方法。

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 ...

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8.4K 次观看.  Naval Research Laboratory. 提出了一种在生理相关条件下测量活体微生物生物膜电导率的方法。

视频: Characterizing Electron Transport through Living Biofilms

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

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

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

流式细胞仪对水生生物膜的表征

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

原位萨尔和 TOF-SIMS希瓦氏菌 oneidensis MR1 生物膜的表征

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

化学

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

细胞和生物膜细胞外成分的定量并发

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

免疫与感染

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

生物工程

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

<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.

通过活生物膜表征电子传输

摘要

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

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

流式细胞仪对水生生物膜的表征

原位萨尔和 TOF-SIMS希瓦氏菌 oneidensis MR1 生物膜的表征

细胞和生物膜细胞外成分的定量并发

方法表征的共同发展生物膜和生境异质性

方法学研究

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

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

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