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 border="0" cellpadding="0" cellspacing="0" width="688">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Carbon felt sheet</td>
			<td style="width:147px;">n/a</td>
			<td style="width:115px;">n/a</td>
			<td style="width:212px;">Used for anode and cathode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Titanium wire</td>
			<td>The Nilaco Cooporation</td>
			<td>TI-451485</td>
			<td>Used to construct fuel cell system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Graphite epoxy</td>
			<td style="width:147px;">Electrolytica lnc.</td>
			<td style="width:115px;">n/a</td>
			<td style="width:212px;">Used to connect the 
			electrodes and Ti wire</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Drying oven</td>
			<td style="width:147px;">Yamato</td>
			<td style="width:115px;">DY300</td>
			<td style="width:212px;">bake the electrode to 
			solidify conductive graphite epoxy</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Digital multi meter</td>
			<td style="width:147px;">Fluke</td>
			<td style="width:115px;">616-1454</td>
			<td style="width:212px;">to check the ohmic value 
			of resistance</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Dissolved oxygen probe</td>
			<td style="width:147px;">Sper Science</td>
			<td style="width:115px;">#&#160; 850045</td>
			<td style="width:212px;">to check the oxygen 
			concentration in the environments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Resistor</td>
			<td style="width:147px;">Sodial</td>
			<td style="width:115px;"></td>
			<td style="width:212px;">Used to construct fuel cell 
			system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Conducting wire</td>
			<td style="width:147px;">Pico</td>
			<td style="width:115px;">81141s</td>
			<td style="width:212px;">Used to construct fuel cell 
			system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Voltmeter and Data logger</td>
			<td style="width:147px;">T&#38;D corporation</td>
			<td style="width:115px;">VR-71</td>
			<td>Used for data recording</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Hydrogen Hexachloroplatinate(IV) Hexahydrate</td>
			<td style="width:147px;">wako</td>
			<td style="width:115px;">18497-13-7</td>
			<td>Used for electropolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Citric acid</td>
			<td style="width:147px;">Wako</td>
			<td style="width:115px;">038-06925</td>
			<td>Used for electropolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sulfuric acid</td>
			<td style="width:147px;">Wako</td>
			<td style="width:115px;">192-04696</td>
			<td>Used for electropolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HCl</td>
			<td style="width:147px;">Wako</td>
			<td style="width:115px;">083-01095</td>
			<td>Used for electrode washing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Glass cylinder</td>
			<td style="width:147px;">N/A</td>
			<td style="width:115px;">N/A</td>
			<td style="width:212px;">Custom-made, used as the electrochemical reactor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">PTFE cover and base</td>
			<td style="width:147px;">N/A</td>
			<td style="width:115px;">N/A</td>
			<td style="width:212px;">Custom-made, used as a cover and a foundation of the electrochemical reactor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Buthyl rubber</td>
			<td style="width:147px;">N/A</td>
			<td style="width:115px;">N/A</td>
			<td style="width:212px;">Custom-made, inserted between each component of electrochemical reactor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Septa</td>
			<td style="width:147px;">GL Science</td>
			<td style="width:115px;">3007-16101</td>
			<td style="width:212px;">Used as an injection port of electrochemical reactor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Indium tin-doped oxide (ITO) electrode</td>
			<td style="width:147px;">GEOMATEC</td>
			<td style="width:115px;">No.0001</td>
			<td style="width:212px;">Used as a working electrode, 5&#937;/sq</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Ag/AgCl KCl saturated electrode</td>
			<td style="width:147px;">HOKUTO DENKO</td>
			<td style="width:115px;">HX-R5</td>
			<td style="width:212px;">Used as a reference electrode, &#934;0.30mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Platinum wire</td>
			<td style="width:147px;">The Nilaco Cooporation</td>
			<td style="width:115px;">PT-351325</td>
			<td style="width:212px;">Used as a counter electrode</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">NaHCO3</td>
			<td style="width:147px;">Wako</td>
			<td style="width:115px;">191-01305</td>
			<td style="width:212px;">Used for The Cedars Media (CMS)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">CaCO3</td>
			<td style="width:147px;">Wako</td>
			<td style="width:115px;">030-00385</td>
			<td style="width:212px;">Used for CMS</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">NH4Cl</td>
			<td style="width:147px;">Wako</td>
			<td style="width:115px;">011-03015</td>
			<td style="width:212px;">Used for CMS</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">MgCl2&#160;&#8226; 6H2O</td>
			<td style="width:147px;">Wako</td>
			<td style="width:115px;">135-00165</td>
			<td style="width:212px;">Used for CMS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NaOH&#160;</td>
			<td style="width:147px;">Wako</td>
			<td style="width:115px;">198-13765</td>
			<td style="width:212px;">Used for CMS</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">Na2SO4</td>
			<td style="width:147px;">Wako</td>
			<td style="width:115px;">194-03355</td>
			<td style="width:212px;">Used for CMS</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">K2HPO4</td>
			<td style="width:147px;">Wako</td>
			<td style="width:115px;">164-04295</td>
			<td style="width:212px;">Used for CMS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CABS</td>
			<td style="width:147px;">SANTA CRUZ</td>
			<td style="width:115px;">SC-285279</td>
			<td style="width:212px;">Used for CMS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Incubator</td>
			<td style="width:147px;">TOKYO RIKAKIKAI CO. LTD.</td>
			<td style="width:115px;">LTI-601SD</td>
			<td style="width:212px;">Used for precultivation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Autoclave machine</td>
			<td style="width:147px;">TOMY SEIKO CO. LTD.</td>
			<td style="width:115px;">LSX-500</td>
			<td style="width:212px;">Used for sterilization of the electrochemical reactor and the medium</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:215px;">Clean bench</td>
			<td style="width:147px;">SANYO</td>
			<td style="width:115px;">MCV-91BNF</td>
			<td style="width:212px;">Used to prevent the contamination of the electrochemical reactor and the medium with other microbes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Centrifuge separator</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:115px;">5430R</td>
			<td style="width:212px;">Rotational speed upto 6000&#215;g is required</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Nitrogen gas generator</td>
			<td style="width:147px;">Puequ CO. LTD.</td>
			<td style="width:115px;">PNTN-2</td>
			<td style="width:212px;">Nitrogen gas cylinder can also be used instead of gas generator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">UV-vis spectrometer</td>
			<td style="width:147px;">SHIMADZU</td>
			<td style="width:115px;">UV-1800</td>
			<td style="width:212px;">Used for optimization of cell density</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Potentiostat</td>
			<td style="width:147px;">BioLogic</td>
			<td style="width:115px;">VMP3</td>
			<td style="width:212px;">Used for biofilm formation and kinetic isotope effect experiments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Thermal water circulator</td>
			<td style="width:147px;">AS ONE</td>
			<td style="width:115px;">TR-1A</td>
			<td style="width:212px;">Used for maintanance of temperature of electrochemcial reactor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Faraday cage</td>
			<td style="width:147px;">HOKUTO DENKO</td>
			<td style="width:115px;">HS-201S</td>
			<td style="width:212px;">Used for electrochemical experiments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Anaerobic Chamber</td>
			<td style="width:147px;">COY</td>
			<td style="width:115px;">TypeB (Vinyl)</td>
			<td style="width:212px;">TO conduct experiments 
			under anaerobic condition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultraclean DNA Extraction kit</td>
			<td style="width:147px;">MoBio</td>
			<td style="width:115px;"></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

7.5K 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

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 ...

科研

环境科学

A Protocol for Conducting Rainfall Simulation to Study Soil Runoff

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The objective of this study was to characterize the effects of antecedent soil moisture content on the fate and transport of surface applied commercial urea, a common form of nitrogen (N) fertilizer, following a rainfall event that occurs within 24&#160;hr&#160;after fertilizer application. Although urea is assumed to be readily hydrolyzed to ammonium and therefore not often available for transport, recent studies suggest that urea can be transported from agricultural soils to coastal waters where it is implicated in harmful algal blooms. A rainfall simulator was used to apply a consistent rate of uniform rainfall across packed soil boxes that had been prewetted to different soil moisture contents. By controlling rainfall and soil physical characteristics, the effects of antecedent soil moisture on urea loss were isolated. Wetter soils exhibited shorter time from rainfall initiation to runoff initiation, greater total volume of runoff, higher urea concentrations in runoff, and greater mass loadings of urea in runoff. These results also demonstrate the importance of controlling for antecedent soil moisture content in studies designed to isolate other variables, such as soil physical or chemical characteristics, slope, soil cover, management, or rainfall characteristics. Because rainfall simulators are designed to deliver raindrops of similar size and velocity as natural rainfall, studies conducted under a standardized protocol can yield valuable data that, in turn, can be used to develop models for predicting the fate and transport of pollutants in runoff.

A rainfall simulator was used to apply a consistent rate of uniform rainfall to packed soil boxes in a study of the fate and transport of urea, a nonpoint source environmental contaminant. Under uniform soil and rainfall conditions, antecedent soil moisture content exerted strong control over urea loss in surface runoff.

一个协议，用于开展降雨模拟研究土壤径流

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

一个铁（II）的实验室模拟的前寒武纪富海洋上升流系统探索光合细菌生长

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

生物毒理学方法与海洋细菌<em&gt;鳗弧菌</em&gt;评估环境污染物的急性毒性

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

用于研究铁 (氧) 氢氧化物、微量元素和细菌之间厌氧生物地球化学相互作用的实验柱装置

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

细菌附着在实验室测量的植物表面

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

用便携式实时 PCR 系统进行土壤载体 Phytopathogens 的现场分子检测

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

利用高效液相色谱-电化学检测仪检测大气环境中的 3-硝基酪氨酸

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

不同环境条件下生物气溶胶的组成及分布分析

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...