We would like to acknowledge Roger Raiche and David McCrory for allowing us access to the Cedars and consulting on locations for long term incubation. We also thank the Cedars field crew during the 2013-2014 season: Shino Suzuki, Shunichi Ishii, Greg Wanger, Grayson Chadwick, Bonita Lam and Matthew Schechter. Additional thanks to Shino Suzuki and Gijs Kuenen for insightful research and culturing support. This work was funded through a Grant-in-Aid for Young Scientists A and B from the Japan Society for Promotion of Science (JSPS) KAKENHI Grant Number 17H04969 and 26810085, respectively, and the Japan Agency for Medical Research and Development (17gm6010002h0002). US funding provided by the US Office of Global Naval Research (N62909-17-1-2038), and the Center for Dark Energy Biosphere Investigations (C-DEBI) (OCE0939564) and the NASA Astrobiology Institute - Life Underground (NAI-LU) (NNA13AA92A). Part of this work was conducted as part of a Japan Society for the Promotion of Sciences: Short-term postdoctoral fellowship for Annette Rowe (PE15019) at the University of Tokyo in the lab of Kazuhito Hashimoto.

1. Construction of a Two-electrode System for Environmental Incubation
<ol>
	<li>Preparation of the anode material and treatment of Carbon felt electrode (Figure 2).
	<ol>
		<li>Cut the carbon felt to equal dimensions depending on desired biomass enrichment. Soak each electrode in 90% ethanol for 30 min, then rinse at least 8 times with deionized water, sonicating for 1 min after each rinse.</li>
		<li>Wash the electrodes twice in 1 M HCl, stirring for a minimum of 12 h for each wash.</li>
		<li>Dry the electrodes in a warm (37 &#176;C) oven for 6&#8211;12 h or until free of liquid.</li>
		<li>Attach electrodes to titanium wire using graphite epoxy per manufacturer&#39;s protocol on a polytetrafluoroethylene plate (non-stick surface). 
		NOTE: We used a titanium wire because of its high tolerance to aerobic corrosion.</li>
		<li>Bake the electrode at 120 &#176;C for 6 h.</li>
		<li>Test the resistance between titanium wire and carbon felt with an ohmmeter and confirm that the resistance between the wire and felt electrodes is less than 5 ohms.</li>
	</ol>
	</li>
	<li>Electroplation of platinum on the carbon felt electrode for preparation of the cathode material
	<ol>
		<li>Submerge carbon felt electrodes, prepared in step 1.1, in 2 M KOH for a minimum of 12 h in a glass container.</li>
		<li>For electrochemical cleaning, place the electrode as working electrode (WE) in a three-electrode reactor, which also accommodates a reference (RE) and counter electrode (CE). Connect WE, RE, and CE to the potentiostat by alligator clips. Confirm all the connections with an ohmmeter. 
		NOTE: We used an Ag/AgCl (KCl saturated) electrode and a platinum wire as RE and CE, respectively.</li>
		<li>Poise the electrode at 1.0 V vs. Ag/AgCl for 600 s in electrolyte solution containing 2 M KOH (using a sufficient amount to submerge the whole electrode). Take out the electrode from the electrochemical reactor (which is made of glass). Rinse the electrode&#160;in deionized water at least 8 times, sonicating for 1 min after each rinse. Dry electrodes at 100 &#176;C for at least 12 h.</li>
		<li>To prepare plating solution, add 100 g of citric acid, 5 g of sodium sulfate, and 2 g of dihydrogen hexachloroplatinate (IV) hexahydrate to 1 L of 2 M sulfuric acid.</li>
		<li>Weigh cleaned and dried electrodes as prepared in steps 1.2.1&#8211;1.2.2, and then cover the electrode in a plating solution as prepared in step 1.2.3. Sonicate the electrode in the plating solution three times for 30 s each.</li>
		<li>Electroplate the electrodes by poising the electrode potential at -0.2 V vs. Ag/AgCl for 460 s in plating solution. Rinse electrodes twice in deionized water and discard the platinum waste.</li>
		<li>Rinse electrodes in deionized water at least 3 times, sonicating for 20 s after each rinse. Rinse without sonication at least three more times.</li>
		<li>Dry electrodes at 100 &#176;C for at least 12 h. Weigh electrode to quantify the electroplated platinum on the carbon felt electrode.</li>
	</ol>
	</li>
</ol>
2. Construction and Installation of Two-electrode System
<ol>
	<li>Investigation of&#160;installation site for each electrode in the natural environment.
	<ol>
		<li>Determine oxygen concentration using a dissolved oxygen (DO) probe.</li>
		<li>Check the depth profile of DO in the site. 
		NOTE: The desired environmental conditions for the anode are consistent hydration and anoxia. If desired, remove the influence of oxygenic photosynthesis by shielding the anode from light. The ideal conditions for cathode placement are consistently hydrated, and near surface waters to be oxic. If necessary, attach&#160;floats to maintain&#160;surface contact in the cathode.</li>
	</ol>
	</li>
	<li>Construction of fuel cell type 2-electrode incubation system
	<ol>
		<li>Connect the insulated wire of desired length and a lead of titanium wire from the electrodes (one anode and one platinum plated cathode) by twisting the two lines. Cover the connections with water-resistant wax and further protect using marine grade heat shrink tubes.</li>
		<li>Connect two wires with a cathode and an anode by a resistor of known resistance. 
		NOTE: For biological systems, lower resistors (10 to 1,000 &#937;) result in more consistent biological activity. If desired, a high resistance resistor will prevent biological activity,&#160;as a negative control. To prevent a corrosion of any connections between resistor and leads, we protected them with heat shrink tubes.</li>
	</ol>
	</li>
	<li>Measurement for voltage and temperature logging over time.
	<ol>
		<li>Check the voltage between the ends of the resistor for estimating current production from the fuel cell reaction.</li>
		<li>Measure the voltage difference over time using a data logging voltmeter with the appropriate connections leading to the anode and cathode (see manufacturer&#39;s protocol). 
		NOTE: Simultaneous temperature data logging is optional, but this information can help relate changes in current to abiotic as opposed to biological fluctuations.</li>
	</ol>
	</li>
	<li>Protection of data logger and electric connections
	<ol>
		<li>Use a stationary&#160;and/or plastic bag to protect the logger and all the electric connections from rain.</li>
		<li>Fix the plastic bag and cables tightly to protect from strong wind. An example is shown in Figure 1.</li>
	</ol>
	</li>
</ol>
3. Collection of the Electrode Sample from the Natural Environment
<ol>
	<li>To prevent the quality of the anode sample from being damaged due to oxygen contamination, collect the electrode under anaerobic condition.</li>
	<li>At least 30 min prior to collecting the electrode sample, put a test tube in an anaerobic location. For example, put the test tube and lid separately at the bottom of the pond to make the bottle inside anaerobic.</li>
	<li>Cut the titanium lead from the electrode with a wire cutter, gently collect the electrode sample into the test tube, and seal it in the anaerobic water zone. To keep the sample fresh, store the sample at 4 &#176;C immediately after the sample collection. 
	NOTE: Alternatively, electrodes can be transferred directly to anoxic (N2 purged) medium. We used a Cedars medium (described by&#160;Suzuki et al.11) that was designed from the aqueous geochemistry measured at the site and amended to provide sufficient nutrients for microbial growth. This media was modified for different laboratory experiments.</li>
</ol>
4. Laboratory Confirmation for Current Production and DNA&#160;Analysis
<ol>
	<li>Electrochemical confirmation for current production capability of microbial consortia attaching to the electrode.
	<ol>
		<li>Construct an electrochemical reactor14,15 with the sampled electrode, a&#160;platinum wire, and a&#160;Ag/AgCl (KCl saturated) electrode as WE, CE, and RE, respectively, in an anaerobic chamber. Fill the electrochemical reactor&#160;with Cedars medium containing soluble carbohydrate electron donors.</li>
		<li>Poise the electrode potential at +0.2 V vs. Ag/AgCl and measure the current production.</li>
	</ol>
	</li>
	<li>DNA extraction from the electrode sample using a microbial DNA kit (see Table of Materials).
	<ol>
		<li>Clean the inside of anaerobic glove box with 70% ethanol and put a sterilized dish on aluminum foil. 
		NOTE: Anaerobic chamber&#160;keeps oxygen concentration less than 1 ppm by maintaining a hydrogen&#160;atmosphere at around ~2&#8211;3% to scavenge oxygen in the presence of a palladium catalyst.</li>
		<li>Open the electrochemical reactor in the&#160;glove box, put the sample electrode on the dish, and cut to a size to fit the tube used in the DNA kit. Proceed with the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Nealson, K. H., Saffarini, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+manganese+in+anaerobic+respiration:+environmental+significance,+physiology,+and+regulation.">Iron and manganese in anaerobic respiration: environmental significance, physiology, and regulation.</a> Annual Reviews of Microbiology. 48, 311-343 (1994).</li><li>Lovley, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bug+juice:+harvesting+electricity+with+microorganisms.">Bug juice: harvesting electricity with microorganisms.</a> Nature Reviews Microbiology. 4 (7), 497-508 (2006).</li><li>Rabaey, K., Rozendal, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+electrosynthesis+-+revisiting+the+electrical+route+for+microbial+production.">Microbial electrosynthesis - revisiting the electrical route for microbial production.</a> Nature Reviews Microbiology. 8 (10), 706-716 (2010).</li><li>Lovley, D. R., Coates, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioremediation+of+metal+contamination.">Bioremediation of metal contamination.</a> Current Opinion in Biotechnology. 8 (3), 285-289 (1997).</li><li>Dinh, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+corrosion+by+novel+anaerobic+microorganisms.">Iron corrosion by novel anaerobic microorganisms.</a> Nature. 427 (6977), 829-832 (2004).</li><li>Myers, C. R., Nealson, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+manganese+reduction+and+growth+with+manganese+oxide+as+the+sole+electron+acceptor.">Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor.</a> Science. 240 (4857), 1319-1321 (1988).</li><li>Lovley, D. R., Phillips, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+mode+of+microbial+energy+metabolism:+organic+carbon+oxidation+coupled+to+dissimilatory+reduction+of+iron+or+manganese.">Novel mode of microbial energy metabolism: organic carbon oxidation coupled to dissimilatory reduction of iron or manganese.</a> Applied and Environmental Microbiology. 54 (6), 1472-1480 (1988).</li><li>Arnold, R. G., DiChristina, T. J., Hoffmann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductive+dissolution+of+Fe(III)+oxides+by+Pseudomonas+sp+200.">Reductive dissolution of Fe(III) oxides by Pseudomonas sp 200.</a> Biotechnology and Bioengineering. 32 (9), 1081-1096 (1988).</li><li>Rowe, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+electrochemical+enrichment+and+isolation+of+a+magnetite-reducing+bacterium+from+a+high+pH+serpentinizing+spring.">In situ electrochemical enrichment and isolation of a magnetite-reducing bacterium from a high pH serpentinizing spring.</a> Environmentakl Microbiology. 19 (6), 2272-2285 (2017).</li><li>Suzuki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+diversity+in+The+Cedars,+an+ultrabasic,+ultrareducing,+and+low+salinity+serpentinizing+ecosystem.">Microbial diversity in The Cedars, an ultrabasic, ultrareducing, and low salinity serpentinizing ecosystem.</a> Proceedings of the National Academy of Science U S A. 110 (38), 15336-15341 (2013).</li><li>Suzuki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+genomic+features+of+highly+alkaliphilic+hydrogen-utilizing+Betaproteobacteria+from+a+continental+serpentinizing+site.">Physiological and genomic features of highly alkaliphilic hydrogen-utilizing Betaproteobacteria from a continental serpentinizing site.</a> Nature Communications. 5, 3900 (2014).</li><li>McCollom, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+trends+for+reaction+rates,+hydrogen+generation,+and+partitioning+of+iron+during+experimental+serpentinization+of+olivine.">Temperature trends for reaction rates, hydrogen generation, and partitioning of iron during experimental serpentinization of olivine.</a> Geochimica et Cosmochimica Acta. 181, 175-200 (2016).</li><li>Morrill, P. 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The authors have nothing to disclose.

In the described study, we show the enrichment of a microbial consortium, linked with in situ current production. The observed patterns in current support microbial activity in this system over short and long time scales. The critical step for constructing a functional two-electrode (fuel cell type) system is identifying and utilizing a location with a stable water-level and oxygen concentration in the environment. The cathode is exposed to oxygen at the air water interface, while the anode is kept under anaerobic condition, and the electrode potential difference promotes anaerobic respiration of EET-capable bacteria.
We observed daily current oscillation in the environmental electrochemical system but not in the laboratory reactor. Because this fluctuation of current was observed during the daylight hours-maximum and minimum currents were observed between dawn and dusk-the effect of sunlight and/or temperature could explain the change in the microbial current production. Measuring temperature, sunlight and/or other environmental variables could further expand understanding of the controls and drivers of microbial electron flow in environmental systems. Alternatively, adding elements to block sunlight could help remove or mitigate the effects of oxygenic photosynthesis and/or potential photoreactions on the electrode, which could serve to better stimulate optimal EET conditions. However, measurement of other environmental factors could better elucidate ecological context in EET-capable microbes, including potential microbial community interactions, as well as the relationships between microbes and the&#160;environment.
Our two-electrode system potentially enriched not only anode-respiring bacteria, but also oxygen-reducing bacteria that harvest energy from electron uptake. Although we did not conduct the community analysis on the cathode, their microbial electron uptake capability is testable in laboratory three-electrode reactor with negatively poising the collected cathode electrode in the presence of oxygen. A stable concentration gradient of electron acceptors from cathode to anode enable our method to theoretically also enrich cathode-respiring bacteria. An alternative enrichment method for the cathode-respiring bacteria is the use of&#160;Fe(0) particles or coupons&#160;as a solid electron donor5. Although hydrogen production can also occur at the surface, successful isolation of bacteria that can directly extract electrons from electrode surface has been reported5,16.
In conclusion, our method successfully enriched EET-capable consortia using a self-sustaining electrochemical system in a low-biomass environment. Several previous cultivation approaches were unsuccessful, which led us to develop an on-site enrichment scheme. In our system, current output reflected the microbial activity, and led to further hypotheses about the microbial ecology of this system. Expanding the isolation of EET-capable microbes&#160;as well as the diversity of environments will enhance our understanding of the mechanism for EET, as well as the role of electron transport in environmental microbiology.

Several mineral-reducing microbes have been shown to utilize solid-phase minerals as terminal electron acceptors, by extracellular electron transport (EET) processes that conduct electrons to the exterior of the cell via redox enzymes1. EET is critical, not only for microbe-mineral processes but also applied energy and environmental technologies, such as microbial fuel cells2, electrode synthesis3, and bioremediation4. New EET-capable bacteria are highly sought after, and have been extensively studied from a fundamental or applied perspective5. However, we only have limited insight into the&#160;ecological or biogeochemical significance of these bacteria. The majority of EET-capable microbes have been isolated following enrichment from aqua, sediment, or anaerobic digesters using solid electron acceptors such as MnO2, Fe2O3 or poised electrodes in laboratory6,7,8. However, these methods often produce similar consortia and potentially miss more sensitive taxa that may dominate low energy or low biomass systems, biasing the ability of these microbes to adapt to the lab or axenic culture environment9. Usually for low biomass environments, large quantities of water from a site are filtered to concentrate bacterial cells. However, EET-capable bacteria often exhibit anaerobic metabolisms and therefore oxygen exposure may further inhibit or prevent their cultivation. Alternative on-site methodologies to concentrate cells without exposing them to oxygen could facilitate the isolation of EET-capable bacteria. Here, we report setup details for an on-site electrochemical technique to enrich EET-capable microbe over a long period of time without the need for an external power source.
Using our electrocultivation experiments from a highly alkaline spring in Northern California, the Cedars10, we describe this on-site electrochemical technique. The geochemistry of the springs at The Cedars are impacted by serpentinization in the subsurface. The springs are highly reductive, with oxygen concentrations below the limit of detection under the air water interface highlighting the potential for microbial energy production via EET in this functionally anoxic environment11. However, there is no evidence to support EET-capable microbes from the Cedars (in either 16S rRNA or Metagenomic analysis). Even though this environment has been characterized as electron acceptor limited, the potential for using insoluble minerals as terminal electron acceptors, including minerals such as the iron baring minerals that result from serpentinization (i.e., magnetite), has not been extensively investigated12. We, therefore, deployed our electrochemical system at Campsite Spring, a high pH spring at the Cedars, to enrich for EET-capable microbes (Figure1)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

Anaerobic respiration coupled with electron transport to insoluble minerals (referred to as extracellular electron transport [EET]) is thought to be critical for microbial energy production and persistence in many subsurface environments, especially those lacking soluble terminal electron acceptors. While EET-capable microbes have been successfully isolated from various environments, the diversity of bacteria capable of EET is still poorly understood, especially in difficult-to-sample, low energy or extreme environments, such as many subsurface ecosystems. Here, we describe an on-site electrochemical system to enrich EET-capable bacteria using an anode as a respiratory terminal electron acceptor. This anode is connected&#160;to a cathode capable of&#160;catalyzing abiotic oxygen reduction. Comparing this approach with electrocultivation methods that use a potentiostat for poising the electrode potential, the two-electrode system does not require an external power source. We present an example of our on-site enrichment utilized in an alkaline pond at the Cedars, a terrestrial serpentinization site in Northern California. Prior attempts to cultivate mineral reducing bacteria were unsuccessful, which is likely due to the low-biomass nature of this site and/or the low relative abundance of metal reducing microbes. Prior to implementing our two-electrode enrichment, we measured the vertical profile of dissolved oxygen concentration. This allowed us to place the carbon felt anode and platinum-electroplated carbon felt cathode at depths that would support anaerobic and aerobic processes, respectively. Following on-site incubation, we further enriched the anodic electrode in the laboratory and confirmed&#160;a distinct microbial community compared to the surface-attached or biofilm communities normally observed at the Cedars. This enrichment subsequently led to the isolation of the first electrogenic microbe from the Cedars. This method of on-site microbial enrichment has the potential to greatly enhance the isolation of EET-capable bacteria from low biomass or difficult to sample habitats.

Current production was successfully measured for approximately 3 months using a voltage data logger as shown in Figure 3. This time was chosen as it was the longest stable incubation period for the spring, due to strong fall rains affecting the spring. A shorter period could be sufficient, though a longer period could provide stronger enrichment of biomass. We confirmed the connection of the two-electrode system after electrochemical incubation and observed no evidence of corrosion in the system. Higher current production was observed in the two-electrode system with lower resistance (1,000 &#937;) compared with negative control with 100 k&#937; resistance. The gradual current production increase in the first month may suggest the growth, accumulation or accommodation of microbes on the surface of the electrode following stable current production for another two months. Interestingly, current production oscillated in an approximately 24 h cycle through the whole period of the electrochemical enrichment.
To confirm the current production capability of microbial consortia attaching on the electrode, we performed chronoamperometry with the collected anode in the laboratory using a 3-electrode electrochemical reactor. We poised the electrode potential at +0.4 V vs. a standard hydrogen electrode (SHE) in the presence of various carbohydrate electron donors. The daily oscillations were no longer observed on the anode when incubated in the laboratory. This suggests that environmental factors impacted the microbial current production, and likely resulted in the observed oscillations.
Comparing the microbial community observed on the enriched electrodes with the attached and planktonic non-electrode communities, we observed distinct differences in the over-arching structure (Figure 4). The electrode microbial community was highly enriched in operational taxonomic units (OTUs) from uncultured lineages, as well as the Firmicute lineages of Bacillus. A shift in the composition of Proteobacteria was also observed; specifically, Betaproteobacteria (predominantly Serpentinamonas sp.) dominated the environmental calcite and planktonic samples, and Gammaproteobacteria dominated the electrode samples10. Differential enrichment of microbial strains between the environment and electrode samples provides support for microbial activity driving the observed experiment. This was further supported through the ultimate isolation of an electrochemically active strain from the enriched Firmictutes OTUs for the Cedars9.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57632/57632fig1.jpg" /> 
Figure 1: Electrochemical system. (a) Schematic image of the on-site electrochemical system to enrich EET-capable bacteria in the environment. An anode of carbon felt accepts respiratory electrons from the microbe and a cathode of Pt-electroplated carbon felt catalyzes oxygen reduction. Current production was monitored by a data logger V&#160;connected in parallel with both ends of a resistor R. (b) Setup example in the Cedars spring where the anode was put at the bottom of spring and cathode near water surface. (c) Protection of data logger and resistor&#160;by a plastic bag and&#160;a rock. The size of the anode is the same as the one shown in Figure 2. <a href="/files/ftp_upload/57632/57632fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57632/57632fig2.jpg" /> 
Figure 2: Carbon felt electrode connected to a titanium wire. <a href="/files/ftp_upload/57632/57632fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57632/57632fig3.jpg" /> 
Figure 3: Current production observed in the two-electrode system for a three-month incubation period.&#160;Data for systems using resistors of 100 k&#937; and 1,000 &#937; are shown in (a). Background current was subtracted to zero the initial current value. Panel (b) corresponds to the square in panel (a). Daily current oscillations were observed across the experiments illustrated in panel (a).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57632/57632fig4.jpg" /> 
Figure 4:&#160;Microbial community sequence distribution for Camp Site springs. DNA extracted from filtered water (CampsiteSpring Planktonic) or 1 g of calcite taken from the pool bottom (CampsiteSpring Calcite Attached) were compared to&#160;DNA extracted from carbon felt electrodes (Electrode Attached) or DNA from cells in the fluid phase of the electrochemical reactor&#160;(Electrode Planktonic). Sequence designations are based on phylum-level or class-level identities for the dominant phyla Firmicutes and Proteobacteria. Abundances are based on percent total reads. Changes in Proteobacterial lineages are outlined in dotted lines. <a href="/files/ftp_upload/57632/57632fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

On-site microbial enrichment or in situ cultivation techniques can facilitate the isolation of difficult-to-culture microbial taxa, especially from low-biomass or geochemically extreme environments. Here, we describe an electrochemical set-up without using an external power source to enrich microbial strains that are capable of extracellular electron transport (EET).

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

Nanyang Technological University

Research

JoVE Journal

Environment

7.5K Views.  National Institute for Materials Science. On-site microbial enrichment or in situ cultivation techniques can facilitate the isolation of difficult-to-culture microbial taxa, especially from low-biomass or geochemically extreme environments. Here, we describe an electrochemical set-up without using an external power source to enrich microbial strains that are capable of extracellular electron transport (EET).

Video: 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.

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

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.

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.

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.

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.

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.

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.

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).

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.

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.

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