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>

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	<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 anaerob...

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

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

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 n...

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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 ...

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7.6K Views.  National Institute for Materials Science. On-site microbiële verrijking of in situ -teelttechnieken kunnen het vergemakkelijken van het isolement van de microbiële taxa moeilijk-aan-cultuur, vooral uit laag-biomassa of geochemically extreme omgevingen. Hier beschrijven we een elektrochemische opstelling zonder gebruik te maken van een externe krachtbron te verrijken van microbiële stammen die kunnen extracellulaire elektronentransport (EET).

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

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

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

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

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

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 ...

Electrochemically and Bioelectrochemically Induced Ammonium Recovery

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

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

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

Chemistry

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

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

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

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

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

Biochemistry

Characterizing Electron Transport through Living Biofilms

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

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

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

In Situ Simulated Anaerobic Method for Culturing Environmental Microbes

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

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

Author Spotlight: Unraveling the Mysteries of Terrestrial Anaerobic Microorganisms in Uncharted Environments by In Situ Culturing

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

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

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

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

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

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

Bioengineering

Chronoamperometry Analysis of EET in Lactiplantibacillus plantarum with DHNA

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

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

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

Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Biology

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and inflammatory mediators, which dynamically alter community composition and microbial metabolic output. Understanding how physiological culture conditions affect microbial communities and diversity and their metabolic capacity will contribute important knowledge of their impact on health and diseases. Here, we present a protocol adapted from the template published by Auchtung et al. to create mini-bioreactor arrays that can cultivate complex fecal communities, define bacterial consortiums or single strains under precise conditions, including nutrient availability, temperature, flow rate, pH, and oxygen content. We describe the process for building the mini-bioreactor system, including adaptations to improve limitations in the published protocol. We also discuss the setup of the mini-bioreactor system under anaerobic conditions with the use of MEGA media (adapted from Han et al.), which is a rich media supporting the growth of diverse bacteria. We describe the inoculation of gut humanized mouse fecal samples into the mini-bioreactor system, followed by the establishment of complex community cultures within the mini-bioreactor system, which can be grown under continuous flow with aseptic sampling to monitor community composition. This system is adaptable to dietary changes and other cultural modifications. The techniques described here allow for the characterization of diverse, fastidious fecal communities under dynamic conditions or in response to perturbation in isolation from host-derived factors.

This protocol details the assembly of mini-bioreactor arrays to be utilized for continuous flow culture of complex fecal communities under anaerobic conditions. We also discuss the methods for assembly, inoculation, and sampling of the reactors for further analysis.

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

Samenvatting

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