Cultivating electroactive microbes—from field to bench

Electromicrobiology is an emerging field investigating and exploiting the interaction of microorganisms with insoluble electron donors or acceptors. Some of the most recently categorized electroactive microorganisms became of interest to sustainable bioengineering practices. However, laboratories worldwide typically maintain electroactive microorganisms on soluble substrates, which often leads to a decrease or loss of the ability to effectively exchange electrons with solid electrode surfaces. In order to develop future sustainable technologies, we cannot rely solely on existing lab-isolates. Therefore, we must develop isolation strategies for environmental strains with electroactive properties superior to strains in culture collections. In this article, we provide an overview of the studies that isolated or enriched electroactive microorganisms from the environment using an anode as the sole electron acceptor (electricity-generating microorganisms) or a cathode as the sole electron donor (electricity-consuming microorganisms). Next, we recommend a selective strategy for the isolation of electroactive microorganisms. Furthermore, we provide a practical guide for setting up electrochemical reactors and highlight crucial electrochemical techniques to determine electroactivity and the mode of electron transfer in novel organisms.


Introduction
Living things conserve energy by translocating electrons from an organic food substrate (electron donor) to a terminal electron acceptor (e.g. oxygen) via redox reactions in a respiratory chain. During classical respiration, these redox reactions are intracellular. In electroactive microorganisms, electron transfer reactions extend beyond the cell surface in a process called extracellular electron transfer (EET) [1][2][3][4]. EET is a unique metabolic feature that enables electroactive microorganisms to use solid-state electron donors or acceptors located outside the cell, which would otherwise remain inaccessible. Electroactive microorganisms have the unparalleled capability to 'release' or 'retrieve' electrons from a solid-state extracellular substrate. Microorganisms 'releasing' electrons onto a solid-state extracellular electron acceptor are electrogens whereas microorganisms that 'retrieve' electrons from an extracellular electron donor are electrotrophs. Electrogens are capable of electron release onto an electrode/anode surface, which is quantifiable as a positive electric current whereas electrotrophs retrieve electrons from a poised electrode/cathode surface, which is quantifiable as a negative electric current [5]. Original content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Electroactive microorganisms (electrogens and electrotrophs) employ different mechanisms of EET, which are direct EET or indirect/mediated EET (figure 1).
Electroactive microorganisms have earned considerable attention in the field of applied microbiology. Accordingly, bioengineering technologies have been developed to match the direction of the electron flow to cells (microbial electrosynthesis) and from cells (microbial fuel cells), independent of the electron transfer mechanism. There are two focus areas in the application of electroactive microorganisms that can be distinguished by the direction of electron flow: electronreleasing bioanodes when cells remove electrons from the feed to be 'released' onto an anode; and electron-retrieving biocathodes when cells 'retrieve' electrons from a cathode to use them as feed.
Some of the earliest bioelectrochemical systems dealt with bioanodes in microbial fuel cells (MFC) where microbes converted chemical energy from food substrates into electrical energy by transferring electrons to an anode [37,38]. In MFCs, microorganisms oxidize simple/complex organics (e.g. glucose) or mixed organics from wastewaters [39], while producing high anodic current densities with coulombic efficiencies as high as 100% [38,[40][41][42]. The effectiveness of MFCs for the production of electrical energy remains a matter of debate [43,44]. Nevertheless, MFCs were successfully applied to purify wastewater [39], to bioremediate toxic chemicals [45], or to adjust the redox balance of a fermentation broth [46][47][48]. These properties make MFCs a technology of interest, especially for remote geographic locations where access to water purification and bioremediation technologies is limited [49].
Until now, electroactivity testing and downstream biotechnology applications rely mostly on laboratory strains isolated and maintained on soluble substrates. Nevertheless, strains adjusted to soluble substrates adapt to a (non-EET) non-selective metabolism by decreasing the expression or by losing components for EET. On the other hand, their environmental analogs maintain EET competence in order to function in a selective EET environment (e.g. mineral rich sediments). For example, two G. sulfurreducens strains isolated with bioelectrochemical methods (table 1) led to higher power outputs than laboratory strains of the same species [73,74]. Moreover, lab cultivation on diffusible substrates (H 2 ) led to diminished cathodic electron use in M. maripaludis, which lost the entire genomic island relevant for EET [36]. In extreme cases, we may be deceived on the electroactivity of a species by studying solely culture collection strains maintained on soluble substrates. This was the case for the culture-collection strain Rhodopseudomonas palustris ATCC 17001, which could not use an anode as electron acceptor whereas a same species isolate from an MFC, R. palustris DX-1, did produce anodic current [75].
Since electroactive microorganisms lose their EET-capabilities when grown under non-specific conditions in the laboratory, enrichment of biotechnologically relevant and effective electroactive microorganisms requires an electrochemical isolation approach. Throughout an electrochemical isolation procedure, electrical current (negative or positive) will provide the selective pressure for the isolation of electroactive microorganisms. In this review, we provide an overview of (table 1) and suggest a strategy for (figure 2) enrichment and isolation of environmental isolates with innate electroactivity. We provide a guide for the isolation of electroactive microorganisms in a standardized microbial electrochemical system (box 1), particularly suited for anaerobes, and finally offer an overview of essential methodologies to detect electroactivity and distinguish between direct and facilitated electron transfer.

Niches for electroactive microorganisms
It is anticipated that electroactive microorganisms occur in environments where a solid-state extracellular electron acceptor or donor is naturally abundant, offering a positive selective pressure for an electroactive metabolism to dominate such a specialized ecological niche. Surprisingly, in a previous review, authors did not find a specific ecological niche for electroactive microorganisms [61]. Most electroactive species described have been isolated on soluble substrates. However, their natural distribution is not suggestive of niche partitioning [61], likely because these species typically do not perform EET in their environment. In other words, species easily isolated on soluble substrates that preserve their EET properties are unsurprisingly not exhibiting EET-niche partitioning, probably because they adopted a generalist behavior and are adjusted to a variety of soluble substrates typical of their environment.
Environments with a predominance of solid-state electron donors and acceptors include: (i) Iron-rich minerals. Iron is the most abundant metal on Earth, so microorganisms have adapted to use iron from minerals such as magnetite or pyrite, as a source or sink of electrons (see below) [76,77]; (ii) Metallic iron (Fe 0 ). Unalloyed Fe 0 is rare in the Earth's crust (e.g. in serpentinite; iron ores), unless mined and enriched for human use (mild-steel infrastructure). Nevertheless, during the Anthropocene microorganisms adjusted to using Fe 0 as an electron donor [78]; (iii) Carbonaceous materials. Some non-metallic materials occur in the environment, have the property to conduct charge and can therefore be used as donors or acceptors by microorganisms. Examples of carbonaceous materials are chars [79] and humic acids, the later includes the majority of undegradable organics in sediments and soils [80]. Chars are especially abundant in areas affected by forest fires [81] or are added to agricultural soils to stimulate plant or the decontamination of toxins [82]; (iv) Other cells act as electron donors and acceptors carrying thermodynamically synchronized metabolic interactions by sharing electrons via extracellular molecular electrical conduits (see below) [56,[83][84][85].
Cell-to-cell interactions in aquatic sediments may also strictly rely on naturally abundant conductive minerals to transfer electrons in between metabolically codependent microorganisms [86].
Some natural occurrences of iron rich-minerals are the conductive structures found in hydrothermal vent chimneys or serpentinizing springs, aquatic sediments and soils [87][88][89]. Environments where conductive minerals abounded dominated throughout Earth's history. Ancient oceanic environments were iron-rich [90] and likewise are present analogs (e.g. lake Matano Indonesia, lake La Cruz, Spain) [91,92]. One environment where electroactive microorganisms may have adapted over long evolutionary time scales [93] to electrically conductive surfaces are hydrothermal chimney walls [94][95][96], which are thought to spontaneously generate electricity [94,95]. In fact, hydrothermal vent isolates were capable of EET with insoluble electron donors like rocks/minerals [97][98][99][100] or electrodes [101,102]. Last but not least, some microbial species can exchange electrons with each other by transferring electrons from an electrogen to an electrotroph via direct interspecies electron transfer (DIET) [56,[83][84][85]. During DIET, the electrogen is provided with an electron donor for oxidative metabolism, but without any of its electron acceptor, whereas the electrotrophs is provided only with an electron acceptor. Thus, by coupling Wastewater EDs and an anode no set potential (TEA)

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Dilution plating on agar-media with glucose (ED) and Fe(III)citrate (TEA) [162] Aeromonas hydrophila PA3 Undefined inoculum Acetate (ED) and an anode no set potential (TEA)

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Serial dilution MFCs with acetate (ED) and an anode no set potential (TEA). 5x

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Anode biomass streaked on agar plates with acetate or lactate (ED) and manganese dioxide (TEA) [175] Citrobacter sp. strain ND-2 Rice paddy soil Note: All the potentials reported here are against the standard hydrogen electrode. MFC microbial fuel cell; AQDS Anthraquinone-2,6-disulfonate; ED electron donor; TEA terminal electron acceptor; FTO fluorine-doped tin oxide. Primary, secondary, tertiary and quaternary refer to the succession during the enrichment/ isolation procedure. a Labrenzia species were inactive on a cathode. Only one showed an FeS-oxidation band for three successive transfers. b Not tested on a cathode, however, it did not maintain FeS-oxidation activity over three successive transfers. c One strain (Marinobacter adherens) was electroactive using a cathode as electron donor only when 2 mM acetate was provided as carbon source. the two processes the dual-species consortia can thrive. During DIET, the electrogen requires the same EET conduit that is required for interactions with electrodes, which includes pili and extracellular MHC-cytochromes [1]. The dependency on the electrical conduit was verified with genetically manipulated partners, incapable to express an EET conduit, and with partners known to use other EET mechanisms (e.g. H 2 rather than DIET) [84,85]. Conversely, it is challenging to demonstrate DIET in the environment, as we do not have a specific molecular or chemical fingerprint. Despite this, DIET has been reported by indirect measurements in environments such as anaerobic digesters [103,104], rice paddies [105], or deep-sea sediments [106,107]. In environmental consortia, DIET is often endorsed by indirect observations such as: (i) high conductivity of the consortia [103,104]; (ii) failure to make use of diffusible formate or H 2 [103]; (iii) high expression of genes associated with EET [105][106][107]; (iv) stimulation of the metabolism by conductive materials [108]; (v) or by phylogenetic affiliation to DIETspecies [109]. However, in these environments, the actual mechanism of interaction and partner co-dependency remain a matter for future inquiry.
Finally, the ability to exchange electrons with the extracellular milieu provides a selective advantage for electroactive microbes in a variety of ecological niches in the environment. Of these pre-adapted electroactive species we can selectively isolate novel strains, characterize them, and use their properties in sustainable technologies relying on bioelectrochemical systems.

Electrochemical enrichment and isolation
The challenge during the isolation of electroactive strains is that isolation on non-selective media was previously shown to lead to loss of electroactivity [110][111][112]. We reviewed the studies that employed electrochemical technologies to obtain electroactive strains (table 1). Sometimes, isolation was possible despite the use of unselective media during the procedure. Nonetheless, below we will focus on those studies, which maintained the selective pressure throughout the steps of enrichment and isolation, with the help of solid-state electron sinks or sources.
A suitable approach to enrich electroactive microorganisms involves the use of in situ electrodes, because it overcomes enrichment bias artifacts [113] that would otherwise lead to changes in cultivability [114] or viability [115], for example due to grazing [116]. In situ enrichment often leads to the isolation of new electroactive strains. For example, an anode inserted directly into a borehole of a deep underground mine provided a niche for the growth of the electroactive Desulfuromonas soudanensis [117]. Different approaches were used to enrich electroactive organisms from groundwater or sediments, in situ. Thus, for in situ colonization, the groundwater from 1478 m depth was passed through a self-designed electrochemical reactor equipped with four electrodes, two poised at oxidizing, and the other two at reducing potentials [118]. For in situ enrichment from sediments, naturally existing redox gradients can be exploited in benthic or sediment MFC (SMFC). SMFCs operate with the anode embedded in the anoxic sediment and the cathode in the oxic water above [44]. The organics in the sediment provide the electron source, while O 2 in the water above acts as the electron sink. SMFCs can selectively enrich native electroactive microorganisms both at the anode and the cathode. This was the case of the electrogen Dietzia sp. RNV-4, which was isolated from the anode of a river sediment SMFC [119], whereas the electrotroph 'Candidatus Tenderia electrophaga' was enriched from the biocathode community of a marine phototrophic SMFC [110,111,120,121].
Generally, the isolation of a species requires growing it from a single cell to ensure a single cell origin. Besides, isolation of a species with unique traits requires sustaining the selective pressure for the entire duration of the isolation. Attempts to isolate electroactive strains often involve unselective media such as solid-LB, due to the simplicity of the isolation procedure, which requires only aerobic streaking to attain single cell colonies [119,[122][123][124]. Conversely, only a few studies upheld selective conditions during enrichment and isolation by adding insoluble electron acceptors to the dilution series [125,126]. Insoluble Fe(III)-oxides have been often used as electron acceptors to isolate electrogenic microbes [117,127]. Nonetheless, by providing insoluble minerals as electrode replacements, we may restrict isolation to microbes skilled for example at insoluble Fe(III)oxiderespiration, but unskilled at electrode-respiration, which was the case of Geobacter bremensis [128].
Isolation of electrotrophs by conventional methods is more challenging than that of electrogens which led to a low number of cathodic isolates (table 1). Electrotrophs are of interest for biotechnology [112,129,130], but are usually isolated with soluble electron donors [131,132]. Some exceptional strains were enriched with metallic iron (Fe0) as an extracellular source of electrons [132][133][134]. However, the researchers discontinued the use of a solid electron donor during the strain purification procedure and instead set up dilution series with H 2 or other soluble/diffusible substrates [120,123,135,136]. Growth on soluble substrates could lead to incapacitation of the strains in using the solid surface at all, as was the case with M. maripaludis strains, which lost the genomic islands relevant for EET-constituents when grown on H 2 [36].
Consistently, many authors applied one ineffective strategy for the isolation of electroactive microorganisms, which is aerobic cultivation with nutrient-rich agar (table 1) [111, 122-124, 131, 136-142]. This strategy favors fastgrowing, oxygen-respiring organisms over electrotrophic ones, obscuring downstream electrochemical studies, and interpretation of data. For example, multiple isolates obtained from a phototrophic SMFC on rich-agar media were not electroactive [111], whereas the actual cathodic microorganism 'Candidatus Tenderia electrophaga' could not be enriched Box 1. Protocol to set up bioelectrochemical reactors for microbial electrosynthesis Principle: An electrochemical reactor consists of one or two chambers with at least two electrodes submerged in a conductive ionic solution (electrolyte). In bioelectrochemical systems, the electrolyte is usually the growth media of the microorganisms without an external electron donor and acceptor. The electrodes used are a working electrode (WE) and counter electrode (CE), with the redox reaction of interest happening at the WE. The circuit (WE/CE) can be closed when the two electrodes get connected to a potentiostat. For precise control of the potential, the WE can be calibrated against a reference electrode (RE) by the potentiostat. The chambers are perferably segregated to keep the oxidation and reduction reactions isolated. Typically, the two compartments are separated by a membrane selective for proton exchange. The following protocol is based on the set up used in our lab and has been tested successfully for cultivation of strict anaerobes such as methanogenic Archaea (e.g. [56]). The list of materials used available in the supplementary materials.
Preparation of the working and counter electrodes 1. Wash the graphite block. Soak in 1 M HCl overnight. Soak in 1 M NaOH overnight. Rinse with deionized water until the pH of the refuse is neutral. Air dry before proceeding 2. Drill a hole on top of each graphite block 2 cm (h) × 2 mm (ø) (figure S1 is available online at stacks.iop.org/NANO/31/174003/ mmedia) 3. Coat one end of a Ti-wire 2 mm (ø) × 12.5 cm (l) with conductive epoxy and insert the wire into the hole of the graphite block. Coat the wire-graphite junction with a biocompatible non-conductive epoxy 4. Cure the epoxy by baking the electrode-wire set up at 80°C for 3 h 5. The electrode-wire connection is tested with a multimeter by examining the resistance between the graphite block and the wire. A good electrical connection gives an internal resistance below 10 Ω 6. To ensure anaerobiosis, reactors are secured with black GL45 rubber stoppers pierced to fit the disconnected end of the Ti-wire. Stoppers can be drilled or pierced with an 18 G heated needle to produce holes of ∼2 mm (ø) (figure S1). Seal the junction between the wire and the stopper with epoxy to avoid possible gas leaks or O 2 -contamination Preparation of the reference electrode 7. Pierce a hole 2 mm (ø) through a blue butyl septum 20 mm (h) using a heated 18 G needle 8. Insert the reference electrode (RE) from the top to protrude ∼4 cm below the stopper and ensure close proximity to the working electrode ∼1 cm (for a 500 ml chamber; see figure S1). Do not seal the junction because sometimes RE must be changed 17. For the chamber with the WE, replace the stopper for the middle port with a sterile RE joined to a rubber stopper ( figure 4). Work quickly and close to a flame to ensure sterility.
18. Degas the chamber for 10 min to reestablish anoxia. 19. Pressurize a bottle of sterile media with sterile N 2 CO 2 ( figure S2). Use the pressure buildup to push ∼550 ml media into the H-cell chamber. Connect the media bottle to the reactor chamber. For this use a sterile tube with Luer-lock adapter ends fitted with needles and controlled by a valve ( figure S2).
on any rich media [121]. Therefore, several researchers developed small-scale electrochemical reactors for the isolation of electroactive microbes. With this approach, Geobacter sp. SD-1 and Ochrobactrum anthropi YZ-1 were isolated via successive liquid dilutions to extinction series in electrochemical reactors exclusive of nutrient-rich media [125,126]. Additionally, an 'electrode-plate method' has been successfully employed to isolate electrogenic microorganisms [141]. The authors used a diluted cell suspension streaked on agarplates containing the soluble electron donor, however with a transparent anode at the top as a solid-state electron acceptor. Besides the anode, a reference and counter electrode were placed inside the agar for precise control of the voltage. It remains to be tested whether this electrode-plating method has applicability in the reverse direction in order to isolate electrotrophs. Published reports revealed challenges in finding an appropriate solid-state electron donor for electrotrophs. Solid-state electron donors like Fe 0 although successfully applied in liquid media [132] pose two problems in solid media-one being the lack of specificity because Fe 0 generates H 2 gas abiotically, and secondly H 2 -gas would induce fractures in the solid media rendering isolation of single cell colonies impossible. As an alternative to Fe 0 , we recommend to use other biocompatible materials that can store charge and be pre-reduced electrically, such as Prussian Blue (a low-cost hexacyano-Fe complex material [144]) or biochar [145], as solid-state electron donors for selective isolation on agarplates.
Below we present an electrochemical cultivation strategy (figure 2) by combining strategies presented in previous studies, including in situ primary enrichment in bioelectrochemical setups (see table 1), followed by laboratory electrochemical enrichment and dilution to extinction series in liquid or solid media with electrodes as electron donor/acceptor, as described by four previous reports [125,126,143,147]. Most electroactive microorganisms are anaerobes [61]; thus, we propose to conduct all steps under anoxia because many anaerobes get inhibited by exposure to O 2 . Anoxia can be achieved by working under a N 2 gas-stream, or ideally inside an anaerobic bag/chamber. For isolation under selective conditions, we propose to follow five (4-optional) liquid electrochemical dilutions to extinction; (5) dilution to extinction on solid-media via electrode-plating.
Step 1: Electrode colonization in situ (e.g. SMFC). The biofilm colonizing the electrode in situ would be the ideal source for single cell direct isolation on solidified media after biofilm detachment. Alternatively, electroactive microorganisms from an environment can be sorted based on polarizability [147] for downstream isolation.
Step 2 (optional): Transfer the electrode-biofilm to a media with a chemical composition similar to the in situ water (enrichment). Enrichment biases are likely [113][114][115], and therefore this step could be discarded for the next step.
Step 3: There are two ways to carry out mechanical detachment by scraping the electrodes or by light sonication (few cycles at <20% intensity to ensure cell integrity) followed by gentle rinsing with a stream of anoxic media. This step should preferably occur in an anaerobic chamber.
Step 4 (optional): The biofilm-suspension can be used for liquid dilutions to extinctions, ideally, in batch bioelectrochemical reactors ( figure 3). In box 1, we illustrate a protocol for anaerobic batch bioelectrochemical reactors. This step could be repeated until one morphotype and 16 S phylotype becomes isolated. Membrane-less, small volume, high throughput electrochemical cells [148] were previously used to enrich anode respiring electrogens and reduce the costs of isolation associated with dual chamber BES. However, membrane-less BES cannot be used to isolate strict anaerobes because inhibitory O 2 is produced at the anode.
Step 5: Ultimately, for isolation of a new species the highest liquid dilution in which growth was observed is used as inoculum for a dilution series in solid media. Solid dilution series should provide us with colonies from a single cell. To maintain the selective pressure, we advise using the electrode-plate method [143]. Another possibility is to place the inoculum at a distance from the electrode, so electroactive cells use taxis towards a solid-state electrode [99,100].

Electrochemical tests
Once isolated, the new strains must have their electrochemical properties tested because the mere association with an electrode is not proof of electroactivity. Nowadays, various types of high throughput methods demonstrate electroactivity, relying on electrochromic approaches with tungsten oxide (WO 3 ) [149], electrochemiluminescence [150], colorimetric [151,152] and dielectrophoretic methods [153]. An example of a high-performance, eco-friendly approach for rapid electrochemical characterization is a paper-based 64-well sensing array containing MFC wirings (anode and cathode connected with a load) [154]. Nonetheless, these methods are not commercially available, so the use of conventional bioelectrochemical techniques is still necessary for standardization between laboratories. Some of the conventional bioelectrochemical techniques are chronoamperometry and cyclic voltammetry. Chronoamperometry helps investigate the ability of a new isolate to facilitate electron transfer to and from an electrode [124,132,155]. Cyclic voltammetry helps distinguish between a direct and facilitated EET mechanism [117]. Hence the two must be used in combination to determine the type of EET mechanism employed by an electroactive microorganism.
Chronoamperometry (CA) is a technique in which the potential of a working electrode (exposed to microorganisms) stepped against a reference standard electrode gives a current response (mA), to be recorded over time ( figure 4). For instance, microorganisms transfer electrons to the working electrode (anodic reactions) leading to the production of positive current, while their uptake of electrons from the working electrode (cathodic reactions) produces a negative one ( figure 4). CA in a batch reactor is usually carried out until the current output stops and falls back to the baseline conditions when the soluble electrode acceptor or donor got depleted. From the current output, we can calculate current density and coulombic efficiency, which can then be used to compare performance with other studies [156]. For example, in an MES-system, the coulombic efficiency describes the recovery of the consumed current in the form of a synthesis product. For a methanogenic MES, the overall coulombic efficiency (ηCE,%) can be calculated from the amount of current consumed (I) for the formation of reduced products (CH 4 , 8 electrons) for the given time (t) according to the equation (1) where F is the Faraday constant ( 96485.332 C mol −1 ), m is the number of moles and n is the During cyclic voltammetry (CV), the potential is cycled between two setpoint potentials (V1 and V2), while the resulting current flow gets measured throughout the scan ( figure 4). CV produces both an oxidative and a reductive current curve for the potential range between V1 and V2 ( figure 4). Electroactive species carrying out reversible reactions between the electrode and the microbe may produce two current peaks, one for each direction (cathodic and anodic).
The CV technique can also be applied to distinguish the mode of electron transfer (direct or facilitated). Direct electron transfer should exhibit electrode-associated electroactivity, which we can assess by comparing current production rates of a microbial culture before and after exchanging the entire liquid volume. If the performance is similar in both conditions, the electroactive agent is localized at the electrode surface and not in solution. In the case of poor biofilm formers, we typically compare the CV of the grown culture to that of the spent cell-free filtered medium. This approach helps identify whether the planktonic cells or a soluble shuttle in the medium are involved in electron exchange with the electrode. For detailed information on how to analyze cyclic voltammograms, as well as data from other electrochemical techniques, we recommend several excellent guides written by other research groups [157][158][159][160].

Conclusions
The field of electromicrobiology is rapidly emerging. Applications utilizing the ability of microorganisms to transport electrons extracellularly have moved far beyond its initial intended use in electricity generation. For example the development of hybrid bioelectrical systems with the ability to reduce carbon bonds with electricity or light [161]. With advances in several interdisciplinary fields, including electrochemistry, material science and biotechnology, microbial electrochemical systems have a real potential to provide meaningful solutions to current energy problems. There is increasing interest in the biotech potential of microorganisms capable of EET. However, electroactive microorganisms could not get isolated via traditional means. Here, we have provided an overview of studies that isolated electroactive microorganisms from the environment; and supplied guidelines for bioelectrochemical isolation methods aspiring to promote the discovery of additional electroactive species for biotechnology.