Weak electricigens: A new avenue for bioelectrochemical research

Bioresource Technology 258 (2018) 354–364

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Review

Weak electricigens: A new avenue for bioelectrochemical research a

Lucinda E. Doyle , Enrico Marsili a b

a,b,⁎

T

Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, 637459, Singapore

G RA P H I C A L AB S T R A C T

A R T I C L E I N F O

A B S T R A C T

Keywords: Electricigens Biofilms Bioelectrochemistry Extracellular electron transfer Microbial fuel cells

Electroactivity appears to be a phylogenetically diverse trait independent of cell wall classification, with both Gram-negative and Gram-positive electricigens reported. While numerous electricigens have been observed, the majority of research focuses on a select group of highly electroactive species. Under favorable conditions, many microorganisms can be considered electroactive, either through their own mechanisms or exogenously-added mediators, producing a weak current. Such microbes should not be dismissed based on their modest electroactivity. Rather, they may be key to understanding what drives extracellular electron transfer in response to transient limitations of electron acceptor or donor, with implications for the study of pathogens and industrial bioprocesses. Due to their low electroactivity, such populations are difficult to grow in bioelectrochemical systems and characterise with electrochemistry. Here, a critical review of recent research on weak electricigens is provided, with a focus on the methodology and the overall relevance to microbial ecology and bioelectrochemical systems.

1. Introduction Microorganisms capable of electronically interacting with conductive surfaces have been described by various names; electroactive microorganisms, exoelectrogens, electric bacteria and electricigens, as they will be referred here (Koch & Harnisch, 2016; Logan, 2009; Lovley, 2006; Nealson, 2017). Electricigens are defined by their ability to carry

⁎

out extracellular electron transfer (EET). As the name hints, EET involves the movement of electrons between the internal cellular environment and a conductive solid beyond the cell boundary, which can act as either an electron acceptor or donor. The conductive solid usually takes the form of either an electrode, as is common in the laboratory, or a metal, which typifies the natural environment that has driven the evolution of this mode of respiration (Lovley, 2008). EET can be viewed

Corresponding author at: Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore. E-mail address: [email protected] (E. Marsili).

https://doi.org/10.1016/j.biortech.2018.02.073 Received 10 December 2017; Received in revised form 15 February 2018; Accepted 16 February 2018 Available online 20 February 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.

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converting organic matter to current, the more interesting it may prove to the researcher or engineer keen to develop MFCs. However, this approach limits observation of what can be termed weak electricigens; microbes engaging in EET on a smaller scale. There is not yet an agreed definition of weak electricigens. However, viewing electroactivity as existing along a spectrum may be a good starting point. On the extreme end lay non-electricigens, who die in the absence of a soluble electron acceptor. Beyond this are weak electricigens, who typically rely on soluble electron acceptors in their natural environment, but who can avail of solid electron acceptors while under stress, likely with an associated limitation in growth during this mode of respiration. Finally, at the other extreme end lay strong electricigens. These species typically rely on solid electron acceptors in their environment, growing well under such conditions, and can usually avail of soluble acceptors if required. Operatively, a microorganism can be classed as a weak electricigen if it produces a small current or has low coulombic efficiency (CE). Small, in this case, is relative, as even between the strongest electricigens, Geobacter and Shewanella, a large difference in current production is seen. As Geobacter can produce up to 10 times the current of Shewanella, the genus can be viewed at the extreme end of the electricigen spectrum. Therefore, Shewanella can be taken as a fair reference point, with weak electricigens deemed as delivering a 10-fold smaller comparative current. However, as current production and CE depend on a multitude of parameters, such as electrode size, carbon source and the extent an anaerobic environment is maintained, the authors have opted not to provide specific cut-off numbers which separate the strong from the weak. This difference in electroactivity between Geobacter and Shewanella may be indicative of the important physiological conditions that drive EET. Geobacter is a strict anaerobe that can use oxidised metals as its sole electron acceptor indefinitely (Caccavo et al., 1994; Lovley et al., 1993). It is likely that the more a species avails of oxygen as its terminal electron acceptor, the less efficient it will be at performing EET. In spite of being conducive to a relatively high level of electroactivity, strict anaerobic conditions are often accompanied by slow growth rates that could pose challenges in real-world applications, e.g., generating sufficient biomass to provide a time-sensitive signal in a biosensor. This may not be a limitation of weak electricigens, who are by definition not as specialised and therefore likely to grow more easily in a range of different environments. As an extension of this, weak electricigens may be able to compensate for a specific metabolic shortcoming of a strong electricigen, enabling a more stable bioprocess in the context of a mixed community. In contrast, Shewanella, a facultative anaerobe with a diverse metabolism (Ong et al., 2014) and easily amenable to genetic manipulation on the lab bench (Coursolle & Gralnick, 2012), is an example of a much less fastidious electricigen. As the deep ocean serves as a common habitat to this genus, psychrophilic (Zhao et al., 2006), thermophilic (Ghosh et al., 2003) and piezophilic (Toffin et al., 2004) strains have been reported, indicating broad metabolic capabilities at the aerobic/ anaerobic interface. Nonetheless, weak electricigens likely have an even broader metabolic diversity and, despite their small current output, warrant further investigation in order to develop novel EETbased devices. In pure culture, industrial applications using this niche of weak electricigens may still be challenging due to the inherent inefficiency of their electron transfer. However, in the absence of a strong counterpart, they may prove very useful. It must be noted that there is always a trade-off by generalists; the weak electricigen may well be a “jack of all trades”, but might not be considered a master of any, and therefore will likely not thrive while forced to carry out EET, with benefits for the cell itself open to discussion (Koch & Harnisch, 2016). Indeed, one possibility worth exploring is that certain weak electricigens engaging in EET may merely be able to survive, and not divide, during these conditions, enabling cell maintenance but not biomass production.

as a survival strategy employed when soluble electron acceptors or donors are not available at sufficient concentrations, thus enabling biomass synthesis and/or cellular maintenance to proceed (Hernandez & Newman, 2001). EET can occur under both anodic conditions (the electrode/metal is reduced) and cathodic conditions (the electrode/ metal is oxidised). Most studies refer to anodic EET, as this mode is common among electricigens and is easily observed in short-term experiments. Conversely, cathodic EET was only reported after the discovery of anodic EET (Gregory et al., 2004), is much less frequent, and is characterised by slow kinetics (Liu et al., 2014). Both anodic and cathodic EET are facilitated by a number of specific mechanisms (Schroder et al., 2015), with electricigens typically employing one or more at a time. Focusing on the anodic flow of electrons from the interior to the exterior of the cell: direct electron transfer (DET) is the movement of electrons across outer-membrane proteins, typically c-type cytochromes, to a solid external electron acceptor (Okamoto et al., 2011). A related mechanism is the conduction of electrons across pilus-like appendages termed nanowires, which extend from the cell membrane and close the distance between the cell and the external electron acceptor (Reguera et al., 2005; Reguera et al., 2006). The reader is referred to a recent comprehensive review by Lovley (2017) on this topic. Finally, the last described mechanism is mediated electron transfer (MET), whereby the cell secretes soluble redox shuttles that carry electrons to a nearby acceptor and diffuse back to the cell upon oxidation, facilitating a revolving door strategy of electron transport (Marsili et al., 2008; von Canstein et al., 2008; Wang et al., 2010). Recent research shows that the lines distinguishing these three modes of EET are somewhat fuzzy, as intermediate strategies such as adsorption of redox mediators to the cell surface (Okamoto et al., 2013) or immobilisation of mediators in the biofilm matrix (Xiao & Zhao, 2017) are also possible. For example, x-ray crystallography of key outer membrane multiheme cytochromes from Shewanella has unveiled not only intramolecular electron transfer pathways, but also the interaction between flavins and the cytochromes (Edwards et al., 2017). While DET, MET and nanowires are the main modes of EET described to date, it is important to note that these findings may not be exhaustive, and could be a consequence of the narrow range of electricigens discovered, with a recent review identifying 94 species described to date (Koch & Harnisch, 2016). Several reports suggest that the electricigens are much more abundant in nature (Cournet et al., 2010) and are present in most ecosystems (Chabert et al., 2015). In spite of this predicted and observed abundance of electricigens, the majority of in-depth bioelectrochemical studies still focus on strong electricigens present in niche environments, with little mechanistic insight available for the majority of alternative electricigens discovered. This trend is likely due to the initially envisioned application of electricigens in microbial fuel cells (MFCs), where strong current producers are highly desirable to maximise power output. However, as researchers have gradually relaxed this criterion, other electricigens have raised interest for their biotechnological and even biomedical applications. In this review, the recent developments in electricigen enrichment and characterisation are summarised, with particular regard to weakly electroactive populations and communities, who may find application in bioprocesses, biosensors and bioremediation; paths related to, but distinct from, the traditional power generation envisioned for strong electricigens. 2. Strong vs. weak electricigens A previous review (Doyle & Marsili, 2015) identified enrichment conditions as a possible reason for the relative lack of diversity seen in microbes capable of EET. The authors would here like to further build upon that discussion by suggesting that screening protocols may also play a large part in underestimating electricigen abundance in a culture, as strains producing a low current may quickly be ruled out. From an applied perspective, this elitist approach to quantifying electroactivity makes sense; the more efficient the microorganism at 355

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3. The role of weak electricigens in mixed microbial communities and potential applications

microbial ecology of weak electricigens in bioelectrofermentation and other reactions will allow control of the final product(s) of such processes, with broader possibilities when viewed from the vantage point of synthetic biology (Song et al., 2014). Industrial applications of weak electricigens and bioremediation can be considered in the context of hostile environments, such as mining wastewater low in pH, with preliminary studies using such microbes yielding promising results (Ni et al., 2016). As a consequence of strong electricigens’ high level of adaptedness to conditions that are conducive to EET, they may not tolerate such environments. For example, cytochrome conformation in the obligate anaerobe Geobacter is pH-sensitive (Morgado et al., 2017), whereas the acidophile Acidithiobacillus successfully produced electricity from tetrathionate at low pH (Sulonen et al., 2016). However, only a small portion of the electron flow was collected at the electrode, demonstrating that low CE is a typical trait of weak electricigens. Other potential applications include biorecovery of metals from electronic waste (Villares, 2017), antibiotic biosensors (Ward et al., 2014), viability sensors (Li et al., 2017; Selim et al., 2017) and of course the need for extremophilic weak electricigens, i.e., those resilient to extremes of pressure, temperature, or pH, as reviewed by Dopson et al. (2016) and Shrestha et al. (2018).

With microbes typically existing as part of a multi-species community in nature, it can be hypothesised that weak electricigens may engage in oxygen scavenging. This would maintain an anaerobic environment for strong electricigens, who may otherwise succumb to an oxygen-induced death. They may also perform complex substrate degradation, providing simple sugars for strong electricigens to oxidise further. Similar metabolic interactions have been previously demonstrated for co-cultures of Escherichia coli and Geobacter sulfurreducens (Bourdakos et al., 2014; Zhang et al., 2013). Once such initial duties have been performed, the weak electricigen may find itself without oxygen and engage its modest EET abilities in order to survive. As such, biotechnological applications are more likely to be possible using mixed communities where both strong and weak electricigens are present. For example, a mixed microbial community successfully degraded cellulose in a sediment MFC (Sajana et al., 2014). Contrary to what was demonstrated in early studies, the use of a strong electroactive community does not always provide an advantage in terms of long-term power production (Ewing et al., 2017). It is likely that a more robust weakly electroactive community will give higher power and increased stability with time, particularly for field applications. For example, synergistic and antagonistic effects among weak electricigens in an MFC treating refinery wastewater have been reported (Guo et al., 2014). In fact, observation of relevant synergistic effects dates back more than a decade, when it was observed that various weak electricigens can use redox mediators produced by Pseudomonas aeruginosa to enhance MFC power output (Rabaey et al., 2005). Potential applications of weak electricigens unrelated to bioelectricity production have been reported, particularly under the term bioelectrosynthesis, in both fundamental (Rabaey & Rozendal, 2010) and applied (Sadhukhan et al., 2016) reviews. Examples include the use of cathodic weak electricigen cultures for methane and acetate synthesis (Deutzmann & Spormann, 2017). In contrast with pure cultures, mixed microbial consortia are likely to perform better in bioelectrosynthesis. However, it is necessary to identify the key contributors to the bioelectrosynthetic process in order to build a synthetic consortium, as pointed out by Marshall et al. (2017). In recent years, the concept of bioelectrosynthesis, initially restricted to microbial electrolysis cells, has been broadened to include the areas of electrical-aided white biotechnology (Harnisch et al., 2015) and bio-electrofermentation (Schievano et al., 2016). These are processes that make use of electric potential or current to steer the metabolic pathway of specific organisms, thus altering the yield and the chemical composition of fermentation products. More than a mere question of semantics, the transition from bioelectrosynthesis to bioelectrofermentation can be seen as a change of perspective, in which electrochemical stimuli are integrated in conventional fermentation processes to achieve benefits such as an increase in carbon efficiency and enhancement of microbial growth. By definition, bioprocesses involving weak electricigens do not have energy as the main output. Notably, many microorganisms commonly used in fermentation processes are also weakly electroactive under well-defined conditions. Beyond traditional electricigens, genetic engineering has also been proposed to confer EET abilities for use with bioelectrosynthesis (Shin et al., 2017). Recently, Moscoviz et al. (2017) demonstrated that both microbial community structure and metabolic patterns were affected when a mixed fermentative community was exposed to cathodic conditions. In another study, application of a negative redox potential changed the fermentation products of Clostridium acetobutylicum, resulting in higher butanol production from fructose-based feedstock (Chen et al., 2017). Similar observations have been reported for wastewater, where application of a positive redox potential caused a shift of the microbial community to electricigens and methanogens, with an associated higher methane production (Guo et al., 2017). Understanding the

4. Discovering weak electricigens As weak electricigens may not occupy a specific ecological niche (in contrast with strong electricigens), their presence could be broadly distributed across nature, with EET becoming a viable, though less energetically favourable, mode of respiration in response to stressful conditions where soluble electron acceptors (or, theoretically, donors) are limited. Possible candidate weak electricigens include aerobic species, pathogens, and even commensals inhabiting the human gut where iron concentrations can be locally high (Abbaspour et al., 2014). As many enrichment studies utilising acetate or lactate as electron donor and carbon source have returned a high abundance of Geobacter and Shewanella, respectively, the hunt for novel weak electricigens may necessitate alternative medium compositions. Since high power outputs will not be a reasonable expectation from a weak electricigen pure culture, investigations could focus on answering why such microbes turn to this mode of respiration at all, in addition to the detection of novel mechanisms of EET which may differ from those employed by strong electricigens. 4.1. Screening of existing pure cultures for EET activity Rather than isolating electricigens from scratch, a novel approach can be to test existing microbial pure cultures for electrogenic abilities. Should EET be detected in these species, it is likely to fall under the umbrella of weak electroactivity, and may be indicative of a survival strategy utilised by the microbe in response to a limitation that occurs occasionally in its natural environment. Examples of such unexpected electricigens are discussed in Section 6. During such screening procedures, it is important to modify the species’ preferred growth conditions so that growth is encouraged but theoretically limited in some way, e.g. soluble electron acceptor removed (or donor, if screening for cathodic EET) and a poised electrode introduced instead. In terms of preparing candidate lists, one example could be to consider which species are associated with metal-rich environments or are resistant to oxidative stress. Such criteria may describe certain pathogens, e.g., those associated with hemochromatosis patients who have high internal iron concentrations (Khan et al., 2007). 4.2. Enrichment of weak electricigens In addition to screening existing pure cultures, novel isolates can be obtained through standard enrichment experiments. While the MFC design is a reactor configuration possible for such experiments, these 356

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devices are optimised for power output from electricigens. Since this, by definition, will not be a viable application for weak electricigens, they will not be discussed in this review. Instead, the recommendation is potentiostat-controlled set-ups capable of detecting small current outputs consistently; a design that has demonstrated reliable results (Pierra et al., 2015). Electrochemical cells consist of a three-electrode set-up where the working electrode is maintained at a controlled potential, measured against a reference electrode, and balanced by a counter electrode (Bard & Faulkner, 2001). The working electrode can be set at either anodic or cathodic potentials, enabling observation of both directions of EET. It has been observed that strong electricigens can perform electron transfer at low electrode potentials, such as −0.2 V (vs Ag/AgCl) and below, either through DET (Bond & Lovley, 2003) or MET (Marsili et al., 2008). In fact, high electrode potential tends not to increase the biomass accumulation in MFCs inoculated with strong electricigens (Wei et al., 2010). Conversely, weak electricigens such as Pseudomonas aeruginosa tolerate high electrode potentials well (0.4 V vs. Ag/AgCl) (Seviour et al., 2015). Therefore, one possibility for selective enrichment of weak electricigens is the use of potentiostat-controlled electrochemical cells where the working electrode is maintained at high oxidative potential. Additionally, redox mediators may be a very effective tool for weak electricigen enrichment and characterisation, as they allow amplification of the small current produced. As seen in Section 3, co-culture experiments can be designed with strong and weak electricigens. These experiments suggest a simple yet effective way of enriching weak electricigens from mixed microbial communities by providing complex substrates that cannot be metabolized by strong electricigens alone. A variation of this method was employed by the authors in a recent study, where two lactate-degrading weak electricigens, Enterobacter sp. EA-1 (Doyle et al., 2018) and Aeromonas sp. CL-1 were enriched in potentiostat-controlled electrochemical cells along with the well-known strong electricigen Geobacter metallireducens (Doyle et al., 2017). Overall, these observations indicate that specific weak electricigens may be enriched through a combination of redox mediator, electrode potential, and medium composition. In terms of the working electrode dimensions, macroelectrodes e.g., 1 × 1 cm carbon felt, provide a large surface area for bacterial attachment, enabling sufficient biomass for DNA and RNA extraction postexperiment (Doyle et al., 2017). The large surface area can also be useful in collecting current, however any variations in electrode size will reduce the repeatability of results due to the inherently small current output. Furthermore, such large electrodes have drawbacks when it comes to certain electrochemical techniques, as discussed in Section 5. Another option is mini-electrodes, such as screen-printed electrodes (SPE), whose working electrodes are a few millimeters in diameter. Advantages of SPE include reliability and small diffusional limitations, which favour the development of a thin, conductive biofilm (Sismaet et al., 2016; Ward et al., 2014). However, this technology is cumbersome for characterisation of anaerobic microorganisms, as the small volume of the sample favors oxygen diffusion, in addition to low biomass yields available for subsequent genetic analysis. Therefore, a combination of both macroelectrodes and SPE may be the optimum experimental set-up.

Fig. 1. Proposed approach for characterisation of weak electricigens in pure culture.

One might be tempted to adopt large electrodes and high surface area materials, like carbon felt or nanotube-coated graphene, to increase current output in a bioelectrochemical device, hoping to improve direct electrochemistry. However, while these materials do maximise current output for weak electricigens, they lead to high capacitive currents during voltammetry, which hides the signature of redox-active species, increases electrochemical noise and lowers repeatability. Thick biofilms are also not desirable when impedance experiments are performed, as the specific conductivity of weak electricigen biofilm is too low to be measured (Kato Marcus et al., 2007). Therefore, to electrochemically characterise weak electricigens it is best to opt for high sensitivity electrochemical methods on small electrodes, which minimise diffusional limitations, while corresponding experiments with larger electrodes can be used to generate sufficient biomass for biological analysis, e.g. DNA extractions. In addition to the electrochemical analyses, the most informative studies will also consist of metabolic or genetic data, in order to deepen existing understanding of fundamental mechanisms of EET. Experiments with single species biofilms do not pose particular challenges, and a proposed approach for such an investigation is provided in Fig. 1. However, current output of complex microbial communities can depend on metabolic networks and even interspecies electron transfer (Villano et al., 2010). Amperometry and chronocoulometry of weak electricigen consortia can be effectively performed together with metagenomics, metatranscriptomics and proteomics, whose results can clarify the role of individual species in the overall current output. In these experiments, particular attention should be used to standardise the reactor volume/electrode surface area ratio and the fluid dynamics, as changes in these parameters result in differing contribution to the EET by the planktonic biomass rather than the biomass in the biofilm. In this section, a summary of some of the techniques at the disposal of the bioelectrochemist is provided.

5. Characterisation of weak electricigens Electrochemical characterisation of weak electricigens differs from that of strong electricigens, as the main measurable output (current) is small and poorly repeatable in many experimental set-ups. Furthermore, with the notable exception of Pseudomonas aeruginosa (Wang et al., 2013), the concentration of microbially-secreted redoxactive species in weak electricigens can be extremely low, if present at all. This leads to low biofilm conductivity, making direct biofilm electrochemistry challenging.

5.1. Use of mediators In contrast with the study of strong electricigens, characterisation of weak electricigens often requires addition of redox mediators and other wiring agents, in addition to modified electrode surfaces to detect small 357

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5.2. Electrochemical methods

currents and to detect the accumulation of non-conductive biofilms. Commonly used redox mediators are quinone-based compounds, including 2-hydroxy-1,4-naphthoquinone (HNQ) and anthraquinone-2,6disulfonate (AQDS). HNQ has been used with E. coli in an MFC as the molecule could freely diffuse through the bacterial membrane (Qiao et al., 2008a). HNQ has low toxicity and low redox potential in aqueous solution (close to 0 mV vs. Ag/AgCl) (Kumar et al., 2014). Furthermore, it shows negligible adsorption to graphite electrodes, and has good photo-stability, which makes for an excellent choice as redox mediator to investigate weak electricigens. The redox couple Fe(III)/Fe(II) can also serve as redox mediator in weak electricigens (Keogh et al., 2018). The disadvantage over other redox mediators is that insoluble Fe oxyhydroxides are formed in the redox process, thus complicating the dynamic of the system. AQDS has a moderately low redox potential (−100 mV vs. Ag/ AgCl) and low toxicity. However, its marked adsorption to carbonaceous materials make it less useful for characterisation of weak electricigens (McDermott et al., 1992). Potassium ferricyanide K3[Fe(CN)6] is more toxic than other redox mediators and has a high redox potential on carbonaceous electrodes (750 mV vs. Ag/AgCl). However, its strong electrochemical signature allows quantification of small electrochemical activity and has been used to follow growth of P. aeruginosa biofilms through surface coverage (Kang et al., 2012). Other commonly used mediators include riboflavin, which is chosen when working at low redox potential. However, riboflavin is known to adsorb to carbon electrodes (Okamoto et al., 2013). A similar compound, flavin adenine dinucleotide (FAD) has been used to characterise MET in Desulfovibrio vulgaris (Zhang et al., 2015). Pyocyanin, a phenazine often associated with P. aeruginosa, could be potentially used as mediator, due to the low redox potential (−200 mV vs Ag/AgCl) and strong electrochemical signature. However, its toxicity to E. coli and other microorganisms and its high cost, together with its marked instability to chemical oxidation, make it less useful when working with weak electricigens. Weak electricigens also include certain eukaryotes, as repeatedly shown in yeast-powered MFCs. Thionine was used as a mediator in a Saccharomices cerevisiae MFC (Rahimnejad et al., 2012). While redox mediators are often needed to detect weak electricigen biofilm formation and activity through amperometry, impedance-based biofilm detection can be carried out without redox mediators as shown for example by Subramanian et al. (2017). In addition to the more traditional mediators described above, other redox active molecules can be used to characterise weak electricigens. For example, osmium redox polymers are effective in promoting electrochemical communication between E. coli and an electrode (Alferov et al., 2009) and have been recently applied to E. faecalis (Pankratova & Gorton, 2017). These redox polymers can be used to study direct EET processes. Careful consideration should be given to the biological or chemical degradation of all redox mediators, particularly in long-term experiments. Recent examples of mediators used with weak electricigens are provided in Table 1.

5.2.1. Amperometry and chronocoulometry Direct measurement of current output remains the principal method to determine electroactivity with time, due to its low cost and amenability to high throughput. Given the low current density of weak electricigens, measurement of charge (i.e. the integration of the current output; a feature typically available in standard electrochemical software) may be more informative (Kim et al., 2013; Wang et al., 2010). Repeatability of the electrochemical signal is of paramount importance, as it enables statistical analysis to detect meaningful differences. Such analysis is extremely important when working with weak electricigens due to the low signal. The required repeatability can be obtained with small electrodes fabricated with standard methods, such as lithographic films, vapour-deposited films, screen printing and associated modifications. As previously noted, weak electricigens may struggle while performing EET, so it is important to ensure the current collected is genuine and not an unspecific signal generated by the lysis of cells. As such, control experiments, such as performing live/dead staining followed by flow cytometry, are recommended at the conclusion of experiments. 5.2.2. EIS with and without redox probe Electrochemical impedance spectroscopy (EIS) of weak electricigens can be very informative, as it allows detection of biofilm formation and other interfaces through the monitoring of interfacial capacitance. In fact, capacitance can be unrelated to electroactivity, as recently demonstrated (Babauta & Beyenal, 2014). EIS is largely considered a nondestructive technique, particularly for low applied potentials, thus it is the ideal tool to monitor biofilms of weak electricigens in long-term experiments. EIS is particularly effective when the electricigen biofilm produces redox mediators or avails of exogenously-added redox mediators for the EET process. In this case, the change in biofilm conductivity can be tracked back to a change in metabolic activity (Paredes et al., 2014) or biofilm matrix composition (van Duuren et al., 2017). EIS returns an average impedance of the whole surface. Since weak electricigen biofilm communities are likely heterogeneous at the µm scale, local EIS might be preferable to determine the microstructure of biofilms in relation to their electroactivity (Moreira et al., 2014). However, local EIS is labour-intensive, thus may not be suitable for routine characterisation of biofilms. EIS can be integrated with other microscopy and spectroscopy methods to provide a more accurate picture of the local electroactivity in biofilms. While little research has been carried out in this direction, it is likely that a higher resolution method will produce detailed knowledge of EET in weak electricigen biofilms. For very weak electricigens, the non-conductive biofilm appears as a large capacitive element, as is typical for non-conductive polymer films. Additional information on the formation of weak electricigen biofilms can be obtained with a strong redox probe, such as K3[Fe(CN)6] (Zhang et al., 2014). The redox probe diffuses to the electrode only in regions free from biofilm, so the process of biofilm formation or removal can be

Table 1 Recent mediators used with weak electricigens. Mediator

Origin

Concentration

References

Notes

Pyocyanin Unidentified hydroquinone HNQ AQDS

P. aeruginosa E. coli Synthetic Synthetic

50–200 nM 150 nM 50 µM 25 mM

Wang et al. (2013) Qiao et al. (2008b) Santoro et al. (2016) Martinez et al. (2017)

Fe(III)/Fe(II) Thionine Cytochrome c Methyl viologen

Exogenous Exogenous Exogenous Exogenous

0.1–5 mM 0.5 mM 1 mM, applied as sol–gel 0.1–10 mM

Keogh et al. (2018) Rahimnejad et al. (2012) Ghach et al. (2014) Choi et al. (2012)

Enhances EET in mixed communities Mediator produced after prolonged growth in MFC Useful within the context of a biosensor Immobilised by electropolymerization, enhances current output in mixed consortia MFCs Enhances EET in E. faecalis; implications for human pathogenicity in the gut Enhances current output in S. cerevisiae and mixed biofilms MFCs Enhances current output in P. fluorescens but not in E. coli Works as cathodic electron donor for C. tyrobutyricum

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hypothesised EET mechanisms. Proteins of interest can be knocked out from the genome and the subsequent mutants tested in the laboratory for a bioelectrochemical signal. Establishing such genetic systems has successfully provided mechanistic insight regarding electricigens (Bretschger et al., 2007; Kim et al., 2008). In addition to sequencing the genome (DNA) of a pure culture, the transcriptome (RNA) can be sequenced, which provides information at the level of gene expression. Simple experimental designs such as comparative profiling of the RNA in the presence of a soluble electron acceptor versus in the presence of a solid electron acceptor could identify specific proteins associated with EET (Fig. 1). Similar stimulusinduced metatranscriptomic studies have been carried out in recent years (Ishii et al., 2013; Ishii et al., 2015).

monitored through the attenuation or increase of the redox probe signal with respect to the control. 5.2.3. Cottrell analysis The analysis of thin Geobacter biofilms through the Cottrell equation has been recently proposed (Zhang et al., 2017). However, it is not clear if this method will also work with weak electricigens or if the Cottrell analysis offers additional insight with respect to EIS. Experimentation with such under-explored electrochemical approaches may improve existing analysis of weak electricigens. 5.2.4. Why voltammetry often does not work Direct voltammetry of weak electricigens is often not very informative, as these microorganisms tend to lack a clear voltammetric signature, either under turnover or non-turnover conditions. There are several examples of weak electricigen voltammetry in earlier studies (Park et al., 2001), but it has mainly been used for qualitative purposes (e.g., showing some electroactivity) rather than for the quantification of electroactive species (which are often unknown) involved in the EET process.

5.3.2. Multispecies genomes and transcriptomes (metagenomics/ metatranscriptomics) In contrast with single species populations, communities consisting of multiple microbial species can also be sequenced en masse. This requires an additional metagenome pipeline to tease out sequences belonging specifically to the weak electricigen of interest from the combined genetic information. A community profile can be generated via use of programmes such as Ribotagger (Xie et al., 2016), which can identify the 16S rRNA gene from the metagenome of a complex microbial consortium. Challenges in community profiling may arise if the species of interest is present in low abundance, as can be the case where weak electricigens reside on an electrode dominated by a strong electricigen such as Geobacter (Doyle et al., 2017). In this case, the genome of the weak electricigen may not be fully recovered or even detected, and may therefore be accidentally overlooked. This potential oversight highlights the importance of isolating individual strains in pure culture when assessing a mixed-species electroactive enrichment, in combination with a screening protocol which detects the EET abilities of resulting isolates. Similarly, the RNA profile of a complex community can be determined, though as with DNA, the relative abundance of the strain of interest will play a large role in how effective this method proves in establishing gene expression in the weak electricigen. From a research community perspective, the deposition of these (meta)genome and (meta)transcriptome sequences in online repositories will enable the true abundance of such weak electricigens to be established, in addition to making the information available for other groups to data mine and use as references.

5.3. Genetic analysis In bioelectrochemical experiments, genetic analysis is often secondary to electrochemical analysis. This trend is likely due to a variety of practical and economic reasons, such as the long timescales, specific skillset and monetary costs associated with genetic sequencing and subsequent bioinformatic analysis. However, in order to confidently identify specific EET mechanisms and the evolutionary origins of weak electricigens, a detailed focus on this underexplored area is required in studies. 5.3.1. Single species genomics and transcriptomics The most straightforward genetic approach consists of sequencing the DNA of a single species population, i.e., a pure culture. Many studies opt to sequence the DNA of the 16S rRNA gene at this point. This is useful for generating a phylogenetic tree to place the strain of interest among its close relatives. However, the rest of the genome beyond this identifier is not recovered using this approach. In order to create a dataset which can be subsequently mined for genes of interest, wholegenome sequencing is necessary. Various sequencing technologies are currently on the market which can achieve this, though sometimes with prohibitive price tags and time-consuming sample preparation steps. However, the development of new platforms such as the relatively low-cost and compact MinION sequencer from Oxford Nanopore, operative both on the bench and in the field, offers an alternative avenue to sequencing (Ashton et al., 2015; Judge et al., 2015). Due to larger error rates when compared with other available platforms (Laver et al., 2015; Mikheyev & Tin, 2014), researchers interested in single nucleotide polymorphisms or the aforementioned phylogenetic analysis based on the 16 s rRNA gene alone may need to opt for more traditional sequencing options. Once the full genome has been recovered, various analytical approaches can be taken. In-silico whole-genome comparisons generating an average nucleotide identity (Richter & Rossello-Mora, 2009) can compare weak electricigens and strong electricigens. Visualisation software, such as the Artemis Comparison Tool (Carver et al., 2005), can be used to quickly view regions of similarity between two genomes, allowing the evolutionary origins of an EET pathway to be investigated. Pairwise protein alignment using software freely available (http:// www.ebi.ac.uk/Tools/psa/) can provide a percentage similarity between regions of interest shared by weak and strong electricigens. Extensive analysis regarding molecular networks, can also be conducted using the KEGG integrated database (Kanehisa et al., 2017). Beyond these mining approaches, whole-genome sequencing provides a template from which to plan genetic mutants to test

5.4. Other considerations Due to the low signal output and conductivity, electrochemical characterisation of weak electricigens must be integrated with other methods borrowed from biofilm microbiology, such as spectroscopy, confocal laser scanning microscopy with metabolic/redox stains, atomic force microscopy and metabolomics (Franks et al., 2010; Reguera et al., 2005; Song et al., 2016). Surface-enhanced Raman scattering was recently proposed for rapid identification of electricigens in small volumes (Wang et al., 2016). The details of the Raman spectrum allow, in principle, to identify strong electricigens from nonelectricigens. However, the applicability of this method to weak electricigens has not been tested. Non-invasive characterisation of strong electricigens has been achieved through confocal Raman spectroscopy (Virdis et al., 2012). However, these and other spectroscopic methods do not measure the electroactivity directly, thus may be more useful for detailed characterisation of known electricigens. Outside of a controlled laboratory environment, weak electricigens might occur as dense and thick microbial mats. CLSM and other microscopy methods are often limited to the analysis of thin biofilms (100 µm). Thus, special methods must be developed to determine the electrochemical characteristics and the 3D structure of these biological formations (Santini et al., 2015). Finally, the duration of experiments with weak electricigens must be 359

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aeruginosa is similar to that of other weak electricigens. Thus, impedance analysis could be coupled with a species-specific separation method (e.g., immunomagnetic beads) to allow precise identification of the weak electricigen (Varshney & Li, 2009). It has been shown that redox cycling of phenazines in P. aeruginosa enhances its anaerobic survival (Glasser et al., 2014), thus providing a possible ecological explanation for weak electroactivity in mixed microbial communities. Considering that phenazines are often toxic to other members of the microbial community, this mechanism might result in increasing P. aeruginosa abundance and increased likelihood of infection. The weak electroactivity of P. aeruginosa can be used for sensing purposes, similar to what has already been reported for strong electricigens. For example, production of phenazines in P. aeruginosa, specifically pyocyanin, was used to determine the toxicity of water containing the contaminant 3, 5-dichlorophenol (Yu et al., 2017). A recent report also identified P. aeruginosa as a potential biocatalyst for nitric oxide removal (Zhou et al., 2016). Electrochemical analysis can also be used to determine the susceptibility of P. aeruginosa to antibiotics. Webster et al. (2015) showed that production of pyocyanin in a thin biofilm grown on a screenprinted electrode was greatly reduced in the presence of antibiotics. While the applicability of similar methods to mixed microbial consortia must be demonstrated, it is likely that electrochemistry can help improve common methods for the determination of antibiotic resistance in clinical settings.

considered carefully. While it is true that long-term experiments increase cumulative charge output, thus facilitating analysis of the EET processes, such experiments result in a dynamic and complex biofilm, with poor repeatability, as is well-known for MFCs (Larrosa et al., 2009). The adoption of specially designed characterisation methods in short-term experiments increases experimental throughput while decreasing the likelihood of these errors. 6. Model weak electricigens While electroactivity has been observed in both Gram-positive and Gram-negative microbes, the thick, non-conductive membrane of Gram-positives makes them generally much less electroactive than Gram negatives. This was demonstrated early on by Matsunaga and Nakajima (1985) where the application of the same (high) redox potential resulted in a higher current for the Gram-negative than for the Gram-positive. As a result of a thicker membrane, it is likely that Grampositive bacteria show DET rather than MET (Modestra & Mohan, 2014). However, electroactivity also depends on biofilm formation and the presence of extracellular redox mediators, eventually immobilised in the biofilm. Here, some of the recent work on weak electricigens, both Gram-negative and Gram-positive, is discussed with particular regard to studies that have provided mechanistic information about the EET process occurring. 6.1. Pseudomonas aeruginosa

6.2. Bacillus Since the discovery of phenazines and their role in EET, P. aeruginosa, a Gram-negative opportunistic pathogen also common in the environment (Alhede et al., 2014), has become a model weak electricigen. This species is often used as a biofilm-forming model organism due to its pathogenicity and antimicrobial resistance (Blair et al., 2015). P. aeruginosa is capable of anodic EET in MFCs (Rabaey et al., 2005). The most investigated EET mechanism avails of phenazines, redox–active pyrazines with high antimicrobial activity. P. aeruginosa produces several phenazines at high concentrations (µM) (Wang et al., 2013). Examples such as pyocyanin are produced mostly during aerobic growth. (Yong et al., 2017). On the one hand, phenazines function as antibiotics, allowing P. aeruginosa to thrive in nutrient-limited environments. On the other hand, phenazines enable shuttle-mediated metabolism of distant substrates and act as oxidants to balance the redox state of cells in the anoxic layers of the biofilm (Okegbe et al., 2017). Furthermore, phenazine production is affected by redox potential (Seviour et al., 2015) and often controlled by quorum sensing (Cabeen, 2014). In the context of EET, the (relatively low) current output is very sensitive to mutations that affect the biofilm matrix, as the matrix determines the retention of phenazines, and hence the MET rate (Qiao et al., 2017). Pseudomonas biofilm formation can be monitored with several electrochemical methods. Among them, EIS provides sensitive results for initial biofilms, where the increase of the double layer capacitance and diffusion element with time corresponds to the accumulation of cells in the planktonic and biofilm phases (Piasecki et al., 2013). Impedance measurement enables non-mucoids and wild type to be distinguished from each other, as the non-mucoid strains show a more conductive phase angle (Ward et al., 2014). While it is certainly true that the impedance spectrum is different across various mutants of P. aeruginosa, it is necessary to design experiments that normalise for phenazine production, as the latter changes broadly across mutants (Wang et al., 2013) and phenazine redox state depends on the oxidation potential of the electrode on which the biofilm is grown (Seviour et al., 2015). After such normalisations, the unicity of EIS P. aeruginosa spectra with respect to other non-electricigens present in biomedical samples may even facilitate identification of the species in a polymicrobial sample. However, the electrochemical signature of P.

Members of the Bacillus genus are Gram-positive, spore-forming bacteria which can thrive in many environments due to their broad physiological abilities in addition to playing a role in pathogenesis (Turnbull, 1996). The weak electroactivity of Bacillus has been repeatedly reported. Bacillus thuringiensis DRR-1 from cow rumen produced a small potential and current when cultivated in an MFC with unspecified medium (Jothinathan & Wilson, 2017). B. cereus was also cultivated on an MFC anode and produced a high current output (Islam et al., 2017). In both studies, the potential was not controlled or measured, as is typical for MFCs. However, the cyclic voltammogram of B. cereus shows a strong peak, either due to the bacterial biomass or to the reduced carbon compounds in the growth medium. B. subtilis in an MFC with glucose as carbon and energy source and 2,4-dichlorophenol as pollutant produced a significant current output (Hassan et al., 2016). Such recent studies demonstrate that Bacillus spp. can perform EET in an MFC. However, accurate experiments in potentiostat-controlled electrochemical cells are needed to understand the mechanism and ecological relevance of EET in the Bacillus genus. Bacillus spp. may also play a role in co-culture bioelectrochemical devices. A recent study showed that Bacillus sp. RH33 produces a high amount of flavins but cannot use them effectively for EET. However, when co-cultured with Shewanella oneidensis MR-1, the power output increased significantly (Liu et al., 2017). Similar observations were made by Wu et al. (2014), who reported another flavin-secreting member of Bacillus capable of EET. Furthermore, a flavin-utilising B. megaterium has been recently reported, demonstrating the versatility of riboflavin as biocompatible redox mediator (You et al., 2018). 6.3. Enterococcus Enterococci are Gram-positive bacteria that frequently inhabit the gastrointestinal tract and can be pathogenic. Some are metabolically versatile and especially resistant to redox stress (Djorić & Kristich, 2015) which is a typical trait of electricigens. A relative of E. gallinarum was demonstrated to use iron as an electron acceptor but the impact on growth was not determined (Kim et al., 2005). More recently, E. faecalis was wired to an electrode maintained at oxidative potential with an 360

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7.3. Metagenomics and metatranscriptomics

osmium redox polymer, showing a sustainable current production (Pankratova et al., 2017). A new study has confirmed EET in E. faecalis, with important implications for pathogenicity in the human gut (Keogh et al., 2018). A form of iron-dependent respiration was observed where, in the absence of heme, EET and increased ATP production enhanced biofilm growth. Such findings indicate that the genus is an interesting candidate for the screening protocols outlined in Section 4.1 and Fig. 1.

An increase of sequencing technologies applied to this area will help resolve whether novel EET mechanisms exist for weak electricigens. Additionally, the role of weak electricigens in supporting strong electricigens in mixed-species bioprocesses can be clarified. 7.4. Relevance of weak electricigens in nature

6.4. Other Gram-positives While the role of strong electricigens in biogeochemical processes has been thoroughly explored, the relevance of weak electricigens has not yet been assessed. It is likely that these microorganisms play a role in oxygen scavenging for anaerobic strong electricigens, and utilise EET only as a survival strategy in times of metabolic emergency.

For the sake of simplicity, the studies investigating EET in Grampositive bacteria can be divided into three groups: a) Gram-positives that avail of soluble redox mediators produced by other bacteria to carry out EET; b) Gram-positives that produce their own mediators or can achieve DET; c) Gram-positives capable of MET through exogenous mediators, either present in the environment or added in the laboratory. Although group a) is less interesting in view of its application, it is relevant in terms of microbial ecology. In fact, mediators produced by a given microorganism can be used by other strains, present as an interspecies collaboration. Notable examples include Brevibacillus sp. PTH1, which was found capable of MET in an MFC when in the presence of microbially-produced phenazines from P. aeruginosa (Pham et al., 2008). Group b) includes weak electricigens such as Lactococcus lactis, which secretes a soluble quinone to achieve EET in MFCs (Freguia et al., 2009). Micrococcus luteus was one of the first Gram-positives found capable of cathodic EET without mediators, likely using membrane redox compounds (Cournet et al., 2010). Another strain capable of anodic EET was Thermincola ferriacetica, which likely transfers electrons through the biofilm without the need for soluble redox mediators (Marshall & May, 2009). In a later study, Wrighton et al. (2011) found that Thermincola potens was capable of direct extracellular reduction of iron, with multiheme c-type cytochromes later implicated (Carlson et al., 2012). Group c) includes many potential electricigens. One example of electroactivity in the presence of exogenously-added mediators was seen when a strain of Corynebacterium produced a current at an MFC anode when supplemented with the artificial redox mediator AQDS (Liu et al., 2010). On the other hand, certain species can use Fe(III) as an electron sink and redox mediator if other electron acceptors are depleted. A Clostridium strain related to C. butyricum could derive a current from glucose in the presence of amorphous ferric oxyhydroxide (Park et al., 2001). This EET mechanism is particularly interesting for its biogeochemical implications, as Fe(III) is a very common chemical species in the environment.

7.5. Applications of weak electricigens Current applications include biosensors, bioremediation and white biotechnology. The latter may prove the most promising, as bioelectrosynthesis/bioelectrofermentation gains momentum. 8. Conclusions Here, weak electricigens have been reviewed, with an emphasis on their ability to enhance existing understanding of EET. The concept of looking beyond traditional power generation in order to consider electroactivity from the vantage point of the microorganism was explored. This approach is likely to clarify what drives evolution of this trait among microbes that may frequently have other modes of respiration at their disposal. Acknowledgements The authors would like to acknowledge financial support from the National Research Foundation, Prime Minister’s Office, Singapore under its Marine Science Research and Development Programme (Award No. MSRDP-P12) and the Singapore Centre for Environmental Life Sciences Engineering (SCELSE), whose research is supported by the National Research Foundation Singapore, Ministry of Education, Nanyang Technological University and National University of Singapore, under its Research Centre of Excellence Programme. The authors thank Kulanan Phanviroj for production of the graphical abstract image. References Abbaspour, N., Hurrell, R., Kelishadi, R., 2014. Review on iron and its importance for human health. J. Res. Med. Sci. 19 (2), 164–174. Alferov, S., Coman, V., Gustavsson, T., Reshetilov, A., von Wachenfeldt, C., Hagerhall, C., Gorton, L., 2009. Electrical communication of cytochrome enriched Escherichia coli JM109 cells with graphite electrodes. Electrochim. Acta 54 (22), 4979–4984. Alhede, M., Bjarnsholt, T., Givskov, M., Alhede, M., 2014. Pseudomonas aeruginosa biofilms: mechanisms of immune evasion. Adv. Appl. Microbiol. 86 (86), 1–40. Ashton, P.M., Nair, S., Dallman, T., Rubino, S., Rabsch, W., Mwaigwisya, S., Wain, J., O'Grady, J., 2015. MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. Nat. Biotechnol. 33 (3), 296–300. Babauta, J.T., Beyenal, H., 2014. Mass transfer studies of Geobacter sulfurreducens biofilms on rotating disk electrodes. Biotechnol. Bioeng. 111 (2), 285–294. Bard, A.J., Faulkner, L.R., 2001. Electrochemical methods: Principles and applications. Principles and Applications Electrochemical Methods. Blair, J.M., Webber, M.A., Baylay, A.J., Ogbolu, D.O., Piddock, L.J., 2015. Molecular mechanisms of antibiotic resistance. Nat. Rev. Microbiol. 13 (1), 42–51. Bond, D.R., Lovley, D.R., 2003. Electricity production by Geobacter sulfurreducens attached to electrodes. Appl. Environ. Microbiol. 69 (3), 1548–1555. Bourdakos, N., Marsili, E., Mahadevan, R., 2014. A Defined co-culture of Geobacter sulfurreducens and Escherichia coli in a membrane-less microbial fuel cell. Biotechnol. Bioeng. 111 (4), 709–718. Bretschger, O., Obraztsova, A., Sturm, C.A., Chang, I.S., Gorby, Y.A., Reed, S.B., Culley, D.E., Reardon, C.L., Barua, S., Romine, M.F., 2007. Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants. Appl. Environ. Microbiol. 73 (21), 7003–7012.

7. Outlook 7.1. Further diversity to explore The recent research reviewed here demonstrates that electricigen diversity is probably higher than what has been discovered to date. In fact, it is likely that many species avail of EET to increase their energy transfer and chance of survival under challenging environmental conditions. In this review, examples of enrichment strategies tailored to weak electricigens have been provided. 7.2. Developing experimental methods Conventional MFC-based set-ups cannot be applied to the analysis of weak electricigen biofilms because of the low power output. Methods common in surface analysis and redox film interfaces should be adapted to biofilms to gain insight into the EET process and mechanisms. Integration with molecular biology and systems biology to create artificial electricigen biofilms will play a key role in this research. 361

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