Author’s Accepted Manuscript Microscale microbial fuel cells: Advances and challenges Seokheun Choi

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To appear in: Biosensors and Bioelectronic Received date: 24 December 2014 Revised date: 10 February 2015 Accepted date: 12 February 2015 Cite this article as: Seokheun Choi, Microscale microbial fuel cells: Advances and challenges, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.02.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Microscale microbial fuel cells: advances and challenges Seokheun Choi * Bioelectronics & Microsystems Laboratory, Department of Electrical & Computer Engineering, State University of New York-Binghamton, Binghamton, NY 13902, USA *Corresponding Author. Email: [email protected] Abstract: The next generation of sustainable energy could come from microorganisms; evidence that it can be seen with the given rise of Electromicrobiology, the study of microorganisms’ electrical properties. Many recent advances in electromicrobiology stem from studying microbial fuel cells (MFCs), which are gaining acceptance as a future alternative "green" energy technology and energy-efficient wastewater treatment method. MFCs are powered by living microorganisms with clean and sustainable features; they efficiently catalyse the degradation of a broad range of organic substrates under natural conditions. There is also increasing interest in photosynthetic MFCs designed to harness Earth’s most abundant and promising energy source (solar irradiation). Despite their vast potential and promise, however, MFCs and photosynthetic MFCs have not yet successfully translated into commercial applications because they de monstrate persistent performance limitations and bottlenecks associated with scaling up. Instead, microscale MFCs have received increasing attention as a unique platform for various applications such as powering small portable electronic elements in remote locations, performing fundamental studies of microorganisms, screening bacterial strains, and toxicity detection in water. Furthermore, the stacking of miniaturized MFCs has been demonstrated to offer larger power densities than a single macroscale MFC in terms of scaling up. In this overview, we discuss recent achievements in microscale MFCs as well as their potential applications. Further scientific and technological challenges are also reviewed. Key words: Microscale microbial fuel cells, biofuel cells, microfabrication, electromicrobiology

1. Introduction The last three decades have witnessed significant developments and performance improvements in microbial fuel cell (MFC) technology.1 These advances are reflected in an increasing number of scientific publications and patents.2-11 Many see MFCs as a promising alternative technology (clean and green, with self-sustaining potential) that could alleviate energy crises and environmental pollution. For this reason, MFCs have provided a focus for renewable energy production research.12 Despite advances, however, it is difficult to see how MFCs can meaningfully contribute to solving the impending energy crisis and environmental pollution in the short term, due to existing MFCs demonstrating low performance, having expensive core parts and materials, and experiencing bottlenecks in scale-up.10,13 Instead, special applications of microbial energy production at the “microscale” level might be more applicable and potentially realizable; (i) powering battery-reliant devices that consume reasonably small amounts of energy,14-17 and (ii) facilitating studies of microbial behavior at a new level of detail and efficiency.18-20 First of all, microscale (containing a microliter-sized anode chamber) MFCs are rapidly gaining attention for their potential as a micro-power source in a wide variety of military mobile and wireless field applications.21-23 The motivation for this interest is clear: it would be ideal for such field-based devices to operate continuously under a variety of conditions for long periods of time, without the need for regular battery maintenance/replacement/recharge. 1

Moreover, a large number of small scale MFC units connected together will produce more power than a single large unit and will provide the most practical means to scale up MFCs.24-25 Second, microscale MFCs can facilitate studies of microbial behavior with a smaller group of cells with excellent control over the microenvironment, eventually contributing to an in-depth understanding of the interplay between MFC devices and active microbes and revealing fundamental problems in electron transfer at the microbial/anode interface.14,18,26,27 The parallel micro-analyses platform can also create high-throughput electrochemical discoveries of microbial activities, identifying which of bacterial genes trigger higher electricity generation.19,28,29 As microscale MFCs are on the verge of entering the commercial realm, the scarcity of comprehensive review articles on this topic with a special emphasis on potential applications and their future directions creates an opportune time to examine and analyse this new field. This review will cover the full scope of microscale MFCs. We will first discuss the MFC fundamentals and then provide a comprehensive review on recent advances and challenges of potential applications of microscale MFCs. In addition, we will include the information of small-scale photosynthetic MFCs (or bio-solar cells) which employ photosynthetic microorganisms enabling the direct conversion of light energy to electrical power. Other potential applications including toxicity biosensors, capacitors and implantable power sources will also be discussed. We hope that this review article will be helpful to readers who are interested in initiating work in this area as well as to researchers already working in this field who wish to learn of the progress achieved to date. 2. MFCs Fundamentals Electromicrobiology is the study of interactions between microorganisms and electronic devices, and the novel electrical properties of microorganisms.9 Recent advances have come from studying MFCs, devices initially designed to harvest electricity from organic matter. MFCs can harvest bioelectricity from both heterotrophic and photosynthetic microorganisms, or from a combination of the two in a single MFC system.30 2.1 Heterotrophic MFCs Heterotrophic MFCs (or simply called MFCs) generate electrical power through the metabolic products of microbial respiration thus requiring a continual supply of organic carbon for energy.4,31 MFCs are typically comprised of anodic and cathodic chambers separated by a proton exchange membrane (PEM) allowing only H+ or other cations to pass from the anode to the cathode. A conductive load connects the two electrodes to complete the external circuit.32 Microorganisms oxidize organic matter in the anodic chamber, completing respiration by transferring electrons to the anode. During this process, chemical energy is captured throughout the electron transport chain. Nicotinamide adenine dinucleotide (NAD +) and nicotinamide adenine dinucleotide dehydrogenase (NADH) function as coenzymes for the reactions, repeatedly oxidizing and reducing to synthesize adenosine triphosphate (ATP), the biological energy unit of the cell (Fig 1).33 MFC technology uses heterotrophic microorganisms that can transfer electrons produced via metabolism across the cell membrane to an external electrode; this process is called extracellular electron transfer (EET), and it plays a key role in harvesting electrons. The primary goals for developing MFCs include identifying strategies to improve metabolic efficiency and optimize biofilm electron transfer activity to the electrode.5,11,34 MFC microorganisms can be planktonic cells and/or biofilms.11,35 Electroactive biofilms include both electrochemically active and inactive microorganisms. While inactive microorganisms may support a number of functions in the microbial community (including the breakdown of complex organics through fermentation or the utilization of other electron acceptors/donors), electrochemically active microorganisms play an important role in enabling efficient electron transfer which maximizes both current densities and the energy efficiency.11 Three EET mechanisms have been proposed35-37: (1) Direct electron transfer occurs between electron carriers in the microorganism and the external electrode. This mechanism is supported by outer-membrane cytochromes that interact directly with the electrode to carry out respiration. Microorganisms using this mechanism require direct contact with the 2

electrode. (2) A soluble electron shuttle is a compound that uses diffusive transportation to carry electrons from the bacteria to the electrode, with which it can react and discharge its electrons. Microorganisms including phenazines, flavins, melanin and quinones are known to produce compounds that act as electron shuttles. In its oxidized state, the shuttle diffuses back to the bacteria cells, which are recycled to continue the process. (3) A solid, extracellular component in the biofilm matrix, that is conductive for electron transfer between the microbes and the electrode, is supported by recent discoveries about the possible role of cellular pili as nanowires, which are being characterized for their conductive capabilities. Despite their vast potential, however, our ability to harness the potential of MFC technology using heterotrophic microorganisms lags from a lack of in-depth understanding of (i) mechanisms for electron harvesting from the microorganisms, and (ii) fundamental factors that maximize MFCs’ power-generating capabilities. 2.2 Photosynthetic MFCs (or Bio-solar cells) Photosynthetic MFCs (or Bio-solar cells) produce bioelectric power based on the exploitation of biocatalytic reactions of the photosynthetic microorganisms, such as cyanobacteria or algae.30,37 During photosynthesis, the microorganisms capture solar energy to convert carbon dioxide and water into oxygen and carbohydrates, which will subsequently be used for their respiratory reaction, re-generating carbon dioxide and water.38 During those reactions, electrons are released through extracellular electron transfer pathways and flow to the cathode through the external electrical circuit (Fig. 2). Simultaneously, the released protons diffuse from the anodic chamber to the cathode, where they re-combine with electrons and O 2 to re-form H2O. Through the aforementioned processes, photosynthetic MFCs can continuously generate electricity from solar energy without additional organic matter by increasing the electrochemical potential inside the cell to split and recreate water, producing oxygen, protons, and electrons. Requiring only sunlight, water, and carbon dioxide to operate, photosynthetic MFCs offer advantages over potentially competing sustainable power sources such as heterotrophic MFCs or photovoltaic cells because the photosynthetic microorganisms used in photosynthetic MFCs (i) do not require an organic fuel, obviating the need for an active-feeding system, and (ii) are capable of producing power both day and at night. This system resembles Earth’s natural ecosystem, where living organisms work in conjunction with the nonliving components of their environment to offer self-sustainable and self-maintainable features as a system. To date, successive efforts have focused on demonstrating the photosynthetic exoelectrogenic activities of various cyanobacteria or algae.30,39,40 However, despite the vast potential and promise of photosynthetic MFCs, they have not yet been successfully translated into commercial applications, as they possess persistent performance limitations and scale-up bottlenecks. These challenges have relegated photosynthetic MFCs, as with ordinary MFCs, to the status of a laboratory curiosity rather than a viable alternative power source. 2.3 Hybrid MFCs Hybrid MFCs are the collective name for a new bio-fuel cell system that integrate both heterotrophic and photosynthetic microorganisms to generate bioelectricity.37,41 Within the hybrid MFC, heterotrophic microorganisms at the anode oxidize organic matter and transfer electrons to the anode while photosynthetic microorganisms provide in-situ oxygen as efficient and sustainable catalysts for the MFC cathodes. In a dual-chamber MFC described in Fig 3, the cathode half-cell is operated with photosynthetic microorganisms while the anode is inoculated with heterotrophic microorganisms. In the hybrid MFC with a non-catalyst-based cathode, oxygen can be continuously supplied for the reaction, resulting in an increase in current generation, which suggests that photosynthetic microorganisms are very efficient oxygenators. 2.4 Evaluation parameters of MFCs

3

There are several important parameters for the evaluation of microscale MFCs’ performances; energy efficiency, internal resistance, start-up times, open circuit voltage (OCV), current density, and power density.42 Energy efficiency in an MFC can be calculated by the product of its columbic efficiency (CE) and potential efficiency (PE) for the electrons captured as electrical current.43 ü ìV EE = CE ´ PE = {C P / CT ) ´ 100%}´ í P ´ 100% ý þ îVT

(1)

, where CP is the total coulombs calculated by integrating the current over the time for substrate consumption and CT is the theoretical amount of coulombs that can be produced from the complete oxidation of organic substrate. VP is the potential measured between the anode and cathode, and VT is the theoretical potential between them. However, most studies do not provide this parameter only the CE value. The internal resistance can be calculated by using total energy loss in MFCs. The total energy loss can be described by eqn. (2) in terms of maximum available output voltage.22,44 EDevice = OCV - IRini = OCV - I(Ra + Rm + Rc + Re ) (2)

; !

,Rini is internal resistance; and Ra, Rm, Rc and Re are anodic, membrane, cathodic, and electrolyte resistance, respectively. Re can be expressed by: Re =

l A× K

(3)

,where l is electrode distance (cm) and A is the cross-sectional area (cm2) through which ionic conduction occurs, and K is the specific conductivity (Ω -1•cm-1) of the electrolyte. Energy loss in a fuel cell should be minimized to decrease the internal resistance for a given current density. MEMS (Micro-Electro-Mechanical-Systems) technology is a particularly attractive option for a microscopic device because it can reduce electrolyte resistance (Re) by increasing the effective surface area of the electrodes (A) while maintaining short proton diffusion lengths (l). Also, because of the increased surface area for mass transport and reactions, the large surface area-tovolume (SAV) ratio represents a more efficient use of substrates per unit volume. However, when scaling towards microscale MFCs, the internal resistance significantly increases, reducing their power density. Microscale MFCs show several orders of magnitude higher internal resistance (several kΩ) than that of macro-sized MFCs (several Ω).15,17 The start-up time is another important parameter to evaluate the MFC performance because it is highly related to the time for accumulation and acclimation of microorganisms on the anode surface of the MFCs. Conventional macroscale MFCs showed very long start-up stages ranging from 4 to 103 days.15 On the other hand, microscale MFCs exhibit a significantly shorter start-up time compared to macroscopic MFCs. Qian et al., described that the short chamber height of their MFC increased the probability of cell attachment and biofilm formation on the anode surface.21 The other electrical parameters (e.g. OCVs, current and power density) are also very critical to determine the bioelectrochemical performances of the MFCs. The OCV is the cell’s potential difference that indicates the difference between the potential under equilibrium conditions and the thermodynamic losses in MFCs. The power and current generated by the MFC must be normalized by a relevant geometric characteristic of the MFC reactor such as the anode surface area or the anodic chamber volume. 4

3. Recent advances, applications, and challenges of microscale MFCs Most MFCs are in macro-sized forms that serve as prototypes of large power sources or energyefficient wastewater treatment technology.13 Recent activities are focused on miniaturizing MFCs for portable power sources and to study individual bacterial behavoir, as well as for other applications with highly comparable performance characteristics.14,18 MFC miniaturization offers inherently advantageous features such as large surface area-to-volume ratio, short electrode distance, and fast response time, theoretically producing far better performance than macro-sized MFCs.15,17 For the fabrication of microscale MFCs, MEMS (Micro-Electro-Mechanical-Systems) technology becomes attractive primarily due to the potential of miniaturization, mass production, and cost reduction.21,22 Moreover, the MEMS MFCs take advantage of several intrinsic characteristics of microfluidics including laminar flow, low consumption of costly reagents, minimal handling of hazardous materials, short reaction time required for power generation, multiple characterization of bacterial species in parallel, portability, and versatility in design. However, MFC miniaturization has many technical challenges, including microfabricating an ion exchange membrane, which demands manual assembly of the MFCs and hampers batch fabrication.17,22 Moreover, the use of commercial ion exchange membranes cause a retarded transfer of protons from the anode chamber to the cathode chamber, which lowers the system stability and bioelectrochemical performance.45 Also, these commercial membranes became swollen or bent in liquid solutions, leading to significant changes in micro-sized chamber volumes. The second challenge is to develop a spacer compatible with MEMS techniques.17,22 The spacer is very important for microscale MFCs because it defines the anode/cathode chamber volume/thickness, optimizing biofilm thickness, mass transport of the nutrients, proton traveling distance, start-up time for bacterial accumulation and acclimation on the anode and ultimately power generation of the MFCs.21,46 The initial MEMS MFCs were assembled by gluing silicone or adhesive at the edge of the membranes.47,48 Such an approach does not offer a precise control of the spacer dimensions. Recently, Choi et al. and Qian et al. reported a MEMS-compatible spacer by patterning photo-definable PDMS and SU-8.21,46,49 Another challenge is to find better anode materials meeting MEMS process requirements and providing better surface characteristics for bacterial biofilm formation.22,50 Aside from all the other factors affecting the MFC performance such as biocatalysts, chemical media, proton exchange membrane, and device configuration, the anode materials play a pivotal role in influencing the power generation by determining: (i) the actual accessible area for bacteria to adhere; (ii) the extracellular electron transfer efficiencies; and (iii) the diffusion rates of the chemical species/metabolic by-products.50 In particular, the selection of the anode materials is more important for microscale MFCs because a small anode surface area/chamber volume has a small number of bacterial cells, which require a more effective way to increase bacterial metabolic efficiency and optimize biofilm electron transfer activity. Therefore, many studies to date have concentrated on improving anode performance with the search for effective anode material and/or modifications to the anode surface.51-54 Recently, many unconventional three-dimensional micro-/nano-scale and/or micro-fabricated anode materials have been proposed to increase porosity, surface area, conductivity, biocompatibility, and biofilm formation.50,55-59 Challenges in the fabrications and anode materials for the microscale MFCs are well reviewed in other literature.14-17,60 In this paper, we would rather focus on potential applications and future directions of the microscale MFCs.

3.1 Portable and self-sustainable power sources Sustainable micropower sources are essential to a growing variety of mobile and wireless applications, including perimeter defence networks, chemical/biological threat detection, environmental protection sensors and micro-vehicle applications. Microscale MFCs exhibit good efficiency and an ability to sustain consistent power production over time by simply consuming a broad range of organic substrates under natural conditions. 5

3.1.1 High Performance microscale MFCs Despite the originality of intricate designs, the maximum power generation and columbic efficiency of microscale MFCs is still insufficient for use as a practical power supply. The power densities of the early microscale MFCs range between 0.019 and 0.4 μW/cm 2,21,47,48 with columbic efficiencies at less than 20%; this puts their power production at up to five orders of magnitude lower, and approximately four times less efficient, than that of macro-sized MFCs.22,44,61 Very low coulombic efficiencies in prior work with microscale MFCs indicate that as the size of the MFC chamber decreases, electron sinks other than the anode become dominant. Methane formation may be an electron sink in MFCs, however, methane’s electron fractions from acetate are typically less than 4%.22,62 Metabolic by-products are other electron sinks, but their fractions are normally below 11%.22,62 Electron storage inside microorganisms or a capacitance effect in the biofilm anode can also decrease MFC performance,22,63 but these effects are temporary.22,64 For these reasons, the most likely electron sink for a microscale MFC is the H2O from the O2 reduction. O2 diffusion into the anode chamber may compete with extracellular electron transfer to the anode, decreasing power generation. Normally, microorganisms consume O 2 immediately and mitigate abiotic O2 reduction on the anode; this O 2-scavenging reaction occurs in macro-sized MFCs with a high biomass concentration in an anode chamber.65 By contrast, the microscale MFC has a small anode chamber volume, small-area anode, and a small number of O 2-utilizing microorganisms. This indicates that while oxygen leakage in macro-sized MFCs may not be as critical when the reaction chamber is large, it becomes extremely critical in micro-scale chambers; minute leakage completely degrades electron harvesting. In order to reduce O 2 intrusion into an anode chamber, one can use MEMS hermetic-wafer bonding technology via eutectic/anodic compounds or oxygen scavengers.22 Table 1 summarizes specifications and performances of microscale MFC work reported to date, with an emphasis on evaluation parameters discussed in section 2.4. As shown in the table, it is difficult to determine critical design parameters and materials specific for microscale MFCs, and establish a general platform for microfabricating microscale MFCs due to variable chemical/physical factors among these studies. The variables include the MFC architecture (e.g. chamber geometry, singe or duel chamber), electrode spacing, environmental conditions (e.g. temperature, pH, humidity), operating conditions (e.g. batch-mode or continuous mode), electrode materials, solution ionic strength and conductivity of the fuel cells. Despite the fact that research has been done by individuals and research groups and this fragmented approach does not seem to support a balanced view of microscale MFCs as a portable power source, the studies provide valuable information and certain perspective to bridge the gap between the vision for a promising technology and the scientific understanding necessary to realize it. First of all, many prior studies used gold as an electrode material because it is biocompatible, highly conductive and is compatible with conventional microfabrication modalities for the development of a microscale MFC platform.21,22,45-48,50,66-68 The gold-based MFCs showed the best performances in microscale MFCs by decreasing the internal resistance down to around 1KΩ.46,68 This was the smallest value among all prior microscale MFCs, but still three orders higher than that of macro-size MFCs.44 Carbon-based electrodes have typically been the material of choice for the construction of macro-sized MFC anodes because it contains functional groups providing bacteria with a more natural habitat.14,19 However, carbon-based materials are not suitable for microscale MFCs because it is non-uniform and difficult to pattern in micro-scale devices. Therefore, gold has been identified as a potential material for microscale MFC anode development. The higher internal resistance associated with gold electrodes relative to carbon-based ones might be due to poor interactions between microorganisms and gold anode. Gold does not contain functional groups, such as quinoes, a natural electron acceptor for anaerobic resiration.69,70 If alternative electrode materials, such as three-dimensional nanomaterial-based anodes, can be found to provide better surface 6

characteristics for microbial biofilm formation, the power density of the microscale MFC will increase substantially (by several orders of magnitude). Recently, many three-dimensional carbon-based materials have been demonstrated in microscale MFCs including carbon nanotubes,50,58,71,72 polymer-fibers,50,59 and graphenes73 with large surface area and higher electrochemical catalytic activity compared to gold anodes. However, none of these candidates showed better performance than gold-based MFCs mainly because their performance is more likely dependent on other factors, and device architectures/fabrication processes are not optimized for microscale platforms. Furthermore, most microscale MFCs require manual assembly in the fabrication, creating many other variables. Therefore, the performances produced by a certain three-dimensional anode in one study cannot be directly compared with another anode unless the MFC architecture, bacteria, and chemical solution are the same. We first need to establish a standardized platform for microscale MFCs, enhancing microfabrication compatibility/reproducibility rather than off-chip integration of the non-microfabricable components. Second, the performance of the microscale MFCs can be significantly improved through a fundamental device level breakthrough (Fig. 4). Typically, an MFC has a double-chambered configuration, consisting of an anode chamber and cathode chamber separated by an ion exchange membrane. A single-chambered device structure can be developed by using air-cathode configuration to significantly decrease proton travel distance, reducing internal resistance. 58,73 The cathode is one of the most important factors in the performance of an MFC. 74 At the cathode, electron acceptors are required for the reduction process, with the electrons and protons traveling from the anode to maintain charge neutrality. Potassium ferricyanide, potassium permanganate, or manganese dioxide has often been used as cathodic electron acceptors in two-chambered MFC configurations. However, using cathodic chemicals is not suitable for development and operation of the sustainable microscale MFCs, because it is expensive and is not environmentally friendly.19 Furthermore, the ion exchange membrane must be used to prevent chemicals from diffusing into the anodic chamber, increasing device costs and fabrication complication. On the other hand, aircathode MFCs offer the best promise for these applications, since oxygen is readily accessible, sustainable, and environmentally friendly. 75,76 Moreover, the membrane can be removed from the system, which reduces production cost and increases power generation with better proton travel efficiency. Generally, single-chambered MFCs show performance enhancement from the twochambered MFCs with similar environmental and operating conditions. 58,73 However, the aircathode is not compatible with microfabrication and requires expensive catalysts and Nafion solutions. Additionally, the fabrication procedures are very complicated, requiring several high temperature treatments.75,76 Although single-chambered MFC configurations have potential and are becoming popular for practical applications, it will not be applicable to microscale MFCs until technical challenges in fabrications are solved. Another membrane-less MFC configuration can be realized by using laminar flow in microfluidics.45,66,67,77,78 In this configuration, the electrodes, media, and oxidant are all contained in a microfluidic channel and are operated without the use of any ion exchange membrane to separate the media and oxidant streams. The channel sizes and operating conditions must be chosen such that: (i) the two liquid streams flow at a Reynolds number of less than 2100 and (ii) the two liquids flow laminarly in parallel without convective mixing.78 Then, the liquid-liquid interface serves as a virtual membrane for the cell. Considering that microscale MFCs can be readily developed by simple fabrication processes without a physical membrane to separate two chambers, the laminar flow-based MFCs might offer greater potential as an on-chip power source. However, the requirement for constant flow will remain a major drawback because (i) the start-up time and duration may last several days before electricity generation begins and (ii) a large amount of fuel and oxidant is needed for continuous laminar flow conditions. Also, (iii) the performance generated from most of laminar flow-based MFCs is not comparable to that of other reported microscale MFCs.78 This is mainly due to non-anode electron sinks such as oxygen. Oxygen diffusion into the anode chamber may compete with EET to the anode and, therefore, decrease power generation.22 Overall, it is unclear whether this 7

concept is a viable option for practical on-chip power application. However, the laminar flowbased MFCs are able to provide an excellent analytical tool for monitoring live bacterial behavior. This application will be discussed in the next section in more detail. Third, the performance of the microscale MFCs is highly related to the type of biocatalyst. Bacteria requiring soluble mediators for EET such as Saccharomyces cerevisiae and Shewanella oneidensis MR-1 cannot produce high current/power density, because the diffusion rates of the mediators significantly limit the rate of EET by diffusion.21,47,48 Moreover, Saccharomyces cerevisiae are unable to produce shuttling compounds; thus, they must be supplied expensive and often toxic exogenous mediators. In contrast, Geobacter sp. form a conductive biofilm matrix for fast EET, resulting in high current/power densities.22,68 Although other variables (e.g. MFC architecture and operating conditions) can be determinant factors that affect the performance of the microscale MFCs, the type of bacterial species plays a profound role because of the different efficiencies of each EET mechanism, supported by the fact that high performance MFCs can be obtained by using Geobacter sulfurreducens or Geobacteraceae-enriched culture.22,46,66,68 However, from a practical point of view, we need to seriously re-consider mixed bacteria cultures which are readily accessible in the environment. One of the unique advantages of the microscale MFCs is their self-sustainability which provides potential as a micro-power source in remote field locations. Although unique aspects of Geobacter sp. have led to much higher power densities in microscale MFCs, they are not suitable for actual applications in our environment because of their limited metabolic diversity and flexibility. Moreover, Geobacter sp. can grow with O2 as a terminal electron acceptor, and this diverts electrons away from the anode, resulting in lower current density.80 Technically, O2 diffusion into the anode chamber cannot be avoided even if the MFC can be built airtight because O 2 diffusion likely happen through media/oxidant injections. Therefore, mixed bacteria culture will be preferable for the practical applications. To date, Choi et al.,46 and Ren et al.,68 achieved the highest power density and columbic efficiency, respectively, among all existing microscale MFCs, which are comparable to macroscale MFC results. Despite excitement about these results, microscale MFC performance is still insufficient for applied use; no microscale MFCs yet exist that can independently power an electronic device. Fundamental breakthroughs that can improve the microscale MFC performance are needed to change this scenario. A better understanding of electron exchange at the micro-level, and a means for overcoming existing performance limits (low power output and energy efficiency) could significantly advance microscale MFC research, contributing to its potential as a sustainable micro-power source. Many other efforts for fundamental studies will be discussed in section 3.2. 3.1.2 Microscale MFC Stacks The theoretical maximum power generation from a single microscale MFC remains limited. Its voltage cannot exceed a theoretical open circuit voltage of 1.14 V, as determined by the NADH (0.32V) and oxidants (+0.82V) redox potentials.6,24 The highest open circuit voltage thus far reported of 0.80 V illustrates this limitation. The maximum current on the other hand is determined by the MFC design which determines the internal resistance and proton transport limitations. Nevertheless, the total output voltage and current are not sufficient for practical applications. Therefore, the use of series or parallel stacked MFCs are currently essential to increase the voltages and currents. Connecting several fuel cells in series adds the voltages, while one common current flows through all MFC units. In case several power sources are connected in parallel, the voltage averages and the currents are added. Any desired current or voltage could be obtained by combining the appropriate number of series and parallel connected fuel cells or power sources. Wilkinson developed a 6-cell stacked macro-sized MFC energizing a fully autonomous robot,81 and Aelterman et al., demonstrated a macro-scale MFC stack increasing the output voltage to 2.02 V and power to 82 mW.82 As far as we are aware, however, there has been only one attempt to assemble microliter-sized MFCs in series developed by Choi et al., 24 and thus, there is little information on the performance of a stacked microscale MFC. They integrated three MFCs into a sandwich of two glass slides, incorporating two chambers of 50 μL volumetric 8

capacity that were separated by a cation exchange membrane (Fig. 5). The MFCs contained a Geobacter-enriched mixed bacterial culture and ferricyanide, which were independently driven into anolyte/catholyte chambers, respectively. The array produced 10x and 4.5x higher power and voltage, respectively, than the best values of previously reported microscale MFCs.22 However, the individual MFC’s voltages diverged and one of them even reversed polarity with the low external resistor. The voltage reversal is not a unique characteristic of microscaleMFCs. Voltage reversal has been one of the main challenges of macroscale MFC arrays.83-85 It has been reported that an insufficient supply of fuel is a major cause of cell reversal and can occur during a sudden change of fuel demand such as during start-up or switching of the load.82 Recently, it was shown that fuel starvation and different anodic reaction rates led to a mismatch of the each MFC, inducing cell reversal.83,85 For practical applications, further studies must be done with stacked microscale MFCs. 3.1.3 Paper-based MFCs Since the power production of the MFCs is at the microwatt-level, the applications of the MFCs might be more appropriate and applicable for powering small scale devices that consume small amounts of energy, such as biosensors. Recently, biosensors have experienced a fundamental materials transformation from more expensive rigid substrates to low-cost, flexible thin materials.86 In particular, paper becomes more popular as a material for biosensors. 87 Paper offers a number of useful advantages; (i) paper is available everywhere and extremely inexpensive; (ii) paper is economically disposed of by an incinerator; (iii) paper is flexible and thin; (iv) paper is biodegradable and biocompatible, and (v) paper provides a high surface area for biomolecules/chemicals to be stored.88-90 In addition, no external pump/tubing is needed to transport liquid through the patterned fluidic pathways in the paper because paper has the ability to wick fluids through capillary action.91 Disposable, paper-based biosensors have attracted more and more interest due to these advantages, and they show remarkable potential for applications in many areas. Therefore, paper-based power source may become indispensable to creating an all paper-based system that can work independently and self-sustainably. To date, several types of paper-based batteries and energy storage devices have been developed as power sources for various applications. The list includes electrochemical batteries, Lithium-ion batteries, supercapacitors, and nanogenerators.91-93 Although the choice of battery type depends on the application, most paper-based batteries developed are neither environmentally friendly nor economically disposable because the electrode or electrolyte used is unstable, explosive, flammable, and environmentally hazardous. Moreover, battery configuration requires many functional layers to be deposited on paper, which increases design complexity and fabrication processes and, in turn, makes them high-cost tools. The paper-based MFC battery will have many advantages over other types of batteries, as (i) it is capable of generating electricity from various kinds of organic matter, such as glucose, urine, biomass, wastewater, and even commercial beverages. Moreover, (ii) the device structure is much simpler than others, (iii) the material/fabrication is cost effective, and (iv) these devices are environmentally friendly, so they can be economically disposed of by an incinerator. Typically, the MFC devices are fabricated on solid state substrates such as glasses, plastics, or silicon wafers with expensive commercial PEMs.14,15 Recently, Choi’s group found that using a paper anode/cathode chamber or reservoir instead of the usual rigid materials allows for rapid adsorption of bacteria-containing liquid (Fig. 6).22,94,95 This adsorption immediately promotes bacteria cell attachment to the electrode, where bacterial respiration can then transfer electrons from the organic liquid to the electrode. A paperbased MFC can therefore show a very short start-up time relative to conventional MFCs; paper substrates eliminate the time traditional MFCs require to accumulate and acclimate bacteria on the anode. Additionally, conventional MFCs have a relatively large anode chamber depth ranging from several millimetres to hundreds of centimetres while paper-based MFCs use hundreds-ofmicrometre-thick filter paper as a reservoir (anodic chamber). Recently, four paper-based MFCs connected in series provided the desired values of current and potential for powering a red LED 9

(HLMP-P156, Digikey) for over 30 minutes. 95 The paper-based MFC is expected to be a simple and easy-to-use power source for single use diagnostic biosensors because even sewage or soiled water in a puddle can become an excellent source for operating MFCs and harvesting electricity through bacterial metabolism. Moreover, river, ocean, and pond water generally host various microorganisms that can transfer electrons produced via metabolism across the cell membrane to an external electrode. Ideally, therefore, this MFC is to be operated by any liquid readily accessible in the environment. However, several challenges remain for developing a paper-based MFC, including that (i) potassium ferricyanide was added to the device as an electron acceptor and (ii) battery stacking in series and/or in parallel is essential to producing higher power output and operating voltages. Simple electrical connections between single MFC units are not suitable for compact and easily-operable paper systems because the MFC array requires a large footprint and each MFC unit needs a drop of the liquid. 3.1.4 Microscale photosynthetic MFCs and toward next-generation bio-solar panels With increasing concerns about the global energy crisis and global warming, solar energy is gaining traction and attention as an extremely abundant and a carbon-free, renewable energy source. Techniques for harnessing solar energy, however, are still limited primarily to semiconductor-based photovoltaic devices with enduring issues, including sustainability, high material costs and fabrication concerns, and limited operational lifetimes. A new approach to convert solar energy into electricity is needed, and a promising one has evolved with advances in microbial fuel cell technologies. Photosynthetic MFCs are an emerging technology designed to harness Earth’s most abundant and promising energy source (solar irradiation) and selfsustainably produce electrical power both day and night. The photosynthetic MFCs can continuously generate electricity from microbial photosynthetic and respiratory activities under day-night cycles. Given the fact that small-scale biological fuel cells are more energy dense than larger units,96 the miniaturization of the photosynthetic MFCs inherently produces favourable conditions for increasing power density by reducing internal resistance and improving mass transport. Further, small-scale photosynthetic MFCs provide one possible way to scale up MFCs by connecting multiple units in a stack configuration. However, only a very small number of research groups have made efforts to miniaturize standard macro-sized photosynthetic MFCs (Table 2).47,97-99 The first microscale photosynthetic MFC was demonstrated by Lin’s group in 2006 generating a power of 40 pW/cm 2.47 Blue-green algae, Phylum Cyanophyta, was inoculated in the transparent MFC reactor to produce electrons by a photosynthetic reaction under light. During night, the device kept generating power by using the glucose produced under light. Later, the same group demonstrated power generation from sub-cellular thylakoid photosystems isolated from spinach cells.97 However, sub-cellular systems can provide power for only a limited period of time, after which the performance of the photosynthetic reactions degrade.97,100 The photosynthetic MFCs based on whole cells provide a more robust and potentially self-sustainable platform for generating power.31 In 2014, Choi’s group reported a 57uL photosynthetic MFC that can produce sustainable energy through the photosynthetic reactions of cyanobacteria, Synechocystis sp. PCC 6803, in the anode.98 They significantly increased the power density of microscale photosynthetic MFCs by increasing surface area-to-volume ratio, obtaining a maximum power density of 7.09 nW/cm 2. This was 170 times more power than ever previously reported for MEMS photosynthetic MFCs,47,97 however, the device performance was still insufficient for applied use and complete self-sustainable power generation was not yet possible. One of the major issues with their device was that the anode material and the device architecture were inappropriate for adequate solar energy capture, bacterial attachment, and light penetration into the first layer of any biofilm growing on the surface. This resulted in a decrease in power/current generation, mainly because the MFCs had a conventional dual-chamber device configuration with a face-to-face arrangement of electrodes. In addition, the thin gold anode showed poor interaction between the bacteria and anode. Another concern was the need to 10

continuously introduce potassium ferricyanide as an electron acceptor (catholyte). Very recently, Choi’s group developed a novel single-chambered device structure that is different from the conventional, dual-chambered microbial fuel cells with a face-to-face electrode arrangement (Fig. 7).99 They report an entirely self-sustainable and scalable microliter-sized device with significant power enhancement by maximizing solar energy capture, bacterial attachment, and air bubble volume in well-controlled micro-chambers. The cell generated a maximum power density of 0.9 mW/m2 through photosynthetic reactions of Synechocystis sp. PCC 6803, which is the highest power density among all microscale photosynthetic MFCs. However, the generated power density is still several orders of magnitude lower than that of even the smallest power heterotrophic MFCs (c.f. Table 1). The current limitation in the performance of the photosynthetic MFCs is primarily due to the high internal resistance, resulting in reduced power densities. Using the polarization curve generated from the recently developed photosynthetic MFCs,99 their internal resistance can be calculated, which is about 470 kψ during both the day and night, several orders of magnitude higher than that of other heterotrophic MFCs (several ψ). The high internal resistance observed in our experiments might be due to the poor electron transfer from photosynthetic bacteria to the anode surface and from the inefficient interactions between the biological material and anode. In order to increase current/power generation by decreasing the internal resistance, a comprehensive understanding of the metabolic pathways involved in extracellular electron transfer is necessary. More specifically, the physiology of those photosynthetic bacteria (and their biofilm) and their interaction with the electrodes must be studied at a new level of detail. The low power density and operating voltages of the photosynthetic MFCs can be improved by connecting multiple units in series or in parallel. Connecting multiple photosynthetic MFC units may be more complicated than connecting conventional batteries because live bacterial behavior is unpredictable and irreproducible. This limitation can be solved by reducing the effective chamber volumes to microliter scale in a well-controlled manner. This method is effective because it drastically decreases background current from a mm 2 scale anode area, small variations in the growth, microbial competition, and metabolisms of the consortium of microbes in the controllable micro-scale environment. Choi’s group reported, for the first time, a proto-type scalable and stackable bio-solar panel by installing miniature photosynthetic MFCs in an array format (Fig. 8).99 Nine small-scale devices were integrated in a panel along with three common feed microfluidic channels.

3.2 Fundamental studies of bacterial behavior Several significant research gaps must be addressed before MFCs can be considered viable or scalable for power generation, and the need to address these gaps is growing more urgent. First, researchers do not yet fully understand how microorganisms donate electrons to electrodes; factors controlling the rate and extent of the exchange process are still ill-defined. Additionally, more research is needed to ascertain which microbial species or consortia may be best suited to generating optimal power density in MFCs. Several contributing factors add to the need for research, including: the inability of traditional macro scale experimental techniques to perform reliable and reproducible measurements; the difficulty of in-situ visualization of microbial biofilm formation; insufficient control over the microbial environment; and limitations in the highthroughput identification/characterization of electrochemically active microbes. Therefore, there is an urgent demand to develop reproducible and practical microscale analysis platforms to support the fundamental study and characterization of microorganism behavior and physiology, and to understand their interaction with electrodes with greater insight and productivity. 3.2.1 Microscale MFC arrays Despite the vast potential and promise of MFC technologies, they have not yet been applied in practical settings as their performance is insufficiently low compared with other battery technologies. Important strategies for improving MFC performance involve genetically engineering exoelectrogenic microorganisms, optimizing formation of bacterial biofilm, 11

maximizing energy efficiency and improving cultivation practices. 19,20 Thus far, only a limited number of bacteria species and their optimal growth conditions have been studied for use in various MFCs. This reveals a significant deficiency of the essential knowledge needed to ascertain which bacteria species or consortia may be best suited for generating optimal power density in MFCs.101 This deficiency is caused by limitations in current screening methods based on larger scale two-bottle MFCs that require long start-up times (from days to weeks), significant space and materials (hundreds of milliliters to liters), and labor-intensive control for MFC experiments, either in series or in parallel circuits.102 This limitation has motivated efforts to miniaturize MFC arrays, such that the effective chamber volumes are reduced to the microliter scale in a well-controlled manner. Table 3 summarizes specifications of prior microscale MFC array work and compares performances (Fig. 9). The first microscale MFC array was presented by Biffinger et al. in 2009. 101 The multi-anode MFC array had nine 1 mL pipette tips as MFC chambers, with a volume of 500 μL. The array allowed for efficient monitoring of bacterial growth cycles and comparison of carbon source utilization of Shewanella oneidensis MR-1. The second microscale MFC array was demonstrated by Han’s group to have 24 wells by using microfabrication.20 Each well had an anode chamber volume of about 600 μL and functioned as an independent MFC. Using the MFC array, they successfully isolated a bacterial strain that displayed 2.3-fold higher power output relative to wild-type Shewanella oneidensis MR-1. Subsequent to that discovery, the same group added an anolyte/catholyte replenishable microfluidic access to their MFC array for a more long-term and efficient analysis. 29 Recently, Choi’s group also presented a 6-well MFC array with an extremely small anodic/cathodic volume (1.5 μL), leading to short start-up (< 2 hours), small deviation (1.4%) from unit to unit, and independent/reliable results from 6 spatially distinct fluidic compartments while prior arts had large size of the anodic chamber volume (> 400 μL) directly related to its underlying long start-up periods (2~3 days) and large variation (> 6%). 19 Hou et al. and Chen et al. developed air-cathode MFC arrays with a single-chambered configuration, holding the potential for practical operation without using ferricyanide.28,103 Despite great excitement about these miniaturized formats, however, the sensing platform still lacks two key aspects: parallelization and rapid power assessment. Currently, no technology can even conceptually provide independent access to more than 24 spatially distinct microbial sensing units with dramatic reductions in a screening speed.29 This is because of (i) complex MFC configurations with microfluidic tubings/channels and their operation with external pumps, and (ii) long start-up times required for bacterial accumulation and acclimation on the sensing electrodes. Recently, Choi’s group introduced a paper-based microbial sensor array as a highthroughput, rapid screening tool for microbial electricity generation studies. 95 Like paper-based MFCs as a power source discussed in Section 3.1.3, the paper-based MFC array exploits the paper’s ability to quickly wick fluid and promote bacterial attachment to the gold anode pads, resulting in instant current generation upon loading of the bacterial inoculum and catholyte. The presented 6-well device contained vertically stacked anode/cathode paper chambers (or reservoirs) separated by a proton exchange membrane (PEM), and gold anode/cathode interface pads with through-holes in the center to introduce anolyte/catholyte. Within just 50 minutes, they successfully determined the electricity generation capacity of two known bacterial electrogens and another metabolically more voracious organism with four isogenic mutants. This paper-based microbial screening tool did not require external pumps/tubings and represents the most rapid test platform ( 6 60 23 l ch eae00 /2. /2. on al., 20 k 12 67 am enri 0 00 ou 31 0 00 33 45 25 25 11 11 [22] Ω ber ched rs 0 7 cult ure She Ca Ca wan rb rb 1 Dua ella Na 6 ~ 0. on on 0. Qian et 6 l ch fio 10 ~3 H N/ 5 10 62 00 Cl Cl al., 20 k 10 62 am onei n 1 0 00 ou A 6 00 .5 4 oth oth 5 11 [49] Ω ber dens 17 rs 0 is M /0. /0. 4 4 R-1 23

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mb er

Table 2

#o De fa via ctu De tio # al Op vic An A n of ba era Cur Po e s ode no A be cte tio rent wer ub / de no Ca St tw M ria Bioc n/ f den de str cat v de tho ar ee Ref. F l s atal lui sity nsit ate hod ol a lyt tn C pec yst dic leve y le m e m ul re e up ar u ies ac ate ate m a l vel ra ni to ces ria rial e yc ts be s l scr ell s een ed Gra Bat Pot ~ 2. ~0. She ch Pi phit 1. ass < 50 Biffinger wan mo pet e fe 1c iu 2h 8 5 μ 02μ 9 9 0 et al., 20 m m f ou % A/c W/c ella de/ ti lt/ 2 2 09 [101] μL 2 sp. p Car erri rs m m no cya bon 29

ac ces s Bat ch mo She P de/ 2 wan M 24 4 ella M no sp. A ac ces s Co nti nu ous m She od P 2 wan e/ M 6 4 ella M sp. A 4-c om mo n acc

pa per

nid e

Pot Gol ass 0. ~0. d/ iu < ~0.5 38 60 6Hou et al Car 0 c m f 2h 14 μA/ 1μ ., 2009 [ 2 W/c bon μL m erri ou % 2 20] cm rs cya 2 clo m nid th e

Pot Gol ass 0. d/ iu ~2μ Hou et al 40 38 m f N N/ Car 0 c N/A W/c ., 2012 [ 2 erri /A A bon μL m 29] m 2 cya pa nid per e

30

ess Co nti nu She ous wan m ella od sp. e/ P M 6 6 and M Pseu 6 i A dom ndi onas vid sp. ual ac ces ses She Bat wan ch ella mo P sp. de/ M 2 24 4 and M no Arth ac A roba ces cter s

Pot ass 0. iu