Towards Bio-Hybrid Energy Harvesting in the Real-World: Pushing the Boundaries of Technologies and Strategies Using Bio-Electrochemical and Bio-Mechanical Processes

applied sciences Review Towards Bio-Hybrid Energy Harvesting in the Real-World: Pushing the Boundaries of Technologies and Strategies Using Bio-Electrochemical and Bio-Mechanical Processes Abanti Shama Afroz 1, Donato Romano 1,2,* , Francesco Inglese 1and Cesare Stefanini 1,2,3   Citation: Afroz, A.S.; Romano, D.; Inglese, F.; Stefanini, C. Towards Bio-Hybrid Energy Harvesting in the Real-World: Pushing the Boundaries of Technologies and Strategies Using Bio-Electrochemical and Bio-Mechanical Processes. Appl. Sci. 2021,11, 2220. https://doi.org/ 10.3390/app11052220 Academic Editor: Borja Velazquez-Marti Received: 7 January 2021 Accepted: 24 February 2021 Published: 3 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1The BioRobotics Institute, Scuola Superiore Sant’Anna, Viale Rinaldo Piaggio 34, 56025 Pontedera, Italy; [email protected] (A.S.A.); [email protected] (F.I.); [email protected] (C.S.) 2Department of Excellence in Robotics & AI, Scuola Superiore Sant’Anna, Piazza Martiri della Libertà33, 56127 Pisa, Italy 3Healthcare Engineering Innovation Center (HEIC), Khalifa University, Abu Dhabi 127788, United Arab Emirates *Correspondence: donato.r[email protected] Abstract: Sustainable, green energy harvesting has gained a considerable amount of attention over the last few decades and within its vast field of resources, bio-energy harvesters have become promising. These bio-energy harvesters appear in a wide variety and function either by directly generating energy with mechanisms similar to living organisms or indirectly by extracting energy from living organisms. Presently this new generation of energy harvesters is fueling various low-power electronic devices while being extensively researched for large-scale applications. In this review we concentrate on recent progresses of the three promising bio-energy harvesters: microbial fuel cells, enzyme-based fuel cells and biomechanical energy harvesters. All three of these technologies are already extensively being used in small-scale applications. While microbial fuel cells hold immense potential in industrialscale energy production, both enzyme-based fuel cells and biomechanical energy harvesters show promises of becoming independent and natural power sources for wearable and implantable devices for many living organisms including humans. Herein, we summarize the basic principles of these bio-energy harvesting technologies, outline their recent advancements and estimate the near future research trends. Keywords: bio-hybrid systems; bio-energy sources; energy; bioengineering; microbial fuel cells; bionics 1. Introduction Development of sustainable and zero-carbon-emission energy resources is considered one of the most demanding goals for the current world [ 1 ]. Within the vast field of green energy harvesting, an important one is bio-energy harvesting [ 2 , 3 ]. Although such possibilities were proposed much earlier [ 4 ], this started to gain prominence in the early 2000s [ 5 – 7 ]. This relatively new research field is opening up opportunities for producing energy from the wide range of living creatures, including microorganisms [ 6 ] and macro-organisms [ 8 ], as well as from bio-hybrid organisms [ 9 ]. The application fields of these bio-energy harvesters are equally elaborated, ranging from industrial-scale energy production [ 10 ] to environmental monitoring [11,12] and biomedical applications [13,14]. The first bio-energy harvesters reported in this review are known as microbial fuel cells (MFCs) which offer the possibility to become one of the next big industrial energy solutions [ 15 , 16 ]. MFCs utilize catabolic metabolism of different microorganisms on a wide range of organic substrates [ 17 ] and turn them into micro bio-reactors generating electrical energy [ 18 ]. Extensive works have been performed on summarizing its various aspects like development in electrode configurations [ 19 – 22 ], dedicated power management systems [ 23 , 24 ], and cell separators [ 25 – 27 ]. With the prospects of becoming a next-generation Appl. Sci. 2021,11, 2220. https://doi.org/10.3390/app11052220 https://www.mdpi.com/journal/applsci Appl. Sci. 2021,11, 2220 2 of 44 large-scale power generation technology, they are already being used for powering up remote marine sensors [ 23 ], long-distance marine communications [ 28 ], simultaneous wastewater processing with auxiliary power supply [ 29 ], metal recovery processes [ 30 ] and in biosensors [31]. The next biochemical energy harvester that has gained a considerable amount of popularity are enzyme-based biofuel cells (EBFCs) where synthetically produced enzyme molecules are immobilized on the electrodes for performing glucose oxidation and in turn power generation [ 32 ]. This particular type falls under the bio-hybrid energy harvesters [ 33 ] and is considered the promising natural resource for powering up implants and prosthetic devices [ 34 ]. Research on these implantable fuel cells is still at the animal trial level [ 35 ]. Simultaneously, investigations have also been started on testing the applicability of such cells from external body-fluids [13]. The third and final bio-energy harvester discussed in this review is mechanical in nature. These biomechanical energy harvesters have a remarkable growing market for powering smart and wearable devices [ 36 ]. These harvesters are preferred for their non-invasive nature and easy-applicability in monitoring [ 37 ], diagnostic [ 38 ] and therapeutic [ 39 ] functions. They utilize mechanical energy produced from living animals and convert them into power solutions [ 40 ]. While their use for human application is eminent [ 41 ], they have also been used for powering other living bio-hybrid animals [42]. In this work, we aim to offer a concise, functional summary of these three promising bioenergy harvesters and their progress in the recent years. 2. Literature Search Method A hierarchical survey was performed where “microbial fuel cell”, “enzymatic fuel cell” and “biomechanical fuel cell” were utilized as primary keywords. At the secondary level keywords “architecture”, “classification” and “applications” were used with all the three primary keywords. “Electrode” keyword was used for both MFCs and EBFCs. Keywords “exoelectrogens”, “photo-reactors”, “membranes”, “waste management” were used only for MFCs. Similarly, “enzyme immobilization” was used for EBFCs while “triboelectric nano generator”, “heap”, “ankle”, ”knee”, ”foot” and “upper limb” were used for biomechanical cells. A pictorial description on the use of the keywords’ hierarchy is given in Figure 1. Appl. Sci. 2021, 11, x FOR PEER REVIEW 2 of 47 electrical energy [18]. Extensive works have been performed on summarizing its various aspects like development in electrode configurations [19–22], dedicated power management systems [23,24], and cell separators [25–27]. With the prospects of becoming a next-generation large-scale power generation technology, they are already being used for powering up remote marine sensors [23], long-distance marine communications [28], simultaneous wastewater processing with auxiliary power supply [29], metal recovery processes [30] and in biosensors [31]. The next biochemical energy harvester that has gained a considerable amount of popularity are enzyme-based biofuel cells (EBFCs) where synthetically produced enzyme molecules are immobilized on the electrodes for performing glucose oxidation and in turn power generation [32]. This particular type falls under the bio-hybrid energy harvesters [33] and is considered the promising natural resource for powering up implants and prosthetic devices [34]. Research on these implantable fuel cells is still at the animal trial level [35]. Simultaneously, investigations have also been started on testing the applicability of such cells from external body-fluids [13]. The third and final bio-energy harvester discussed in this review is mechanical in nature. These biomechanical energy harvesters have a remarkable growing market for powering smart and wearable devices [36]. These harvesters are preferred for their non-invasive nature and easy-applicability in monitoring [37], diagnostic [38] and therapeutic [39] functions. They utilize mechanical energy produced from living animals and convert them into power solutions [40]. While their use for human application is eminent [41], they have also been used for powering other living bio-hybrid animals [42]. In this work, we aim to offer a concise, functional summary of these three promising bioenergy harvesters and their progress in the recent years. 2. Literature Search Method A hierarchical survey was performed where “microbial fuel cell”, “enzymatic fuel cell” and “biomechanical fuel cell” were utilized as primary keywords. At the secondary level keywords “architecture”, “classification” and “applications” were used with all the three primary keywords. “Electrode” keyword was used for both MFCs and EBFCs. Keywords “exoelectrogens”, “photo-reactors”, “membranes”, “waste management” were used only for MFCs. Similarly, “enzyme immobilization” was used for EBFCs while “triboelectric nano generator”, “heap”, “ankle”, ”knee”, ”foot” and “upper limb” were used for biomechanical cells. A pictorial description on the use of the keywords’ hierarchy is given in Figure 1. Figure 1. Hierarchical keywords to summarize recent progresses in bio-energy solutions. Figure 1. Hierarchical keywords to summarize recent progresses in bio-energy solutions. Appl. Sci. 2021,11, 2220 3 of 44 3. Research Progress in Energy Harvesting from Microorganisms MFCs extract electrical energy from different microorganisms by utilizing their catabolic metabolism over various organic components [ 17 , 18 ]. Bacteria are the most used microbes for this purpose, whose activity can be boosted with the aid of other microorganisms like algae [43,44]. The following paragraphs depict research progress in MFCs, their structural development, a pragmatic classification of the MFCs, adaptations performed to optimize their operations in different scenarios and their diverse conjugated applications along with electricity generation. 3.1. Principles of Electricity Generation with Microbial Fuel Cell (MFC) Structurally, an MFC is composed of a cathode, an anode, the microorganism and the oxidizing substrate, which in most cases, is composed of organic matter. The structure can be of single or dual chamber type depending on the absence or presence of a separator. In a classic configuration, the microorganisms decompose the organic substrate in the anaerobic anode chamber through catabolic processes to obtain energy and generate electrons and protons/cations as a by product. The generated protons flow to the cathode chamber through the cation (permeable) exchange membranes (CEMs) and thus create a potential difference between the electrodes. The excess electrons flow from the anode to cathode via external circuit and generate a current flow [18,45,46]. A classic dual cell MFC is depicted in Figure 2. Bio-electrochemical systems started to gain attention as a possible green energy source in the early years of the 21st century [ 29 ] since microorganisms possess the flexibility to generate energy from a very wide range of biomass varieties [ 47 ]. Bacterial electron transfer mechanisms and their dependence on mediators and biofilms are three major considerations of MFC performance. Appl. Sci. 2021, 11, x FOR PEER REVIEW 3 of 47 3. Research Progress in Energy Harvesting from Microorganisms MFCs extract electrical energy from different microorganisms by utilizing their catabolic metabolism over various organic components [17,18]. Bacteria are the most used microbes for this purpose, whose activity can be boosted with the aid of other microorganisms like algae [43,44]. The following paragraphs depict research progress in MFCs, their structural development, a pragmatic classification of the MFCs, adaptations performed to optimize their operations in different scenarios and their diverse conjugated applications along with electricity generation. 3.1. Principles of Electricity Generation with Microbial Fuel Cell (MFC) Structurally, an MFC is composed of a cathode, an anode, the microorganism and the oxidizing substrate, which in most cases, is composed of organic matter. The structure can be of single or dual chamber type depending on the absence or presence of a separator. In a classic configuration, the microorganisms decompose the organic substrate in the anaerobic anode chamber through catabolic processes to obtain energy and generate electrons and protons/cations as a by product. The generated protons flow to the cathode chamber through the cation (permeable) exchange membranes (CEMs) and thus create a potential difference between the electrodes. The excess electrons flow from the anode to cathode via external circuit and generate a current flow [18,45,46]. A classic dual cell MFC is depicted in Figure 2. Bio-electrochemical systems started to gain attention as a possible green energy source in the early years of the 21st century [29] since microorganisms possess the flexibility to generate energy from a very wide range of biomass varieties [47]. Bacterial electron transfer mechanisms and their dependence on mediators and biofilms are three major considerations of MFC performance. Figure 2. A classic dual chamber microbial fuel cell (MFC) with cathode, cation exchange membrane and anode (from left to right). The intracellular electron transport (ET) mechanisms are of fundamental concern for MFC systems. The MFC compatible microorganisms can transfer the electrons to the anode either through direct contact or with the help of mobile electron shuttles or mediators [31,45]. These mediators are compounds that act as biocatalysts and shuttle electrons from the intracellular space to the extracellular environment within the MFC and could either be externally added or produced by the living cells [46]. The ability of microorganisms to utilize the soluble mediators as electron shuttles was highly beneficial in the early stages of MFCs. In these first-generation or mediator-dependent MFCs, presence of Figure 2. A classic dual chamber microbial fuel cell (MFC) with cathode, cation exchange membrane and anode (from left to right). The intracellular electron transport (ET) mechanisms are of fundamental concern for MFC systems. The MFC compatible microorganisms can transfer the electrons to the anode either through direct contact or with the help of mobile electron shuttles or mediators [31,45] . These mediators are compounds that act as biocatalysts and shuttle electrons from the intracellular space to the extracellular environment within the MFC and could either be externally added or produced by the living cells [ 46 ]. The ability of microorganisms to utilize the soluble mediators as electron shuttles was highly beneficial in the early stages of MFCs. In these first-generation or mediator-dependent MFCs, presence of a suitable electron shuttle or mediator was mandatory [ 31 ]. In addition, efficient MFCs Appl. Sci. 2021,11, 2220 4 of 44 without requiring artificial mediators also started to emerge. However, not all bacteria are suitable for generating electricity in a mediator-less configuration MFC. Microorganisms that can effectively generate electricity in MFCs without additional mediators include a few classes of Proteobacteria in addition to some microalgae, yeast, and fungi species [ 48 ]. These microorganisms, capable of extracellular electron transfer are often referred to as exoelectrogens as well as electrochemically active bacteria, anode-respiring bacteria and electricigens [ 47 ]. They are capable of generating proteins or molecules, followed by oxidizing procedures, for transferring the electrons exogenously [48–50] . A sub group of these bacteria are nanowire generators. Geobacter sulfurreducens was one of the first organisms shown to produce conductive nanowires. These nanowires were latter referred to conductive pilA [ 48 ] because of their composition with the pila protein. The bacterium Shewanella oneidensis can also generate electrical nanowires [ 51 , 52 ] under special condition and as extensions of their outer-membrane, allowing the transfer of electrons to the anode without requiring soluble electron shuttles. The presence of bacterial biofilm can be highly advantageous for MFCs because of their electroactive nature aids to generate electricity more efficiently. This biofilm is a complex, organic, polymeric matrix, produced by the bacteria themselves at any biotic or abiotic surface and these organic films can be formed by a single bacterial (pure-culture) or multiple bacterial species (mixed-culture) [ 48 ]. It has been proven that bacteria capable of forming thick anodic biofilms generate higher current densities than those who can not [ 53 ]. A pictorial summary of bacterial electron transport processes is given in Figure 3. Appl. Sci. 2021, 11, x FOR PEER REVIEW 4 of 47 a suitable electron shuttle or mediator was mandatory [31]. In addition, efficient MFCs without requiring artificial mediators also started to emerge. However, not all bacteria are suitable for generating electricity in a mediator-less configuration MFC. Microorganisms that can effectively generate electricity in MFCs without additional mediators include a few classes of Proteobacteria in addition to some microalgae, yeast, and fungi species [48]. These microorganisms, capable of extracellular electron transfer are often referred to as exoelectrogens as well as electrochemically active bacteria, anode-respiring bacteria and electricigens [47]. They are capable of generating proteins or molecules, followed by oxidizing procedures, for transferring the electrons exogenously [48–50]. A sub group of these bacteria are nanowire generators. Geobacter sulfurreducens was one of the first organisms shown to produce conductive nanowires. These nanowires were latter referred to conductive pilA [48] because of their composition with the pila protein. The bacterium Shewanella oneidensis can also generate electrical nanowires [51,52] under special condition and as extensions of their outer-membrane, allowing the transfer of electrons to the anode without requiring soluble electron shuttles. The presence of bacterial biofilm can be highly advantageous for MFCs because of their electroactive nature aids to generate electricity more efficiently. This biofilm is a complex, organic, polymeric matrix, produced by the bacteria themselves at any biotic or abiotic surface and these organic films can be formed by a single bacterial (pure-culture) or multiple bacterial species (mixed-culture) [48]. It has been proven that bacteria capable of forming thick anodic biofilms generate higher current densities than those who can not [53]. A pictorial summary of bacterial electron transport processes is given in Figure 3. Figure 3. Electron transfer mechanisms, mediators and biofilm for MFC technology. 3.1.1. Electrodes One of the major concerns in MFC developments has been developing efficient and economically viable electrodes [16,54]. Multiple review works have been reported with the intention of summarizing developments in MFC cathodes [19,55] as well as anodes [20,56] and in general electrode materials [21,22]. The anode accepts the electrons generated by the microbial community and hence promoting bacterial adhesion at MFC anodes is of utmost importance [57]. Additionally, ensuring an anoxic environment is required to ensure that anode is the only electron acceptor in the vicinity [58]. MFC anode materials should possess key physical features including biocompatibility [20], corrosion resistance [54], high electrical conductivity [54,59], Figure 3. Electron transfer mechanisms, mediators and biofilm for MFC technology. 3.1.1. Electrodes One of the major concerns in MFC developments has been developing efficient and economically viable electrodes [ 16 , 54 ]. Multiple review works have been reported with the intention of summarizing developments in MFC cathodes [ 19 , 55 ] as well as anodes [ 20 , 56 ] and in general electrode materials [21,22]. The anode accepts the electrons generated by the microbial community and hence promoting bacterial adhesion at MFC anodes is of utmost importance [ 57 ]. Additionally, ensuring an anoxic environment is required to ensure that anode is the only electron acceptor in the vicinity [ 58 ]. MFC anode materials should possess key physical features including biocompatibility [ 20 ], corrosion resistance [ 54 ], high electrical conductivity [ 54 , 59 ], wettability [ 58 ] and chemical strength to withstand the wastewater environment with diverse Appl. Sci. 2021,11, 2220 5 of 44 organic and inorganic contents [ 56 ], other wastes, electrolytes and soil contaminating components. While multiple metallic anode configurations for MFCs have been tested, stainless steel was found to be a suitable one because of its capacity to withstanding corrosion [ 60 ]. Earlier works utilized different forms of carbon anodes that provided better microbial adhesion than metal ones including carbon paper, carbon cloth, activated carbon, carbon felt, graphite felt, tungsten carbide, graphite foil and others while still not being the optimal solution because of their intrinsic hydrophobic nature [ 20 ]. Facilitating biofilm growth at the anode [ 48 , 57 ] has been an important consideration. It has been found that high porosity and increased surface area facilitate biofilm growth and anode surface texture also plays an important role in promoting bio-catalytic activity [ 61 ]. While biofilm growth on anode is promoted, ensuring absence of other electron acceptors except anode material itself becomes an important issue in maintaining MFCs’ performance. This performance is often affected by dissolved oxygen in water that gains access to local anode spots caused by burrowing organisms [62]. Based on these research trends, we have updated the MFC anode classification based on materials by [60] into the following hierarchy as in Figure 4. Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 47 wettability [58] and chemical strength to withstand the wastewater environment with diverse organic and inorganic contents [56], other wastes, electrolytes and soil contaminating components. While multiple metallic anode configurations for MFCs have been tested, stainless steel was found to be a suitable one because of its capacity to withstanding corrosion [60]. Earlier works utilized different forms of carbon anodes that provided better microbial adhesion than metal ones including carbon paper, carbon cloth, activated carbon, carbon felt, graphite felt, tungsten carbide, graphite foil and others while still not being the optimal solution because of their intrinsic hydrophobic nature [20]. Facilitating biofilm growth at the anode [48,57] has been an important consideration. It has been found that high porosity and increased surface area facilitate biofilm growth and anode surface texture also plays an important role in promoting bio-catalytic activity [61]. While biofilm growth on anode is promoted, ensuring absence of other electron acceptors except anode material itself becomes an important issue in maintaining MFCs’ performance. This performance is often affected by dissolved oxygen in water that gains access to local anode spots caused by burrowing organisms [62]. Based on these research trends, we have updated the MFC anode classification based on materials by [60] into the following hierarchy as in Figure 4. Figure 4. Classification of MFC anodes according to their structural configuration. The electrons generated at the anode of an MFC cell flows through the external circuit to the cathode and completes the oxygen reduction reaction (ORR) in the presence of electron acceptors and ORR catalysts [19]. Classically, it consists of a conductive base material core and an ORR catalyst layer. Ideally, the MFC cathode should be very reactive, capable of supporting ORR catalysts as well as remaining at low cost [63], although such an optimal MFC cathode configuration has not yet been achieved. In the first generation of MFCs, expensive ORR catalysts like platinum (Pt) and copper-oxide (CuO) were widely used [61] despite their biofouling tendency and reduced capacity due to bio-poisoning caused by microorganisms [16] even in the presence of membrane [64,65]. An effective cathode configuration also requires the continuous presence of electron acFigure 4. Classification of MFC anodes according to their structural configuration. The electrons generated at the anode of an MFC cell flows through the external circuit to the cathode and completes the oxygen reduction reaction (ORR) in the presence of electron acceptors and ORR catalysts [ 19 ]. Classically, it consists of a conductive base material core and an ORR catalyst layer. Ideally, the MFC cathode should be very reactive, capable of supporting ORR catalysts as well as remaining at low cost [ 63 ], although such an optimal MFC cathode configuration has not yet been achieved. In the first generation of MFCs, expensive ORR catalysts like platinum (Pt) and copper-oxide (CuO) were widely used [ 61 ] despite their biofouling tendency and reduced capacity due to bio-poisoning caused by microorganisms [ 16 ] even in the presence of membrane [ 64 , 65 ]. An effective cathode configuration also requires the continuous presence of electron acceptors like oxygen in the vicinity [ 31 ] and oxygen is still the primary choice for the terminal electron Appl. Sci. 2021,11, 2220 6 of 44 acceptor [ 66 ]. However, it has also been reported that in deep water column MFC configurations, there may appear to be an anoxic environment and in such cases, the ORR reaction is completed by other electron acceptors like nitrates, sulfates or iron oxides [ 67 ]. A classification of MFC cathode configurations is given in Figure 5. Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 47 ceptors like oxygen in the vicinity [31] and oxygen is still the primary choice for the terminal electron acceptor [66]. However, it has also been reported that in deep water column MFC configurations, there may appear to be an anoxic environment and in such cases, the ORR reaction is completed by other electron acceptors like nitrates, sulfates or iron oxides [67]. A classification of MFC cathode configurations is given in Figure 5. Figure 5. Classification of MFC cathodes according to their functionalities. For sediment or benthic-type MFCs where submerged cathodes are used, the dissolved oxygen in water serves the purpose of electron acceptor. For such open water configurations like marine MFCs, supply of dissolved oxygen is not a big issue [6,58] and still floating marine MFCs [68] have also been implemented. For close water systems like waste-water processing plants ensuring of continuous oxygen supply becomes a common challenge [69]. Researchers have worked on providing additional air circulation at the cathodes, especially with innovative mechanical solutions. In many cases such additional units increase the production cost. While multiple examples of mechanical aeration procedures are reported, use of such units require additional cost [61,70–72]. Many works have been undertaken on improving the flexibility of such system including use of comb type [73], brush type [74] and rotating type [75] cathodes. Air cathodes [70] have emerged as a popular low-cost solution providing sustainable aeration at cathodes. In this configuration the cathode surface partially remains open to air and continuously receives oxygen supply. Janicek et al. [76] reported a generalized air cathode configuration which consists of a catalyst layer that faces the solution side of the cathode, a gas diffusion layer that faces air, and a conductive base material layer. The conductive layer also acts as a current collector as well as a mechanical support provider. Biocathodes also became an efficient solution for continuous oxygenation requirement for MFCs. Biocathodes are defined as cathodes with attached microorganisms serving as ORR biocatalysts [77]. Biocathodes emerged as a solution where bio-fouling at cathodes was utilized as an advantage rather than a disadvantage where bacterial and micro-algae [78] were grown intentionally to aid ORR catalyst operations instead of using additional expensive catalysts. A second generation of these cathodes also include a natural oxygen generating mechanism by incorporating growth of photosynthetic bioreactors [79] on the same platform. Figure 5. Classification of MFC cathodes according to their functionalities. For sediment or benthic-type MFCs where submerged cathodes are used, the dissolved oxygen in water serves the purpose of electron acceptor. For such open water configurations like marine MFCs, supply of dissolved oxygen is not a big issue [ 6 , 58 ] and still floating marine MFCs [ 68 ] have also been implemented. For close water systems like waste-water processing plants ensuring of continuous oxygen supply becomes a common challenge [ 69 ]. Researchers have worked on providing additional air circulation at the cathodes, especially with innovative mechanical solutions. In many cases such additional units increase the production cost. While multiple examples of mechanical aeration procedures are reported, use of such units require additional cost [ 61 , 70 – 72 ]. Many works have been undertaken on improving the flexibility of such system including use of comb type [ 73 ], brush type [ 74 ] and rotating type [ 75 ] cathodes. Air cathodes [ 70 ] have emerged as a popular low-cost solution providing sustainable aeration at cathodes. In this configuration the cathode surface partially remains open to air and continuously receives oxygen supply. Janicek et al. [ 76 ] reported a generalized air cathode configuration which consists of a catalyst layer that faces the solution side of the cathode, a gas diffusion layer that faces air, and a conductive base material layer. The conductive layer also acts as a current collector as well as a mechanical support provider. Biocathodes also became an efficient solution for continuous oxygenation requirement for MFCs. Biocathodes are defined as cathodes with attached microorganisms serving as ORR biocatalysts [ 77 ]. Biocathodes emerged as a solution where bio-fouling at cathodes was utilized as an advantage rather than a disadvantage where bacterial and micro-algae [ 78 ] were grown intentionally to aid ORR catalyst operations instead of using additional expensive catalysts. A second generation of these cathodes also include a natural oxygen generating mechanism by incorporating growth of photosynthetic bioreactors [ 79 ] on the same platform. Appl. Sci. 2021,11, 2220 7 of 44 3.1.2. Membranes In a classic dual-chamber configuration, the MFC anode and cathode are divided with a physical separator called membrane [ 26 ] where protons generated in the anode chamber travel across the membrane [ 80 ] towards the cathode chamber for the final reaction with oxygen and electrons. An optimal MFC membrane should: 1. Provide high ion, especially cation, conductivity [81]; 2. Inhibit oxygen diffusion from the cathode side to the anode side for facilitating redox reaction at the cathode and maintaining anaerobic condition at the anode [82]; 3. Reduce the impacts of pH slitting [83]; 4. Reduce biofouling occurrence at cathode [ 84 ] and the membrane [ 85 ] by inhibiting substrate crossover [26]; 5. Provide chemical stability [26]. Proton exchange membranes (PEMs) are extensively [ 86 ] used as separators in MFCS and of them Nafion is the most popular one [ 21 , 26 , 86 ]. Nonetheless, due to the excessive cost of Nafion multiple alternatives, sulfonated polymer materials [ 16 ] have been exclusively tested. Here, we propose an integrated classification of all these MFC membranes from the concepts combined form [16,27,87]. Structurally the membranes can be classified into two major groups: (i) non-porous polymer membranes and (ii) porous membranes. Non-porous membranes can be further classified into cation (CEM) and anion (AEM) exchange membranes. The famous proton exchange membranes fall under the CEM group including Nafion [ 88 ], Hyflon [ 89 ] and Ultrex [ 90 ]. These CEMs are further classified into perfluorinated membranes, hydrocarbon membranes and composite membranes. Perfluorinated membranes are of special interest as Nafion and its derivatives fall under this group. Instead of cations, AEM membranes conduct hydroxide or carbonate anions from the cathode to the anode chamber while acting as proton carriers [ 91 ]. They are preferred over Nafion and similar ones where reducing the impact of pH splitting is of importance [ 92 – 94 ]. Porous membranes offer higher chemical, thermal and mechanical stability and lower cost over non-porous ones [ 27 ]. They can be further classified into ceramic [ 95 – 97 ] and fiber types [ 98 ]. A hierarchy of MFC membranes can be seen in Figure 6. Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 47 3.1.2. Membranes In a classic dual-chamber configuration, the MFC anode and cathode are divided with a physical separator called membrane [26] where protons generated in the anode chamber travel across the membrane [80] towards the cathode chamber for the final reaction with oxygen and electrons. An optimal MFC membrane should: 1. Provide high ion, especially cation, conductivity [81]; 2. Inhibit oxygen diffusion from the cathode side to the anode side for facilitating redox reaction at the cathode and maintaining anaerobic condition at the anode [82]; 3. Reduce the impacts of pH slitting [83]; 4. Reduce biofouling occurrence at cathode [84] and the membrane [85] by inhibiting substrate crossover [26]; 5. Provide chemical stability [26]. Proton exchange membranes (PEMs) are extensively [86] used as separators in MFCS and of them Nafion is the most popular one [21,26,86]. Nonetheless, due to the excessive cost of Nafion multiple alternatives, sulfonated polymer materials [16] have been exclusively tested. Here, we propose an integrated classification of all these MFC membranes from the concepts combined form [16,27,87]. Structurally the membranes can be classified into two major groups: (i) non-porous polymer membranes and (ii) porous membranes. Non-porous membranes can be further classified into cation (CEM) and anion (AEM) exchange membranes. The famous proton exchange membranes fall under the CEM group including Nafion [88], Hyflon [89] and Ultrex [90]. These CEMs are further classified into perfluorinated membranes, hydrocarbon membranes and composite membranes. Perfluorinated membranes are of special interest as Nafion and its derivatives fall under this group. Instead of cations, AEM membranes conduct hydroxide or carbonate anions from the cathode to the anode chamber while acting as proton carriers [91]. They are preferred over Nafion and similar ones where reducing the impact of pH splitting is of importance [92–94]. Porous membranes offer higher chemical, thermal and mechanical stability and lower cost over non-porous ones [27]. They can be further classified into ceramic [95–97] and fiber types [98]. A hierarchy of MFC membranes can be seen in Figure 6. Figure 6. Classification of actively used MFC membranes. Appl. Sci. 2021,11, 2220 8 of 44 3.2. Classification of MFCs An MFC cell consists of an anaerobic, biotic anode component and an aerobic biotic/abiotic cathode component. Depending on the external environment, MFCs can broadly be classified into lab-scale (inside lab) and in situ (outside lab) configurations. In situ lab configurations can be further classified into aquatic and terrestrial configurations. Aquatic MFCs consist of sediment docked and non-sediment docked configurations and sediment docked configuration can be further classified into open water configurations and closed water configurations. A hierarchy of this placement dependent MFC classification can be found in Figure 7. Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 47 Figure 6. Classification of actively used MFC membranes. 3.2. Classification of MFCs An MFC cell consists of an anaerobic, biotic anode component and an aerobic biotic/abiotic cathode component. Depending on the external environment, MFCs can broadly be classified into lab-scale (inside lab) and in situ (outside lab) configurations. In situ lab configurations can be further classified into aquatic and terrestrial configurations. Aquatic MFCs consist of sediment docked and non-sediment docked configurations and sediment docked configuration can be further classified into open water configurations and closed water configurations. A hierarchy of this placement dependent MFC classification can be found in Figure 7. Figure 7. Classification of MFCs according to their placements. 3.2.1. Laboratory-Scale MFCs Laboratory-scale or in-lab MFCs are smaller and are usually in the order of milliliters in volume [99–101]. These lab-scale MFCs are the most investigated ones and have been extensively utilized to identify fundamental conditions like anode-materials [102,103] and configurations [104,105], improved catalyst/cathode configurations [106,107] as well as for the study of microbial communities [101]. For scaling up operations, it is also conventional to implement a lab-scale MFC and achieve an optimal output, mimicking the desired outdoor conditions [108–110] and then transfer it to the external environment for pilot studies [99,111]. However, this methodology poses huge challenges since complex configurations for a large scale are difficult to implement [100] and increase expense. Bio-fouling and clogging become more severe in the case of in situ long-term MFC operations [99]. Thus, in multiple cases it has been found that volumetric power densities reduce with increasing MFC reactor size [112,113]. Figure 7. Classification of MFCs according to their placements. 3.2.1. Laboratory-Scale MFCs Laboratory-scale or in-lab MFCs are smaller and are usually in the order of milliliters in volume [ 99 – 101 ]. These lab-scale MFCs are the most investigated ones and have been extensively utilized to identify fundamental conditions like anode-materials [ 102 , 103 ] and configurations [ 104 , 105 ], improved catalyst/cathode configurations [ 106 , 107 ] as well as for the study of microbial communities [101]. For scaling up operations, it is also conventional to implement a lab-scale MFC and achieve an optimal output, mimicking the desired outdoor conditions [108–110] and then transfer it to the external environment for pilot studies [ 99 , 111 ]. However, this methodology poses huge challenges since complex configurations for a large scale are difficult to implement [ 100 ] and increase expense. Bio-fouling and clogging become more severe in the case of in situ long-term MFC operations [ 99 ]. Thus, in multiple cases it has been found that volumetric power densities reduce with increasing MFC reactor size [112,113]. 3.2.2. In Situ MFCs A classic in situ MFC has a biotic anode and an abiotic cathode and is placed in an external environment outside the lab and hence requires a robust architecture. They can be further classified into aquatic and terrestrial MFCs. Appl. Sci. 2021,11, 2220 9 of 44 3.2.3. Aquatic MFCs In aquatic MFCs the anode is buried in the anaerobic soil facilitating bacterial growth and metabolism while at the aerobic cathode, either submerged in water or exposed to air, the cell redox action is completed. Sediment docked MFCs Benthic MFCs or sediment MFCs are possibly the most studied configuration where bacterial metabolic activity occur at anaerobic, buried anodes and therefore we refer to them in our classification as sediment-docked MFCs [ 114 ]. The resulting electrons from oxidation of organic components are received by the anode and then travel via an external circuit to an aerobic, submerged cathode [ 115 ]. The limiting factors in developing this in situ benthic MFC configuration include: 1. Low output voltage and power [116]; 2. Depth dependent performance of both anode [104] and cathode [68]; 3. High resistance value of mass and electron transport in sediments [117]; 4. Degradation of electrode materials quality due to: a. electrochemical deposition, b. corrosion, c. impacts of water flow, d. fish grazing [118], and e. burrowing anodes [62]. A major section of benthic MFC based research works include development of benthic MFCs for distant marine environment [ 11 , 119 , 120 ] while others include power generation from environments like lakes [ 28 , 121 ], mangrove lands [ 122 ], rice paddy fields [ 123 ], aquatic ponds [ 124 ], and conjugate application of power generation and biodegradation from contaminated sediments of waste water treatments [29,125,126]. Based on the condition that the sediment for burial of the anodes could be manipulated or not, we propose to classify benthic MFCs into two more subcategories: open-water and close-water benthic MFCs. The major feature of open-water in situ MFCs is that the anode burying underwater sediment can not be manipulated for ensuring the continuous operation of MFCs. Over the last decade this particular type of energy harvester has emerged as a viable solution for distant ocean monitoring by providing electricity to low power sensors [ 11 ] as well as communication devices with special concentration toward acoustic modems [119,120,127,128] . Additionally, they are also being used as biosensors for detecting organic carbon in sea water [129]. The selection of anode and cathode materials for marine benthic MFCs has been an important field of research so that they provide structural stability [ 109 ] as well as facilitate biofilm growth [ 130 ]. There are reports of using multiple engineered configuration of carbon materials like activated carbon fiber felt [ 131 ], granulated activated carbon [109,132] , modified polyaniline-graphene nanosheets [ 116 ] and composite multi wall carbon nanotube [58,130,133] materials. Benthic MFC electrodes can be of small [ 11 , 128 ] (within 4 m), medium [ 115 , 134 ], (between 5 to 8 m) and large (above 8 m) [ 127 ]. For the large-scale MFCs, development and deployment of large-scale graphene felt cathode/anode [ 108 ] has remained as another active research area. Different electrode configurations [ 132 ] and combinations as well as application-specific power management systems (PMSs) are also of great concern for these remote-powering MFCs. Distributed benthic MFCs are considered to be a practical solution for harsh marine environment. This is due to the fact that in case of the failure of one MFC anode/cathode, the other electrodes still remain operational and thus provide enhanced durability [62,100,105,119,132,135]. The sediment MFC is the first popular version of MFCs and in addition to open water configurations, they have also been used in closed water configurations where the Appl. Sci. 2021,11, 2220 16 of 44 cessing, please see the review study by Gude [ 215 ] and for the impact of wastewater substrate composition, please see the works of Pandey et al. [ 216 ]. To go into more detail of how to integrate wastewater processing with MFC technologies, please see the works of He et al. [ 55 ]. Do et al. [ 29 ] classified conventional wastewater treatment-MFC power-generation systems into 5 major groups including sediment MFCs, constructed wetland MFCs, membrane bioreactor MFCs (MBR-MFCs), desalination MFCs (DS-MFCs) and others. The authors also performed comparison between the classes in terms of their substrate, power density, and chemical oxygen demand rate. Logroño et al. [ 217 ] reported their work on simultaneous electricity production real dye textile wastewater processing. Further work on textile waste water processing with different MFC configurations has also been reported. For example, decolorization of azo dye with biocathode configuration [ 218 – 220 ]. Use of algae in MFC systems has been another promising trend, whether being used as biocathode material [217] or as substrate [10]. Constructed wetland is a man-made wetland used for organic degradation for the wide range of agricultural to industrial wastewater [221]. Various efforts can be found on integrating the organic process of constructed wetland with MFCs. Oon et al. [ 221 ] built an up-flow constructed wetland where anaerobic and aerobic regions were naturally developed in the lower and upper bed and the system obtained a 100% chemical oxygen demand removal efficiency. Yadav et al. [ 222 ] reported the use of another vertical flow constructed wetland system to remove different dye from synthetic wastewater and generate electricity as well. The configuration achieved the maximum value of 93.15% dye removal efficiency following 96 h of treatment from the wastewater with 500 mgl −1 initial dye concentration. Villaseñor et al. [ 223 ] tested the applicability of horizontal subsurface flow constructed wetland for performing simultaneous organic waste processing and power generation. This study offered two major observations: the photosynthetic activity of the macrophytes, Phragmites australis was dependent affected on the light/darkness changes, and this caused voltage fluctuations and affected stable performance of the system. Liu et al. [ 224 ] demonstrated that use of Ipomoea aquatica plant in their constructed wetland MFC system provided a higher power density and nitrogen removal in comparison to their contemporary systems. The authors also worked on optimizing the vertical constructed wetland with 3 different electrode materials (stainless steel mesh, carbon cloth, granular activated carbon) and found both stainless steel mesh and granular activated carbon’s suitability for such configurations. Fang et al. [ 219 ] reported another successful combination of Ipomoea aquatica plantation constructed wetland-MFC system for azo dye decolorization. The planted system achieved the decolorization rate of maximum 91.24% with a voltage output of about 610 mV. Additionally, the system promoted growth of Geobacter sulfurreducens and β -Proteobacteria while inhibited Archaea growth in anode. Srivastava et al. [ 225 ] found from their study that a coupled constructed wetlandβ MFC system performs better in removing organic substances than normal constructed wetland systems. For a more detailed review of coupled constructed wetland and MFC system, please see the review by Doherty et al. [ 190 , 226 ]. In [ 190 ], the authors particularly stressed the importance of maintaining anaerobic anode and oxygenated cathode configuration separated. Corbella and Puigagut [ 226 ] indicated that constructed wetland systems naturally offer aerobic conditions in the upper layers and anaerobic in the deeper ones and results in a favorable environment for MFC power generation system implementation. Xu et al. [ 191 ] identified high internal resistance as one of the limiting factors of such coupled systems. The researchers tested a new strategy, called capacitator engaged duty cycling, with an open air bio-cathode constructed wetland MFC system and obtained 19.81% more electric charge than the conventional continuous loading system. MFC integrated wastewater treatment processes have been used to remove sulfur [ 227 ], sulfide [ 106 , 199 , 228 , 229 ], nitrogen [ 230 ], chromium [ 10 ] and salt components [ 172 ]. While metal contaminated waste possess great health and environmental risks, it also provides possibilities of precious metal recovery [ 231 ]. Combination of MFCs and microbial electrosynthesis have emerged as a method of choice for such metal recovery systems [ 30 ]. Appl. Sci. 2021,11, 2220 17 of 44 Fradler et al. [ 232 ] reported that heavy metal ions (for example Zn 2+ , Ni 2+ , Cr 2+ , V 5+ or Co 2+ ) are often present in industrial wastewater and can be extracted using similar combined configurations. Li e al. [ 233 ] reported a self-sustained combination of MFC and microbial electrosynthesis capable of extracting three heavy metals including chromium (Cr), lead (Pb) and nickel (Ni). 3.4.3. Bioremediation An additional application of MFC technology has been bioremediation and this is also one of the most investigated applications of sediment MFCs [ 234 ]. In this process microorganisms are utilized to treat polluted sites to break down environmental pollutants, to regain their original condition [ 235 ]. This has long remained as an alternative natural process of waste removal from land [ 236 ]. For more details on recent developments on bioremediation of sediments, please refer to the works of [ 67 ]. There have been particular examples of this in removing in removing organic [ 237 ] hydrocarbon [ 238 ] and metal [ 239 ] based pollutants. MFCs have been reported to recover Ag(I), Au(III), Co(II), Cd(II), Cr(VI), Cu(II), Hg(II), Pb(II), Se(IV), V(V), U(VI), and Zn(II) [ 30 ]. Yun-Hai et al. [ 240 ] reported their study on silver recovery from silver alkaline wastewater and simultaneous electricity production in a dual chamber bio-electrochemical cell. In their review, Dominguez-Benetton et al. [ 241 ] summarized the latest mechanisms of metal recovery using MFCs. In another review, Wang et al. [ 231 ] classified mechanisms of metal recovery using MFCs in 4 different categories: direct metal recovery with abiotic cathodes; metal recovery with externally powered abiotic cathodes, metal conversion with bio-cathodes and metal conversion with externally powered bio-cathodes supplemented by external power sources. 3.4.4. Solid Waste Processing Mohan et al. [ 242 ] reported a solid phase MFC system, developed to evaluate the potential of bioelectricity production by fermentation of food waste that gave promising results. The configuration utilized open air cathode sediment MFC configuration with graphite electrodes. They identified distance between the electrodes and PEM had a significant influence on power output, and the amendment of sodium carbonate improved system’s power buffering capacity. Lee et al. [ 243 ] reported another MFC-based system for handling solid wastes as a feedstock. The authors evaluated the system with two configurations: (1) a single chamber, combined membrane-electrode configuration; and (2) a dual chamber, proton-membrane-less configuration with brush-type anode and double air cathode. They have used cow manure for feedstock and the second configuration provided better results with higher power output. Wang et al. [ 244 ] also reported a solid state MFC for processing cow manure with a single-chamber, air-cathode MFC configuration. The authors reported that a moisture content higher than 80% was suitable for current generation. Moreover, an addition of small amount of platinum catalyst improved the power density by 10-fold and output voltage by twice as much. Damiano et al. [ 245 ] reported their study of feasibility analysis of two MFC-based electricity generation configurations that could simultaneously treat municipal solid waste landfill leachate. They identified that for such cases, smaller configurations perform better in power generation. Pendyala et al. [ 246 ] tested the practicality of using solid municipality waste as substrate for an MFC-based system where they categorized the organic waste components in 3 main groups including food waste, paper–cardboard waste and garden waste and concluded that the organic fraction of municipal solid waste is a promising feedstock for MFC-based waste processing. Detailed data analysis from their observations indicate that the microbial composition of the anodic biofilm became a function of the feed composition. Moreover, they also found that regulating the protein content and removing furfurals and phenolic compounds from feedstock could increase the percentage of chemical oxygen demand removal rate. Appl. Sci. 2021,11, 2220 18 of 44 3.4.5. Biosensing A biosensor can be defined as a system that utilizes specific biochemical reactions to identify the presence, absence or concentration change of chemical components by electrical, thermal or optical signals [ 31 ]. The MFC current output is directly related to the metabolic activity of the electroactive microbial community in the anode zone [ 247 ], thus making the system readily usable for multiple biosensing applications. Multiple review works also targeted summarizing biosensor applications of MFCs [31,192,247–251]. Two most common applications of this category are biochemical oxygen demand (BOD) sensing and various toxicity sensing [ 31 , 248 , 249 ]. Jiang et al. [ 252 – 254 ] reported a copper, i.e., Cu (II) toxicity sensor, a formaldehyde detection sensor and an overall water-quality monitoring system including organic matter, heavy metal (Cu2+), and acidic toxicity detection. Quek et al. [ 129 ] reported an intuitive MFC-based biosensor configuration for detection of biofouling occurring at reverse osmosis membranes of marine desalination systems. Zhou et al. [ 255 ] reported an MFC-based carbon monoxide detecting biosensor depending on the hypothesis that carbon monoxide inhibits bacterial activity in the anode would also decrease electricity. 4. Research Progress in Energy Harvesting from Enzyme-Based Biofuel Cells (EBFCs) EBFCs have emerged as the most practical form of chemical energy harvesting technology [ 33 , 256 ] for living organisms in implantable form and are also referred to as glucose biofuel cells. These EBFCs are the second largest group within the bioenergy harvesters. Similar to MFCs, they also depend on the electro-catalytic activity of enzymes for energy generation [ 4 ]. However, by contrast with MFCs, these cells are not intended for industrial energy production and their focus is on powering up various micro-scale [ 7 ], biomedical devices [34] that are used for clinical purposes. 4.1. Principles of Electricity Generation from EBFCs EBFCs are bio-electrochemical cells that can extract energy from glucose and alcohol based organic substances [ 257 ]. In a classic EBFC configuration, glucose oxidase or glucose dehydrogenase (GDH) are immobilized at the bio-anode for glucose oxidation while oxygen is reduced at the biocathode using immobilized laccase or bilirubin oxidase and generating power [33]. Although their principle of operation is similar to MFCs, both anode and cathode of such bio-fuel cells are prepared by embedding pure enzymes [ 257 ]. Thus, at a lower level, EBFC electrodes are inherently different from MFC electrodes since they require the presence of specific enzymes without the presence of the microorganisms that generate them. However, from a top view, EBFCs are equivalent to a classic two-electrode MFC configuration since they perform oxidation of glucose at the anode and oxygen reduction at the cathode to generate electrical power [ 32 ] and remain connected via an external load resistance. A conceptual implementation of an EBFC is shown in Figure 10. These enzyme-immobilized electrodes are an indispensable part of EBFCs where synthetically produced enzyme catalysts are assembled on electrode surfaces [ 259 ]. The development of enzyme immobilization techniques has remained as an active research area for the last 20 years [ 25 ]. Common enzyme immobilization techniques include enzyme covalent binding or “wiring”, sandwiching between a protective polymer layer and the electrode surface and entrapment inside the polymeric membrane coating of the electrode [ 260 ]. This technology offers an opportunity to miniaturize the cell structure since the configuration does not require separation of fuel and oxidant [ 259 ]. A pictorial summary of various immobilization techniques has been given in Figure 11. Appl. Sci. 2021,11, 2220 19 of 44 Appl. Sci. 2021, 11, x FOR PEER REVIEW 19 of 47 Although their principle of operation is similar to MFCs, both anode and cathode of such bio-fuel cells are prepared by embedding pure enzymes [257]. Thus, at a lower level, EBFC electrodes are inherently different from MFC electrodes since they require the presence of specific enzymes without the presence of the microorganisms that generate them. However, from a top view, EBFCs are equivalent to a classic two-electrode MFC configuration since they perform oxidation of glucose at the anode and oxygen reduction at the cathode to generate electrical power [32] and remain connected via an external load resistance. A conceptual implementation of an EBFC is shown in Figure 10. Figure 10. A futuristic concept of a biofuel cell for implementation within a blood vessel with enzyme immobilized electrodes, redrawn from the schematic shown in [258]. These enzyme-immobilized electrodes are an indispensable part of EBFCs where synthetically produced enzyme catalysts are assembled on electrode surfaces [259]. The development of enzyme immobilization techniques has remained as an active research area for the last 20 years [25]. Common enzyme immobilization techniques include enzyme covalent binding or “wiring”, sandwiching between a protective polymer layer and the electrode surface and entrapment inside the polymeric membrane coating of the electrode [260]. This technology offers an opportunity to miniaturize the cell structure since the configuration does not require separation of fuel and oxidant [259]. A pictorial summary of various immobilization techniques has been given in Figure 11. Figure 11. Various enzyme immobilization techniques for enzyme-based biofuel cells (EBFCs). Figure 10. A futuristic concept of a biofuel cell for implementation within a blood vessel with enzyme immobilized electrodes, redrawn from the schematic shown in [258]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 19 of 47 Although their principle of operation is similar to MFCs, both anode and cathode of such bio-fuel cells are prepared by embedding pure enzymes [257]. Thus, at a lower level, EBFC electrodes are inherently different from MFC electrodes since they require the presence of specific enzymes without the presence of the microorganisms that generate them. However, from a top view, EBFCs are equivalent to a classic two-electrode MFC configuration since they perform oxidation of glucose at the anode and oxygen reduction at the cathode to generate electrical power [32] and remain connected via an external load resistance. A conceptual implementation of an EBFC is shown in Figure 10. Figure 10. A futuristic concept of a biofuel cell for implementation within a blood vessel with enzyme immobilized electrodes, redrawn from the schematic shown in [258]. These enzyme-immobilized electrodes are an indispensable part of EBFCs where synthetically produced enzyme catalysts are assembled on electrode surfaces [259]. The development of enzyme immobilization techniques has remained as an active research area for the last 20 years [25]. Common enzyme immobilization techniques include enzyme covalent binding or “wiring”, sandwiching between a protective polymer layer and the electrode surface and entrapment inside the polymeric membrane coating of the electrode [260]. This technology offers an opportunity to miniaturize the cell structure since the configuration does not require separation of fuel and oxidant [259]. A pictorial summary of various immobilization techniques has been given in Figure 11. Figure 11. Various enzyme immobilization techniques for enzyme-based biofuel cells (EBFCs). Figure 11. Various enzyme immobilization techniques for enzyme-based biofuel cells (EBFCs). To obtain a summary of the progresses in the enzyme based biofuel cells over the last 30 years, the readers are referred to the works of Rasmussen et al. [ 261 ]. According to the authors, the biggest challenge in this field is the low stability and electrochemical performances of EBFCs. Babadi et al. [ 33 ] predicted that enzyme immobilization of EBFC electrodes can be greatly improved with novel nano carbon materials. For more details on EBFC-targeted carbon nano materials and functionalization, the readers are referred to their work [ 33 ]. Gonzalez-Solino et al. [ 262 ] summarized the most commonly used enzymes for EBFC anodes, wearable EBFC solutions, EBFC-based biosensors and provided a comparison of implantable EBFCs in terms of their output power. They hypothesize that these developments of cost-effective and safe EBFCs can power up key biomarker monitoring and help saving millions of lives. Gamella et al. [ 263 ] have focused on the interfacing technologies between EBFCs. They hypothesize that these cells, operating either internally or externally on a human body, could pave the way of bionic humanmachine hybrid and this scope can extend to cyborg animals as well and can greatly contribute to environmental monitoring, homeland security and military applications. Jeerapan et al. [ 264 ] have summarized the key bottlenecks of wearable EBFC technology. They focus on the fact that the sustainable development of wearable EBFCs must be able to dynamically adjust with uncontrolled body changes that occur due to regular movements. Appl. Sci. 2021,11, 2220 20 of 44 Carbon nanomaterials are extensively used in fabrication of enzyme-immobilized electrodes for EBFCs owing to the thin diameter, a feature that makes electrodes accessible to the enzyme active sites [ 33 ]. For example, Göbel et al. [ 265 ] reported their work where the anodes were fabricated with carbon nanotubes (CNTs) and modified with a polyaniline film and the cathode was made with PQQ (pyrroloquinoline quinone)-modified carbon nanotubes. Chung et al. [ 266 ] developed a carbon nano-flower structure which can be successfully utilized for immobilizing enzymes for EBFC application. Another study reported the use of spray coating for producing flexible biocathodes [ 267 ]. In this study , the target of effective enzyme wiring was achieved with flexible biocathodes. These cathodes were spray coated using a conductive ink composed of carbon nanotubes dispersion. Thin continuous layers of CNTs were successfully coated on to of a gas diffusion layer paper with a variable thickness 1 and 7.8 µm and were later with Laccase enzyme. A very concerning feature for implantable components, and thus for EBFCs, is their host immune response. This is of utmost importance for any implants where living bodies react to unfamiliar materials through a series of reactions that would lead to the formation of a capsule of collagen around any external component. However, EBFCs require regular exchange of analytes through the cell and any protective collagen layer around the implantable device limits this mandatory transition of analytes [ 268 ]. Moreover, there are also possibilities of extracellular matrix infiltration inside the EBFC which can cause degradation of the immobilized enzymes [ 269 ]. A pictorial description of the concept is given in Figure 12. Appl. Sci. 2021, 11, x FOR PEER REVIEW 21 of 47 Figure 12. Common reasons for implanted EBFC failures. 4.2. Classification of EBFCs The motivation for developing EBFCs was backed by the demand of electrical power requirements for monitoring [270] and maintaining [258] physiological parameters in living macro-organisms. So far, successful operations of EBFCs have been employed in plants [270–273], insects [9], mollusks [8], lobsters [34] and mammals [35]. We can classify EBFCs into four major groups: in vitro, plant-powered, animal-powered and human-powered. Animal-powered EBFCs can be further classified into externally and internally implantable sub-groups. Nearly all presently available human-powered EBFCs fall under the wearable sub-group. This classification is depicted in Figure 13. Figure 13. Classifying EBFCs according to their placement. Studies on in vitro EBFCs, i.e., EBFCS which have been tested outside any living organism and in a lab environment, have been widely reported. One such experiment was presented by Castorena-Gonzalez et al. [274] where bucky paper electrodes were used. The anode was modified with PQQ-dependent glucose dehydrogenase, which is a quinoprotein enzyme with PQQ cofactor for facilitating glucose oxidation [275], and the cathode used laccase. The cell was initially tested in human serum solution and then on exposed rat cremaster tissue. Szczupak et al. [276] informed about an EBFC with a bienzymatic trehalose glucose oxidase trehalose anode and a bilirubin oxidase dioxygen Figure 12. Common reasons for implanted EBFC failures. 4.2. Classification of EBFCs The motivation for developing EBFCs was backed by the demand of electrical power requirements for monitoring [ 270 ] and maintaining [ 258 ] physiological parameters in living macro-organisms. So far, successful operations of EBFCs have been employed in plants [ 270 – 273 ], insects [ 9 ], mollusks [ 8 ], lobsters [ 34 ] and mammals [ 35 ]. We can classify EBFCs into four major groups: in vitro , plant-powered, animal-powered and humanpowered. Animal-powered EBFCs can be further classified into externally and internally implantable sub-groups. Nearly all presently available human-powered EBFCs fall under the wearable sub-group. This classification is depicted in Figure 13. Appl. Sci. 2021,11, 2220 21 of 44 Appl. Sci. 2021, 11, x FOR PEER REVIEW 21 of 47 Figure 12. Common reasons for implanted EBFC failures. 4.2. Classification of EBFCs The motivation for developing EBFCs was backed by the demand of electrical power requirements for monitoring [270] and maintaining [258] physiological parameters in living macro-organisms. So far, successful operations of EBFCs have been employed in plants [270–273], insects [9], mollusks [8], lobsters [34] and mammals [35]. We can classify EBFCs into four major groups: in vitro, plant-powered, animal-powered and human-powered. Animal-powered EBFCs can be further classified into externally and internally implantable sub-groups. Nearly all presently available human-powered EBFCs fall under the wearable sub-group. This classification is depicted in Figure 13. Figure 13. Classifying EBFCs according to their placement. Studies on in vitro EBFCs, i.e., EBFCS which have been tested outside any living organism and in a lab environment, have been widely reported. One such experiment was presented by Castorena-Gonzalez et al. [274] where bucky paper electrodes were used. The anode was modified with PQQ-dependent glucose dehydrogenase, which is a quinoprotein enzyme with PQQ cofactor for facilitating glucose oxidation [275], and the cathode used laccase. The cell was initially tested in human serum solution and then on exposed rat cremaster tissue. Szczupak et al. [276] informed about an EBFC with a bienzymatic trehalose glucose oxidase trehalose anode and a bilirubin oxidase dioxygen Figure 13. Classifying EBFCs according to their placement. Studies on in vitro EBFCs, i.e., EBFCS which have been tested outside any living organism and in a lab environment, have been widely reported. One such experiment was presented by Castorena-Gonzalez et al. [ 274 ] where bucky paper electrodes were used. The anode was modified with PQQ-dependent glucose dehydrogenase, which is a quinoprotein enzyme with PQQ cofactor for facilitating glucose oxidation [ 275 ], and the cathode used laccase. The cell was initially tested in human serum solution and then on exposed rat cremaster tissue. Szczupak et al. [ 276 ] informed about an EBFC with a bienzymatic trehalose glucose oxidase trehalose anode and a bilirubin oxidase dioxygen cathode. Yin et al. [ 273 ] reported their study on a needle-type biofuel cell and tested it on artificial blood glucose and from the blood glucose in a mouse heart. Initially and until now in the primary phase, the EBFCs have been tested in plant mediums. Flexer et al. [ 270 ] utilized cactus leaf and a light source to implement a biosensor for continuous monitoring of O 2 and glucose generation rate. MacVittie et al. [ 271 ] reported their enzyme-based bioreactor for orange pulp and the generated power was used to power a wireless data transmission system. A similar experiment using orange pulp for powering a wireless transmitter was reported by Holade et al. [ 272 ]. The authors worked on an EBFC in which electrodes were modified with inorganic nanoparticles deposited and with carbon black. Yin et al. [ 273 ] reported a needle type biofuel cell whose performance was tested in various fruits and 55, 44, and 33 µ W of power output was obtained from specimens of grape, kiwifruit, and apple, respectively. There is an interesting bio-hybrid EBFC implanted animal group that includes experiments where the EBFC (main cell) is placed outside the energy-harvesting organism. It is inspired by the observation that placement of a complete cell inside the receiving living organism becomes difficult for small-sized organisms like snails [ 8 ]. A similar configuration has also been used in live American lobsters [ 34 ]. A cyborg insect, Blaberus discoidalis, was reported by Schwefel et al. [ 9 ] where the insect hemolymph was used for the substrate for a trehalose/oxygen biofuel cell. As a continuation, a similar system was implemented in a moth for powering up a wireless sensor for environmental monitoring [12]. There are also examples of internally implanted EBFCs in animals. Cinquin et al. [ 277 ] reported the first in vivo implantation employed in the retroperitoneal space of moving rats. Zebda et al. [ 32 ] reported a similar configuration in rats, capable of producing an average open-circuit voltage of 0.57 V. Nadeau et al. [ 278 ] reported a cell implanted in the gastrointestinal tract of pigs that could perform in vivo temperature sensing and wireless communication. Ichi-Ribault et al. [ 35 ] reported implantation of another abdominal implant in a rabbit that operated for 2 months. A new group of EBFCs are now being investigated which can be implanted externally. For example, Toit et al. reported power generation from a transdermal extract of pig skin [279]. Appl. Sci. 2021,11, 2220 22 of 44 Scopes of utilization for such external or wearable EBFCs for human use are also being explored. For example, studies on the use of an enzymatic biofuel cell on a contact lens [ 280 – 282 ] and patches [ 283 , 284 ] have been reported. Generation of electrical power from human perspiration are also reported [13,285]. Because of the present limitations in invasive cell implantation, research scopes took a turn towards the possibilities of using EBFCs with physiological fluids as an alternative to blood [262]. 5. Research Progress in Biomechanical Energy-Harvesting Technologies There has been an increasing demand for biocompatible and environment friendly alternative energy sources for next generation of low-power, wearable electronic components [ 1 ] and biomechanical energy-harvesting technology has emerged as a promising solution. They offer an eco-friendly and non-invasive power solution for monitoring, diagnostic and therapeutic operations. These devices utilize mechanical energy produced from living animals and convert them into power solutions. 5.1. Biomechanical Energy-Harvesting Mechanisms According to Dong et al. [ 286 ], the four major bio-mechanical energy harvesters are piezoelectric, electromagnetic, electrostatic, and triboelectric mechanisms. In most cases, these mechanisms fall either within the vibration-based energy-harvesting technologies or motion-based energy-harvesting technologies. Piezoelectric materials with cantilever geometry have been the classic choice [ 287 ] for mechanical energy harvesting. These materials are a subset of ferroelectric materials and produce an electrical charge when being mechanically deformed [ 288 ]. Deterre et al. [ 14 ] reported a micro-spiral piezoelectric energy harvester for extracting energy from regular blood pressure variation, Shafer et al. [ 289 ] reported an energy-harvesting system from the wings of flying birds and bats that could be used to power environmental monitoring systems. Vibration-based piezoelectric energy-harvesting mechanism is more applicable for insects or animals with flapping wings than from human joint movements. Currently with piezoelectric technology, it is possible to generate around 20 watts from foot strikes as well as from movement of the body’s center of mass, 60 watts from ankle movements, 30 watts from both knee and heap movements, 2 watts from both elbow and shoulder movements [ 290 ]. A generalized piezoelectric-based energy-harvesting system is depicted in Figure 14. Appl. Sci. 2021, 11, x FOR PEER REVIEW 23 of 47 Piezoelectric materials with cantilever geometry have been the classic choice [287] for mechanical energy harvesting. These materials are a subset of ferroelectric materials and produce an electrical charge when being mechanically deformed [288]. Deterre et al. [14] reported a micro-spiral piezoelectric energy harvester for extracting energy from regular blood pressure variation, Shafer et al. [289] reported an energy-harvesting system from the wings of flying birds and bats that could be used to power environmental monitoring systems. Vibration-based piezoelectric energy-harvesting mechanism is more applicable for insects or animals with flapping wings than from human joint movements. Currently with piezoelectric technology, it is possible to generate around 20 watts from foot strikes as well as from movement of the body’s center of mass, 60 watts from ankle movements, 30 watts from both knee and heap movements, 2 watts from both elbow and shoulder movements [290]. A generalized piezoelectric-based energy-harvesting system is depicted in Figure 14. Figure 14. Piezoelectric based biomechanical electricity generation principle, redrawn from the schematic shown in [291]. An electromagnetic induction principle has also been utilized to extract biomechanical energy. Zurbuchen et al. [292] reported their prototype for harvesting energy from endocardial heart motion by electromagnetic coupling and the system has been tested in vivo in domestic pigs. The proposed energy-harvesting device consists of serially aligned copper coils, surrounded by a linear arrangement of permanent magnets are suspended between two spiral springs. The permanent magnet stack oscillates in the case of motion, which in this case is the heart’s endocardial motion. Nakada et al. [293] developed an electromagnetic generator, suitable for inserting in the abdominal cavity of the birds and tested their performance in chickens and pheasants and obtained a power average of 0.47 mW. The objective of this work was to power up a biosensor to test the antigen-antibody reaction of avian influenza. Powering up the sensor was performed by a tiny generator with an electromagnetic induction coil implanted in the bird’s abdominal cavity. The generator supplied power when chickens walk and pheasants flew. Almansouri et al. [294] efficiently used a magneto-acoustic resonator for tracking aquatic animals. The authors developed a system that converts low-frequency fish motion to excite high-frequency acoustic pulses. The prototype was able to generate an average acoustic sound of 55 dB sound pressure level at 1 m of distance with a resonant frequency of 15 kHz. Electrostatic energy harvesters transform energy from changing in capacitance according to Coulomb’s law for two parallel plate capacitors [295]. Although some examples can be found [296,297], these components did not gain much popularity for biomechanical energy harvesting. Triboelectric nano generators (TENG) were first introduced by Fan et al. [298] and rely on the principle of electric charge separation at the friction of two surfaces, different in terms of nano-scale roughness and thus creating an electric charge layer, by imposing variation in the capacitance electric energy that can be obtained from such small, flexible systems. This tribo-electrification is the working principle creating the natural phenomena of amber effect and lightning [299]. Combining the principle of tribo-electrification Figure 14. Piezoelectric based biomechanical electricity generation principle, redrawn from the schematic shown in [291]. An electromagnetic induction principle has also been utilized to extract biomechanical energy. Zurbuchen et al. [ 292 ] reported their prototype for harvesting energy from endocardial heart motion by electromagnetic coupling and the system has been tested in vivo in domestic pigs. The proposed energy-harvesting device consists of serially aligned copper coils, surrounded by a linear arrangement of permanent magnets are suspended between two spiral springs. The permanent magnet stack oscillates in the case of motion, which in this case is the heart’s endocardial motion. Nakada et al. [ 293 ] developed an elec- Appl. Sci. 2021,11, 2220 23 of 44 tromagnetic generator, suitable for inserting in the abdominal cavity of the birds and tested their performance in chickens and pheasants and obtained a power average of 0.47 mW. The objective of this work was to power up a biosensor to test the antigen-antibody reaction of avian influenza. Powering up the sensor was performed by a tiny generator with an electromagnetic induction coil implanted in the bird’s abdominal cavity. The generator supplied power when chickens walk and pheasants flew. Almansouri et al. [ 294 ] efficiently used a magneto-acoustic resonator for tracking aquatic animals. The authors developed a system that converts low-frequency fish motion to excite high-frequency acoustic pulses. The prototype was able to generate an average acoustic sound of 55 dB sound pressure level at 1 m of distance with a resonant frequency of 15 kHz. Electrostatic energy harvesters transform energy from changing in capacitance according to Coulomb’s law for two parallel plate capacitors [ 295 ]. Although some examples can be found [ 296 , 297 ], these components did not gain much popularity for biomechanical energy harvesting. Triboelectric nano generators (TENG) were first introduced by Fan et al. [ 298 ] and rely on the principle of electric charge separation at the friction of two surfaces, different in terms of nano-scale roughness and thus creating an electric charge layer, by imposing variation in the capacitance electric energy that can be obtained from such small, flexible systems. This tribo-electrification is the working principle creating the natural phenomena of amber effect and lightning [ 299 ]. Combining the principle of tribo-electrification and electrostatic induction, TENG were developed and have gained huge popularity for harvesting biomechanical energy. A generalized TENG energy-harvesting system is depicted in Figure 15. Appl. Sci. 2021, 11, x FOR PEER REVIEW 24 of 47 and electrostatic induction, TENG were developed and have gained huge popularity for harvesting biomechanical energy. A generalized TENG energy-harvesting system is depicted in Figure 15. Figure 15. Triboelectric nano electricity generation (TENG) principle. These nano generators, referred to as triboelectric nano generators (TENG) became very popular in a short time period, due to their flexibility [300] and cost-effectiveness. Zheng et al. [301] reported an in vivo biomechanical-energy harvesting using a TENG for the first time. Dong et al. [302] reported development of wearable, large-length, energy-harvesting textiles by incorporating TENG mechanism. 5.2. Energy Harvesting from Humans Biomechanical energy-harvesting solutions gained much popularity thanks to their easily wearable features. Choi et al. [40] summarized the applicability of biomechanical energy harvesting from various form of human actions and motions including foot, knee, hip as well as upper limb motions and gave special focus to high power density, back-pack like wearable rotary energy harvesters. Similar wearable harvesters have also been reported by Xie et al. [303], Yuan et al. [304] and Martin et al. [41] which generate watt level power density. Gurusamy et al. [305] reported the identification of lower limb joints which would be better suitable for energy harvesting. Many research groups have concentrated on utilizing biomechanical energy from foot strikes through shoe soles [306–308]. Works are also found on biomechanical energy harvesting from ankle [309], knee [310–313] and hip [314] movements. A generalized wearable backpack based biomechanical energy-harvesting system is depicted in Figure 16. Figure 15. Triboelectric nano electricity generation (TENG) principle. These nano generators, referred to as triboelectric nano generators (TENG) became very popular in a short time period, due to their flexibility [ 300 ] and cost-effectiveness. Zheng et al. [301] reported an in vivo biomechanical-energy harvesting using a TENG for the first time. Dong et al. [ 302 ] reported development of wearable, large-length, energyharvesting textiles by incorporating TENG mechanism. 5.2. Energy Harvesting from Humans Biomechanical energy-harvesting solutions gained much popularity thanks to their easily wearable features. Choi et al. [ 40 ] summarized the applicability of biomechanical energy harvesting from various form of human actions and motions including foot, knee, hip as well as upper limb motions and gave special focus to high power density, backpack like wearable rotary energy harvesters. Similar wearable harvesters have also been reported by Xie et al. [ 303 ], Yuan et al. [ 304 ] and Martin et al. [ 41 ] which generate watt level Appl. Sci. 2021,11, 2220 24 of 44 power density. Gurusamy et al. [ 305 ] reported the identification of lower limb joints which would be better suitable for energy harvesting. Many research groups have concentrated on utilizing biomechanical energy from foot strikes through shoe soles [ 306 – 308 ]. Works are also found on biomechanical energy harvesting from ankle [ 309 ], knee [ 310 – 313 ] and hip [ 314 ] movements. A generalized wearable backpack based biomechanical energyharvesting system is depicted in Figure 16. Appl. Sci. 2021, 11, x FOR PEER REVIEW 25 of 47 Figure 16. Rack-pinion gear based biomechanical electricity generation used in backpacks for utilizing up and down movements of the center of mass, redrawn from the schematic shown in [315]. Development of smart, energy-harvesting textiles with TENG components that would facilitate both electrical powering and wearable sensing applications has gained a considerable amount of interest over recent years. Example of such works include development of TENG-incorporated stainless steel/polyester fiber-blended yarns [302] and washable hybrid fibers with a piezoelectric-enhanced TENG mechanism [316]. Efforts have also been made to implement skin like transparent sheets that could harvest biomechanical energy. Relevant examples include, a textile-based TENG that can be actuated with skin touch [317]; a soft skin-like triboelectric nanogenerator that works both as an energy harvester and tactile sensor [318]; a transparent triboelectric, piezoelectric, pyroelectric combined hybrid nanogenerator with silver nanowires [319]; and an optically transparent, textile compatible TENG fiber with silk protein and silver nanowires [320]. 5.3. Energy Harvesting from Non-Human Living Organisms In addition to human-centered applications, biomechanical harvesters have also been tested for powering up cyborg-animal applications, targeted either toward environmental monitoring or observation of physiological parameters of the host animal. Aktakka et al. [42] reported a vibration energy scavenger for Cotinis nitida (Linnaeus) that could extract energy from the beetle’s wing vibrations without affecting its movement. Zheng et al. [301] implanted a TENG-based energy harvester in a living rat for powering up a pacemaker. Nakada et al. [293] reported developing an electromagnetic generator implantation in the subcutaneous area or abdominal cavity of chicken-like birds and obtained a maximum 7 V peak-to-peak signal at 560/min of flapping of wings. Li et al. [321] presented an energy-harvesting module containing a flexible piezoelectric beam for extracting energy from fish movement to power up an acoustic transducer. Similarly, Almansouri et al. [294] utilized a “magneto-acoustic” resonator that converts low-frequency motions, ranging from 0.15 to 100 Hz into high-frequency acoustic signals. Shearwood et al. [322] reported a mountable energy harvester for bees that extracts energy from wing vibrations and powers up a radio frequency bee tracker with minimal physical hindrance. 6. Promising Bio-Energy Solutions, Lessons Learned Our review of bio-energy harvesters focuses on three promising green energy resources. Tables 1–3 depict summaries of the key research components in the fields of MFC, enzyme-based fuel cells and biomechanical energy harvesters, respectively, over the last decade. Figure 16. Rack-pinion gear based biomechanical electricity generation used in backpacks for utilizing up and down movements of the center of mass, redrawn from the schematic shown in [ 315 ]. Development of smart, energy-harvesting textiles with TENG components that would facilitate both electrical powering and wearable sensing applications has gained a considerable amount of interest over recent years. Example of such works include development of TENG-incorporated stainless steel/polyester fiber-blended yarns [ 302 ] and washable hybrid fibers with a piezoelectric-enhanced TENG mechanism [ 316 ]. Efforts have also been made to implement skin like transparent sheets that could harvest biomechanical energy. Relevant examples include, a textile-based TENG that can be actuated with skin touch [ 317 ]; a soft skin-like triboelectric nanogenerator that works both as an energy harvester and tactile sensor [ 318 ]; a transparent triboelectric, piezoelectric, pyroelectric combined hybrid nanogenerator with silver nanowires [ 319 ]; and an optically transparent, textile compatible TENG fiber with silk protein and silver nanowires [320]. 5.3. Energy Harvesting from Non-Human Living Organisms In addition to human-centered applications, biomechanical harvesters have also been tested for powering up cyborg-animal applications, targeted either toward environmental monitoring or observation of physiological parameters of the host animal. Aktakka et al. [ 42 ] reported a vibration energy scavenger for Cotinis nitida (Linnaeus) that could extract energy from the beetle’s wing vibrations without affecting its movement. Zheng et al. [ 301 ] implanted a TENG-based energy harvester in a living rat for powering up a pacemaker. Nakada et al. [ 293 ] reported developing an electromagnetic generator implantation in the subcutaneous area or abdominal cavity of chicken-like birds and obtained a maximum 7 V peak-to-peak signal at 560/min of flapping of wings. Li et al. [ 321 ] presented an energy-harvesting module containing a flexible piezoelectric beam for extracting energy from fish movement to power up an acoustic transducer. Similarly, Almansouri et al. [ 294 ] utilized a “magneto-acoustic” resonator that converts low-frequency motions, ranging from 0.15 to 100 Hz into high-frequency acoustic signals. Shearwood et al. [ 322 ] reported a mountable energy harvester for bees that extracts energy from wing vibrations and powers up a radio frequency bee tracker with minimal physical hindrance. 6. Promising Bio-Energy Solutions, Lessons Learned Our review of bio-energy harvesters focuses on three promising green energy resources. Tables 1–3depict summaries of the key research components in the fields of Appl. Sci. 2021,11, 2220 25 of 44 MFC, enzyme-based fuel cells and biomechanical energy harvesters, respectively, over the last decade. Table 1. Summary of reviewed works on MFCs. MFC Focus Topic Sub-Topic(s) Studies MFC Biofilm [53,68,77,159,217,227,229,323–327] Dual chamber MFC [175,200,229,323,328–341] Single chamber MFC [57,194,217,342–348] MFC Anodes [20,49,61,63–65,349–372] MFC Cathodes [73,75,76,90,99,106,107,116,161] Air cathodes [63,65,70,74,77,126,344,348] Bio cathodes [68,77,145,159,179,183,192,220,224,254] Algae/micro-algae bio cathodes [72,177,193,195,196,217] Plant bio cathodes [2,140,158,176,184,186–189,370,373] Membranes [16,27,63,80,88,98,374,375] Cation exchange membranes [85,86] Anion exchange membranes [91–93,336] Porous, ceramic membranes [87,95–97,373,376–379] Supported liquid ion membrane [26,88,232] PMS [23,62,109,211,380] Remote power generation [6,11,23,24,61,65,68,107,110,123,127–129,135–137,140– 142,151,172,197,199] Waste processing Waste-water processing [19,29,58,60,67,73,114,121,132,149,155,194,202,203,216,315,322,325, 328,330,336,359,360] constructed-wetland [111,190,191,219,221–225] textile and dye processing [140,217–220] solid waste processing [242–246] metal recovery [30,231–233,240,241,369] Biosensing [11,28,31,111,129,143,154,194,201,202,247–255,354] Powering robots [381–385] Table 2. 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