Biofilms in Bioelectrochemical Energy Conversion Cells

Abstract

Provided herein is a voltacell comprising a biofilm for facilitating energy conversion in a bioelectrochemical energy conversion cell, where the biofilm comprises one or more microbial populations.

Description

[Background technology]

[0001] [Incorporated by reference] The PCT Request Form is being filed contemporaneously herewith as a part of this application. Each application to which this application claims the benefit or priority of an application identified in a contemporaneously filed PCT Request Form is hereby incorporated by reference in its entirety for all purposes.

[0002] Current voltaic cell and solar panel systems have limited efficiency, require complex materials, and result in significant associated costs. Many solar panels use wafer-based crystalline silicon cells or cadmium or silicon-based thin film cells. These cells are fragile and must be protected from moisture through the addition of multiple protective layers. Panels are deployed in series for increasing voltage and / or in parallel for increasing current. The panels are interconnected via conductive metal wires. An inherent problem with typical systems is the susceptibility of the cells to overheating due to reverse current when one portion of the panel is shaded and another portion of the panel is exposed to direct sunlight. Another inherent problem is that solar cells become less efficient at higher temperatures, which limits the geographical effectiveness of light conversion to electricity. Improvements, such as arrayed lenses and mirrors, improve light focusing and increase efficiency, but have additional manufacturing complexities and higher associated costs.

[0003] While biochemical voltacels may be a suitable alternative, they also face many challenges. Biochemical voltacels rely on organisms capable of generating energy that can be harvested and converted to generate electrical potential energy. However, utilizing these organisms involves maintaining a sufficient level of activity within the cell to keep the organisms alive and operating in a reliable manner. In some instances, the organisms may interfere with the operation of the cell itself, for example, by generating electricity at currents and / or voltages that are less than or greater than levels desired for the particular cell. Furthermore, maintaining a suitable environment to allow sustainable conditions for organisms to grow within a biochemical voltacel can be difficult and costly.

[0004] The description of the background art provided herein is for the purpose of generally presenting the context of the present disclosure. The inventors' work cited in this application is not admitted, explicitly or implicitly, as prior art to the present disclosure, to the extent that it is described in this Background section, and aspects of the description that may not otherwise qualify as prior art at the time of filing. Summary of the Invention

[0005] One embodiment includes a voltacell comprising: (a) an anode for receiving electrons and providing electrons to an external circuit or load; (b) a cathode for donating electrons to an electrochemical reaction; (c) a biofilm having microorganisms, the biofilm in electrical contact with the anode or cathode; (d) a buffer having an ionically conductive medium in contact with the anode and cathode; and (e) a vessel that at least partially contains the biofilm and the buffer.

[0006] In various embodiments, the voltaic cell also includes an ion-permeable, electron donor-impermeable barrier that separates the buffer into an anodic compartment and a cathodic compartment, thereby preventing the electron donor population from contacting the cathode, hi some embodiments, the barrier is electronically conductive.

[0007] In some embodiments, the barrier contacts the anode.

[0008] In various embodiments, the biofilm is in contact with at least one of the anode and the cathode, hi some embodiments, the biofilm is in contact with at least one of the anode, the cathode, and the ion-permeable, electron donor-impermeable barrier.

[0009] In various embodiments, the biofilm comprises two or more microorganisms.

[0010] In various embodiments, the biofilm is formed on a substrate within the voltaic cell. In some embodiments, the substrate is either the anode or the cathode. In some embodiments, the substrate is in contact with a surface of the anode or the cathode.

[0011] In various embodiments, the biofilm comprises positively charged moieties.

[0012] In various embodiments, the biofilm comprises negatively charged moieties.

[0013] In various embodiments, the biofilm comprises a synthetic portion.

[0014] In various embodiments, the biofilm comprises a non-synthetic portion.

[0015] In various embodiments, the biofilm comprises one or more filamentous appendages.

[0016] In various embodiments, the biofilm comprises one or more classes of microorganisms that are one or more of anaerobic, aerobic, and facultative anaerobic microorganisms.

[0017] In various embodiments, the biofilm comprises sulfur-oxidizing and sulfur-reducing microorganisms.

[0018] In various embodiments, the biofilm is selected from the group consisting of Rhodoferax ferrireducens, Lactobacillus acidophilus, Rhodospirillum rubrum, Desulfovibrio desulfuricans subsp. desulfuricans, Peptostreptococcus anaerobius, Rhodospirillum centenum, Catonella morbii, Lachnospiraceae species, Photobacterium leiognathi, Arochromatium vinosum, Lactobacillus casei, Fusobacterium nucleatum subsp. polymorphum, Helcococcus kunzii, and the like. The microorganisms include one or more microorganisms selected from the group consisting of: Cutibacterium acnes, Rhodospirillum rubrum, Hercococcus kunzii, Allochromatium vinosum, and Ferrovum myxofaciens.

[0019] In various embodiments, the biofilm comprises a matrix comprising a natural polymer, a synthetic polymer, a DNA hydrate, a protein hydrate, or a carbohydrate hydrate.

[0020] In any of the above described embodiments, the voltaic cell may also include a current collector in electrical communication with the anode.

[0021] In any of the embodiments described above, the first species of microorganisms and / or the second species of microorganisms include light-harvesting antennas. In various embodiments, the first species of microorganisms are excited by electromagnetic radiation in a first band and at least one other species of microorganisms in the buffer are excited by electromagnetic radiation in a second band, and the first band and the second band do not substantially overlap.

[0022] In any of the embodiments described above, the first species of microorganisms comprises phototrophic or chemotrophic microorganisms.

[0023] In any of the embodiments described above, the first species of microorganisms are chemotrophs and the second species of microorganisms are phototrophs.

[0024] In any of the embodiments described above, the first major metabolic pathway oxidizes a compound containing carbon, nitrogen, phosphorus, or sulfur, and the second major metabolic pathway reduces the oxidized compound produced by the first major metabolic pathway.

[0025] In any of the embodiments described above, the first species of microorganism has a fimbrial, fibrous, flagellar, and / or filamentous shape.

[0026] In any of the embodiments described above, the first species of microorganism has multiple metabolic pathways.

[0027] In any of the embodiments described above, the first species of microorganism is a naturally occurring microbial species.

[0028] In any of the embodiments described above, the first primary metabolic pathway and the second primary metabolic pathway each involve cellular respiration.

[0029] Another aspect includes a method of converting chemical and / or light energy into electrical energy, the method comprising: operating a voltaic cell as described in any preceding embodiment.

[0030] Another embodiment includes a voltacell comprising: (a) cathode airflow hardware; (b) cathode gas diffusion layer; (c) cathode agar layer; (d) electrolyte layer comprising an ionically conductive medium in contact with the anode and cathode; (e) an anode layer for receiving electrons and providing electrons to an external circuit or load; (f) an anode agar layer; (g) a window layer; and (h) a biofilm comprising microorganisms.

[0031] In various embodiments, the microorganisms are present in one or more of the layers.

[0032] In various embodiments, the anode layer includes any one or more of the following materials: aluminum nanoparticles, aluminum microparticles, transparent conductor particles, hydrophilic polymers, and hydrophilic gels.

[0033] In various embodiments, the window layer comprises glass.

[0034] In various embodiments, the biofilm is in contact with at least one of the anode and the cathode, hi some embodiments, the biofilm is in contact with at least one of the anode, the cathode, and the ion-permeable, electron donor-impermeable barrier.

[0035] In various embodiments, the biofilm comprises two or more microorganisms.

[0036] In various embodiments, the biofilm is formed on a substrate within the voltaic cell. In some embodiments, the substrate is either the anode or the cathode. In some embodiments, the substrate is in contact with a surface of the anode or the cathode.

[0037] In various embodiments, the biofilm comprises positively charged moieties.

[0038] In various embodiments, the biofilm comprises negatively charged moieties.

[0039] In various embodiments, the biofilm comprises a synthetic portion.

[0040] In various embodiments, the biofilm comprises a non-synthetic portion.

[0041] In various embodiments, the biofilm comprises one or more filamentous appendages.

[0042] In various embodiments, the biofilm comprises one or more classes of microorganisms that are one or more of anaerobic, aerobic, and facultative anaerobic microorganisms.

[0043] In various embodiments, the biofilm comprises sulfur-oxidizing and sulfur-reducing microorganisms.

[0044] In various embodiments, the biofilm comprises one or more microorganisms selected from the group consisting of Rhodoferax ferrireducens, Lactobacillus acidophilus, Rhodospirillum rubrum, Desulfovibrio desulfuricans subsp. desulfuricans, Peptostreptococcus anaerobius, Rhodospirillum centenum, Catonella morbii, Lachnospiraceae species, Photobacterium leiognatii, Allochromatium vinosum, Lactobacillus casei, Fusobacterium nucleatum subsp. polymorphum, Helicococcus kunzii, Cutibacterium acnes, Rhodospirillum rubrum, Helicococcus kunzii, Allochromatium vinosum, and Ferrovum mixofaciens.

[0045] In various embodiments, the biofilm comprises a matrix comprising a natural polymer, a synthetic polymer, a DNA hydrate, a protein hydrate, or a carbohydrate hydrate.

[0046] In any of the above described embodiments, the voltaic cell may also include a current collector in electrical communication with the anode.

[0047] In any of the embodiments described above, the first species of microorganisms and / or the second species of microorganisms include light-harvesting antennas. In various embodiments, the first species of microorganisms are excited by electromagnetic radiation in a first band and at least one other species of microorganisms in the buffer are excited by electromagnetic radiation in a second band, and the first band and the second band do not substantially overlap.

[0048] In any of the embodiments described above, the first species of microorganisms comprises phototrophic or chemotrophic microorganisms.

[0049] In any of the embodiments described above, the first species of microorganisms are chemotrophs and the second species of microorganisms are phototrophs.

[0050] In any of the embodiments described above, the first major metabolic pathway oxidizes a compound containing carbon, nitrogen, phosphorus, or sulfur, and the second major metabolic pathway reduces the oxidized compound produced by the first major metabolic pathway.

[0051] In any of the embodiments described above, the first species of microorganism has a fimbrial, fibrous, flagellar, and / or filamentous shape.

[0052] In any of the embodiments described above, the first species of microorganism has multiple metabolic pathways.

[0053] In any of the embodiments described above, the first species of microorganism is a naturally occurring microbial species.

[0054] In any of the embodiments described above, the first primary metabolic pathway and the second primary metabolic pathway each involve cellular respiration.

[0055] These and other aspects are further described below with reference to the drawings. [Brief description of the drawings]

[0056] [Figure 1A] FIG. 1 is a schematic diagram of an energy conversion cell according to certain embodiments.

[0057] [Figure 1B] FIG. 1B is a diagram of a variation of the cell shown in FIG. 1A.

[0058] [Figure 1C] FIG. 1 is a diagram of an exemplary photosystem.

[0059] [Figure 1D] FIG. 1 is a diagram of a microbial containment enclosure having electrodes and a biofilm, according to certain disclosed embodiments.

[0060] [Figure 1E] FIG. 1 is a diagram of two layers that may be implemented within a multi-layer biofilm for a microbial containment enclosure.

[0061] [Figure 1F] 1 is a process flow diagram showing the arrangement of components for a microbial-based methanol fuel cell.

[0062] [Figure 1G] FIG. 1 is a diagram of multiple microorganism containment enclosures, according to certain disclosed embodiments.

[0063] [Figure 1H]FIG. 1 is a diagram of a microbial containment enclosure on an electrode, according to certain disclosed embodiments.

[0064] [Figure 1I] FIG. 1 is a process flow diagram illustrating operations that may be performed in accordance with certain disclosed embodiments.

[0065] [Figure 2A] FIG. 1 is an example of a filament shape formed on a biofilm.

[0066] [Figure 2B] FIG. 1 is a schematic diagram of a biofilm with microorganisms on a surface. [Figure 2C] FIG. 1 is a schematic diagram of a biofilm with microorganisms on a surface. [Figure 2D] FIG. 1 is a schematic diagram of a biofilm with microorganisms on a surface.

[0067] [Figure 2E] FIG. 1 is a schematic diagram of an example of a microorganism. [Figure 2F] FIG. 1 is a schematic diagram of an example of a microorganism.

[0068] [Figure 2G] FIG. 1 is a schematic diagram of connectivity between microorganisms.

[0069] [Figure 2H] FIG. 1 is a diagram of electron flow along a microbial filament.

[0070] [Figure 3A] FIG. 1 is a diagram of various shapes of biofilms on a substrate surface. [Figure 3B] FIG. 1 is a diagram of various shapes of biofilms on a substrate surface. [Figure 3C] FIG. 1 is a diagram of various shapes of biofilms on a substrate surface. [Figure 3D] FIG. 1 is a diagram of various shapes of biofilms on a substrate surface.

[0071] [Figure 4A] FIG. 1 is a cross-sectional view of an energy conversion cell in which a biofilm is formed on a substrate.

[0072] [Figure 4B] FIG. 4B is a diagram of the biofilm and porous surface of the substrate within the energy conversion cell of FIG. 4A.

[0073] [Figure 5A] FIG. 2 is a diagram of an exemplary energy conversion cell in a horizontal format.

[0074] [Figure 5B] FIG. 2 is a diagram of an exemplary three-layer conversion cell format.

[0075] [Figure 5C] FIG. 1 is a diagram of an exemplary “pack” design for energy conversion cells in a horizontal format.

[0076] [Figure 5D] FIG. 1 is a diagram of layers of microorganisms in a biofilm within an energy conversion cell.

[0077] [Figure 5E] FIG. 2 is a side view of an exemplary voltaic cell that may be used in accordance with certain disclosed embodiments.

[0078] [Figure 6] FIG. 1 is a process flow diagram illustrating operations that may be performed in methods according to certain disclosed embodiments.

[0079] [Figure 7] 1A-1D are diagrams of exemplary energy conversion cells having different carbon-containing anodes. [Figure 8] 1A-1D are diagrams of exemplary energy conversion cells having different carbon-containing anodes.

[0080] [Figure 9]FIG. 2 is a diagram of an exemplary stack of layers that may be implemented as a bioelectrochemical voltacell, according to certain disclosed embodiments.

[0081] [Figure 10] 1 is a graph showing measured currents in experiments performed in accordance with certain disclosed embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the presented embodiments. The disclosed embodiments may be practiced without some or all of these specific details. In other instances, well-known process operations have not been described in detail in order not to unnecessarily obscure the disclosed embodiments. While the disclosed embodiments will be described in conjunction with specific embodiments, it will be understood that they are not intended to limit the disclosed embodiments. I. Definition

[0083] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Various scientific dictionaries that include the terms contained herein are well known and available to those skilled in the art. Any methods and materials similar or equivalent to those described are useful in the practice of the disclosed embodiments.

[0084] The terms defined immediately below are more fully understood by reference to the present specification. The definitions are provided to describe only certain embodiments and to aid in understanding the complex concepts described herein. They are not intended to limit the full scope of the present disclosure. In particular, it will be understood that the present disclosure is not limited to the specific compositions, systems, designs, methods, protocols, and / or reagents described, as these may vary depending on the context in which they are used by those skilled in the art.

[0085] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content and context dictate otherwise. For example, reference to "a cell" includes a combination of two or more such cells. Unless otherwise indicated, the conjunction "or" is used in its proper sense as a Boolean logic operator and encompasses both selection of features in an alternative (A or B, where selection of A is mutually exclusive with B) and selection of features in combination (A or B, where both A and B are selected).

[0086] An "electron donor" is a component that donates electrons as part of a process involving the conversion of energy from radiation (e.g., light), chemical moieties, mechanical manipulation, or other processes. In this disclosure, examples of electron donors include photosynthetic and non-photosynthetic microorganisms, light-harvesting antennae, and pigments.

[0087] A "light-harvesting antenna" is a biochemical or chemical structure that can be excited by light energy. In some cases, light can excite the antenna into a state that allows the antenna to generate electrical or electrochemical energy. In some cases, photosynthetic microorganisms contain light-harvesting antennae.

[0088] A "dye" is any composition that is capable of being excited by light energy, usually through wavelength-selective absorption. A dye is one or a component of a light-harvesting antenna. Dyes can be produced synthetically or biologically.

[0089] "Non-photosynthetic microorganisms" are microbial cells that do not require light energy for growth and metabolic processes. Such microorganisms may contain electron transport components, which may be embedded in the cell membrane and / or membrane invaginations and / or membrane vesicles and / or organelles.

[0090] "Photosynthetic microorganisms" or "phototrophic microorganisms" are microbial cells that use light energy for growth and metabolic processes. Such microorganisms usually contain light-harvesting antennae capable of utilizing light energy and electron transport components, which may be embedded in the cell membrane and / or membrane invaginations and / or membrane vesicles and / or organelles.

[0091] "Chemotrophic microorganisms" are microbial cells that use the organic or inorganic oxidation of electron donors in their environment to generate energy for growth and metabolic processes.

[0092] "Heterotrophic microorganisms" are microbial cells that use organic compounds as electron donors in one or more metabolic pathways used for growth and metabolic processes.

[0093] "Liquorotrophic microorganisms" are microbial cells that use inorganic compounds as electron donors in one or more metabolic pathways used for growth and metabolic processes.

[0094] "Heterotrophic microorganisms" are microbial cells that use organic compounds as a carbon source.

[0095] An "autotrophic microorganism" is a microbial cell that uses carbon dioxide as a carbon source.

[0096] A "biofilm" is a microbial film comprising one or more microbial populations capable of adhering to a surface and facilitating the functioning of a bioelectrochemical energy conversion cell. A biofilm may contain non-microbial components (e.g., extracellular matrix). Extracellular components include, but are not limited to, polysaccharides, such as chitosan and carrageenan.

[0097] An "electronically conductive material" is a material that allows the movement of electrons from one location in the electronically conductive material to another. An electronically conductive material can be electronically conductive or semiconducting. It can conduct holes. II. Introduction

[0098] Bioelectrochemical energy conversion cells are a viable alternative for generating energy. Such cells can use a variety of organisms, including but not limited to photosynthetic organisms, phototrophs, chemotrophs, chemoorganotrophs, chemolithotrophs, photoheterotrophs, autotrophs, heterotrophs, and other organisms capable of generating energy that can be captured for use in a voltaic cell. Electron carriers continuously pass excited electrons through the electron transport chain, simultaneously facilitating the coordinated effort of proton separation across the membrane to generate electrical potential energy.

[0099] Photosynthetic microorganisms and plants are very efficient at converting light energy into other usable forms of energy. Photosynthetic microorganisms contain light-harvesting pigments and antenna systems or reaction centers in their membranes to harness the energy provided by photons.

[0100] There are two types of photosynthesis: nonoxygenic and oxygenic. Nonoxygenic photosynthesis was historically thought to precede oxygenic photosynthesis and does not produce oxygen. Oxygenic photosynthesis occurs in plants and cyanobacteria and uses HO as the electron donor for photosynthetic nutrition. Nonoxygenic photosynthesis can utilize hydrogen, sulfur, and certain compounds as electron donors for photosynthetic nutrition.

[0101] A demonstrated ability for maximum light utilization has been identified in green sulfur bacteria that reside in deep-sea thermal vents, nearly a mile below the ocean surface, where minimal light reaches these microorganisms. These microorganisms are able to utilize nearly 100% of the residual light in non-oxygenic photosynthesis.

[0102] The use of photosynthetic microorganisms to produce usable energy has focused primarily on biofuel production.

[0103] In addition to, or instead of, photosynthetic microorganisms, bioelectrochemical energy conversion cells may use chemotrophic organisms, including those that may be found in the deep regions of the ocean. Bioelectrochemical energy conversion cells may also use heterotrophic organisms that are efficient at converting carbon-containing nutrients into usable forms of energy.

[0104] Some bioelectrochemical energy conversion cells may include more than one of the above types of organisms. For example, some bioelectrochemical energy conversion cells may have organisms with energy conversion pathways whose products can be used as an energy source for the energy conversion pathway of another organism in the same bioelectrochemical energy conversion cell.

[0105] Disclosed herein are microbial-based electricity generating cells that utilize biofilms to enhance the performance (e.g., efficiency) of the cells. In some implementations, the incorporation of biofilms allows the cells to provide a low-cost energy production process and high light-to-electricity conversion rates compared to current bioelectrochemical energy generation technologies. Cells with biofilms can use the biofilms to control the energy conversion process within the cell and can be implemented in a variety of geographic locations where such biofilms may naturally occur. The cells can be customizable to accommodate requirements such as topography, climate, season, structural needs, etc. In certain embodiments, the cells have one or more biofilms disposed on one or more surfaces within the bioelectrochemical conversion cell.

[0106] In certain embodiments, the voltacell includes a container that stores a buffer system, a microbial cell population, one or more biofilms carrying at least a portion of the microbial cell population, and a current collector. In some embodiments, the voltacell includes a container that stores a buffer system, a microbial cell population, and a conductive biofilm. In some embodiments, the voltacell includes a container that stores a buffer system, a microbial cell population, one or more biofilms, and a current collector. In some embodiments, the voltacell includes a container that stores a light-harvesting antenna population, a buffer system, one or more biofilms, a mirror or other optical energy directing component, and a regulator system. In some cell designs, the biofilms can facilitate electron transfer, ionic and electronic conductivity, and other functions within the cell. In some aspects, the voltacell includes a container that stores a light-harvesting antenna population, a buffer system, one or more biofilms, an electronically conductive material, a mirror system, and a regulator system. In yet other aspects, the voltacell includes a container that stores a microbial population, a buffer system, one or more biofilms, a regulator system, and a charge storage device. The regulator can have sensing and control feedback functions. In some designs, the cells are capable of generating electricity without the presence of light, hi some implementations, the cells are deployed in solar panels. III. Voltac Cell Embodiments

[0107] 1A illustrates a schematic representation of an energy conversion cell 105 having a containment vessel 107 holding in its interior 109 a fluid in which one or more microbial populations reside. The cell 105 also includes an optional cover element 131 fitted over the vessel 107. The element 131 is transparent to radiation in the wavelength range to which the microbial populations respond. Optionally, the cell 105 includes an ion-permeable barrier or cell separator 111 disposed within the vessel 107 to prevent the passage of the microorganisms, electron donors, and / or other components in the interior region 109 to the compartment 113 on the opposite side of the permeable barrier 111. It should be understood that the permeable barrier 111 is optional and that in some cases only a single solution is provided within the vessel 107.

[0108] Returning to FIG. 1A , cell 105 includes, in compartment 109, and optionally in compartment 113, an anode 115 and a cathode 117 that are electronically separated from each other by an ionically conductive fluid. The fluid can be a liquid, a gel (including a hydrogel), or a matrix. In operation, the microbial population in compartment 109 can generate electrons that are collected at anode 115. These electrons flow through a load 119 in a circuit connecting cathode 117 and anode 115. In some implementations, the microorganisms in compartment 109 accept protons or other positively charged species from anode 115.

[0109] Returning to FIG. 1A, compartment 113, if used, may contain a distinct microbial population. In some implementations, the microorganisms in compartment 113 donate protons or other positively charged species to cathode 117. In some implementations, the microorganisms in compartment 113 accept electrons, protons, or other negatively charged species from cathode 117. The microorganisms in compartment 109 and optional compartment 113 convert energy by various mechanisms. In various embodiments, the microorganisms in at least compartment 109 are phototrophic.

[0110] As shown, the energy conversion cell may include one or more biofilms. FIG. 1A illustrates optional biofilms on various components of the cell 105. Optional biofilms, including biofilms 199a, 199b, 199c, and 199d, are adjacent to, and optionally attached to, or otherwise in contact with, the anode 115, the cathode 117, and the semi-permeable barrier 111, respectively. The biofilm 199a at the anode 115 may improve electronic conductivity and / or perform other functions when electrons are collected at the anode 115. For example, microorganisms within the biofilm 199a may donate electrons or other negatively charged species to the anode 115. Additionally or alternatively, such microorganisms may accept protons or other positively charged species from the anode 115. Similarly, the biofilm 199b at the cathode 117 may promote electronic conductivity and / or perform other functions when electrons are transferred from the cathode 117. Microorganisms present on biofilms 199b, 199c, and 199d may donate protons or other positively charged species to cathode 117. Additionally or alternatively, such microorganisms may accept electrons or other negatively charged species from cathode 117. Microorganisms within biofilms 199a, 199b, 199c, and 199d may also transduce energy by one or more mechanisms. In some embodiments, the microorganisms are phototrophic.

[0111] In certain embodiments, the fluid system 121 is coupled to the container 107 and optionally has separate ports for the compartments 109 and 113. The fluid system 121 may include various elements, such as a reservoir for holding make-up fluids for the compartments 109 and / or 113, one or more pumps, one or more pressure gauges, mass flow meters, baffles, and the like. The fluid system 121 may provide fresh buffer solution and / or microorganisms to the cells 105. It may also provide one or more of a variety of control agents to these fluids. Such control agents may include acids, bases, salts, nutrients, dyes, and the like. One or more salts may function as pH buffer agents for the solution. In some cases, the control agent includes a redox species that may participate chemically, electrochemically, and / or biochemically to control the solution. One example of a redox species is a sulfur-containing species, such as hydrogen sulfide (H2S), sulfur dioxide (SO2), or sulfate ion (SO4). 2- In some embodiments, the microorganisms may be more efficient when contained within a biofilm 199a, 199b, 199c, or 199d than when contained within a buffer solution.

[0112] The cell 105 may also be interfaced with a controller 125 that controls the fluid system 121. The controller 125 may have one or more other functions. For example, it may receive inputs from various components of the system, such as the anode 115, the cathode 117, the fluid system 121, and / or circuitry coupling sensors 127 and 129 located in the compartments 109 and 113, respectively. The sensors may monitor any one or more relevant operating parameters of the cell 105. Examples of such parameters include temperature, chemical properties (e.g., constituent concentrations and pH), optical properties (e.g., opacity), electrical properties (e.g., ionic conductivity), and the like.

[0113] FIG. 1B illustrates a variation of the cell 105. Specifically, the figure illustrates an alternative cell 135 having an anode plate 137, a cathode plate 139, and a compartment 141 defined by a spacer 143 between the plates 137 and 139. Within the compartment 141 is an ionically conductive medium. The anode plate 137 may include or be composed of a semi-permeable material that allows ion transmission between the two sides of the plate but does not allow the passage of microorganisms or microbial components. Provided on the anode plate 137 is a population 145 of phototrophic microorganisms that include photon-harvesting antennae. Optional biofilms 189a and 189b are adjacent to the anode plate 137 and the cathode plate 139, respectively. It will be understood that biofilms may also be present on other surfaces of the cell 135.

[0114] The photoconversion system may include an anode positioned immediately adjacent to the biofilm configured to transfer electrons and generate electrical current in a circuit including the anode and a cathode, the circuit may be coupled to a conversion module for an electrical grid or other system.

[0115] In one form, the disclosed microbial energy conversion cell includes a container that houses a buffer system, a light harvesting antenna population, and one or more biofilms. In some embodiments of the present disclosure, the cell may include a container that houses a light harvesting antenna population, a buffer, one or more biofilms, a mirror system, and a regulator system.

[0116] In some embodiments, the photoconversion system includes a light-harvesting antenna component population and one or more biofilms for improved efficiency of light conversion to electricity with reduced complexity and cost.

[0117] In certain embodiments, the light conversion system includes a buffered electrolyte solution surrounding a population of microbial light harvesting antennae, the population having multiple light harvesting antennae per component, where the component population has the ability to harvest light over a wide range of wavelengths including ultraviolet and far-red light, and can harvest light over a range of intensities, including diffuse light. The population can include one or more microbial species, including a mixture of photosynthetic and non-photosynthetic microorganisms, membrane components from the microorganisms, or vesicles that include light harvesting antenna components and electron carrier components.

[0118] In some embodiments, the light harvesting antenna population includes a light harvesting pigment or a photosystem that includes an electron carrier molecule and a reaction center, hi some implementations, the light harvesting antenna population can include a variety of different light harvesting pigments and photosystems with similar electron carrier molecules.

[0119] In some embodiments, the disclosed microbial energy conversion cells include a vessel housing a buffer system, a microbial population including one or more chemotrophic microorganisms, and one or more biofilms. In some aspects of the present disclosure, the cell may include a vessel housing one or more chemotrophic microorganisms, a buffer, one or more biofilms, a mirror system, and a regulator system.

[0120] In some embodiments, the energy conversion system comprises a chemotrophic microbial population and one or more biofilms for improved efficiency of energy source conversion into electricity with reduced complexity and cost.

[0121] In some embodiments, the energy conversion system comprises an endoplasmic microbial population and one or more biofilms for improved efficiency of carbon conversion to electricity with reduced complexity and cost.

[0122] In certain embodiments, the energy conversion system includes a buffered electrolyte solution surrounding a microbial population, the population having a plurality of microorganisms, where each microorganism has the ability to convert different energy sources in different conditions and using different metabolic pathways. The microbial population can include one or more microbial species including a mixture of photosynthetic, non-photosynthetic, chemotrophic, autotrophic, heterotrophic, and organotrophic microorganisms, membrane components from the microorganisms, or vesicles including electron carrier components.

[0123] Chemical redox reactions occurring at the electrodes convert chemical energy into electrical energy by donating (anode) or accepting (cathode) electrons to an external electrical circuit. Ions of the appropriate charge are consumed or donated (via redox reactions) at the appropriate electrodes to maintain local charge balance and overall electrical current (electrons in the external circuit and ions in the cell medium (electrolyte)).

[0124] Biological organisms (or components thereof) participate in the operation of the cell: they (i) may facilitate electronic and / or ionic conduction required by the cell, (ii) they may participate in energy-generating redox reactions at the electrodes, and / or (iii) they may harvest energy from an external source (e.g., sunlight for photosynthetic microorganisms or chemical energy for chemotrophic organisms) and provide the harvested energy in the form of chemical compounds that can participate in redox reactions at the electrodes.

[0125] In certain disclosed embodiments, the biofilm is capable of facilitating electron and / or ion conduction, participating in energy-generating redox reactions, and / or harvesting energy from external sources.

[0126] One exemplary photosystem may operate as shown in FIG. 1C. In some embodiments, the photosystem resides within a cell membrane of an organism. In some embodiments, the photosystem resides within a membrane derived from an organism, but is no longer part of that organism. In other embodiments, the photosystem is incorporated into a synthetic micellar structure. Such structures may be created by techniques well known in the art, for example, sonicating oils and lipids in a solvent with a detergent. The resulting micellar structure may be spiked with the necessary components of the photosystem. Such components typically include a reaction center, such as the molecule chlorophyll a, a light-harvesting pigment, and an electron-transporting molecule. Certain pigment molecules may function as both a light-harvesting pigment and an electron-transporting molecule. In certain embodiments, one or more elements of the photosystem are provided in a biofilm or similar component of a voltaic cell. For example, one or more dyes may be added to a hydrogel or branched polymer matrix. Examples of such matrices include alginate, agar, agarose, pectin, gelatin, and Sephadex.

[0127] When light hits the light-harvesting pigment in the microbial membrane, the excited electrons are passed unidirectionally to an electron carrier component in the membrane (the antenna accessory pigment in FIG. 1C) and to an electron-transport component that passes the electrons to a terminal electron acceptor. In some cases, the transport component is a biofilm. Electrons exit the microbial membrane and flow onto the biofilm, which may help facilitate electron flow. The electron flow can then be harnessed by a nearby anode, e.g., a metal plate or wire, to maximize the flow of current out of the microbial population. If the net flow of electrons on one part of the cell (at one electrode) is significantly different from another part of the cell (at a different electrode), a current can be generated.

[0128] Electrons can flow from the photosystem to the anode by various means. In some cases, the microorganisms are attached directly to the anode as a biofilm or other adhesive structure. In such cases, electrons generated by the photosystems are transferred directly from the photosystems to the anode. In other cases, the photosystems are not attached to the anode and the electrons flow into the solution, where they can be captured and transferred by media in the solution or by a biofilm on another part of the cell, for example, a biofilm adjacent to the cathode. In similar embodiments, the electrons are provided to a conductive network linking the anode to microorganisms or other photosystem-containing elements in the solution. In certain embodiments, the photosystems represent light-harvesting antennae.

[0129] While photosystems are frequently described as a source of electrons with respect to the disclosed embodiments, non-photosynthetic biochemical processes that produce electrons may be used in place of or in addition to photosystems. Thus, where appropriate, references to photosystems and similar terms may be deemed to include metabolic and other biochemical systems that produce available electrons for donation to an anode in an energy conversion cell. A.Sterilization

[0130] In some embodiments, the bioelectrochemical energy conversion cell is manufactured by a process that includes sterilizing one or more components before or after they are installed in the cell or partially manufactured cell.

[0131] The starting materials for a bioelectrochemical energy conversion cell as described herein should be sterile so that when microorganisms are introduced, only the microorganisms of interest are introduced into the conversion cell and not undesirable microorganisms. During operation of the bioelectrochemical energy conversion cell, the cell is completely sealed and / or closed to prevent harmful microorganisms or conditions outside the conversion cell from entering the cell environment and device.

[0132] As an example, all components of the bioelectrochemical energy conversion cell can be made and manufactured in a sterile fashion. For example, aluminum anodes can be sterilized by boiling water bath followed by ethanol or isopropyl alcohol spray and then drying, or metal anodes can be sterilized by baking in a high temperature oven for 1 hour. Gel media can be made using sterile deionized water, or non-sterile deionized water and then autoclaved. Gel cooling and molding can be made by covering and placing a sterile foil mold in a sterile container and placing it in a cooling chamber. Carbon cloth can be exposed to 70% ethanol or germicidal spray and then dried. Pre-sterilized components including containers and sealants can be packaged and stored in low throughput shelves and opened under the hood during assembly. Leads can be exposed to ethanol or germicidal spray and dried. IV. Gel and polymer electrolyte bioelectrochemical energy conversion cell design

[0133] In certain embodiments, the bioelectrochemical energy conversion cell comprises one or more metal or non-metal containing electrodes that are optionally coated with a biofilm and then an ion-conducting polymer.

[0134] In certain embodiments, the bioelectrochemical energy conversion cell has a gel or polymer electrolyte. In some cases, such cells optionally do not have a separate liquid electrolyte compartment or region. Ion conduction between the anode and the cathode occurs primarily or exclusively within the gel or polymer matrix. In other cases, such cells have a liquid electrolyte portion and a gel or solid electrolyte portion. As an example, such cells may have an electrode coated or partially coated with a biofilm and an ion-conducting polymer coated on the biofilm. The entire structure (electrode / biofilm / polymer) is in contact with a liquid electrolyte that is in ionic contact with the counterelectrode.

[0135] 1D and 1E show schematic diagrams of bioelectrochemical energy conversion cells with gel or polymer electrolytes. FIG. 1D shows a simplified schematic diagram of a voltaic cell 1019 with an anode 1017, an optional biofilm 1099a, a polymer electrolyte 1016, an optional biofilm 1099b, and a cathode 1015. In some embodiments, for example where the microorganisms in the biofilm 1099b are photosynthetic microorganisms, the cathode 1015 is transparent. FIG. 1E shows a simplified schematic diagram of a voltaic cell 1119 with an anode 1117, a biofilm 1199a, a polymer electrolyte 1116, a liquid electrolyte 1105, an optional biofilm 1199b, and a cathode 1115.

[0136] The electrolyte allows the conduction of ions between the anode and the cathode while blocking the conduction of electrons. When the electrolyte is (or includes) a solid or gel, the electrolyte may be referred to as a separator. In addition to conducting ions, the separator may function to provide a hospitable environment for one or more microorganisms involved in the electrochemical process that produces electrical energy. The gel or polymer separator may include pores of suitable dimensions, e.g., micrometer-scale, to accommodate the microorganisms. The pores may carry and / or allow the movement (entrance and exit) of the microorganisms.

[0137] In certain embodiments, the electrolyte or separator includes multiple components, at least one of which is an ion-conducting substrate in a solid or gel state. Such substrates may have any of a variety of compositions. In some embodiments, NaCl dissolved in water is used to provide ion conductivity (i.e., is an electrolyte). This may provide a suitable environment for various microorganisms. In some embodiments, salt dissolved in water provides ion conductivity. In some embodiments, the water-based electrolyte is absorbed by a gel or polymer. The gel or polymer may be ion-conducting in some embodiments. The gel or polymer may not be ion-conducting in some embodiments. In some embodiments, the electrolyte may be used to conduct different types of ions, e.g., H+ and OH-. The electrolyte selected for a particular disclosed embodiment may depend on the chemistry of the particular fuel cell or solar cell embodiment based on the ions to be conducted. For example, if a hydrogen fuel cell embodiment is implemented, an H+ (proton) conducting electrolyte may be used.

[0138] Examples of suitable gel matrix materials include polysaccharide materials such as alginates (e.g., sodium alginate), agarose, agar, acrylamide, polyacrylamide, glycerin, glycerol, hydrogels, gelatin, pectin, PEG, celluloses, nucleic acid chains, polyproteins, synthetic polymers, cellulose, other Winogradski mineral media, and mixtures thereof. The gel matrix material may provide a viable scaffold for bacterial growth. In some embodiments, the biofilm matrix may also have a source of calcium or may be in a calcium-rich buffer solution. A. Manufacturing method

[0139] In some embodiments, the electrodes may be surrounded or coated with a polymer gel. In such embodiments, the electrodes may be laid down into a mold to which the molten gel is applied and allowed to harden. This allows for direct contact between the gel and the electrode surface.

[0140] In some embodiments, the electrodes may be inserted into a hardened polymer gel. In such embodiments, a molten gel is applied to a mold and allowed to harden. The electrodes may then be inserted into the hardened gel once the gel is removed from the mold. This allows for direct contact between the gel and the electrode surface once the gel is inactive.

[0141] In some embodiments, the cell can be manufactured by providing a polymer film and applying electrodes to both sides of the polymer film. The application of the electrodes can be performed by any deposition technique, including but not limited to printing, coating, and spraying.

[0142] In some embodiments, the cell includes a heat source, such as a geothermal source, for generating the microorganisms that can be incorporated into the cell. As with any of the cell embodiments described herein, carbon dioxide consumption from the atmosphere or from carbon dioxide generating sources, such as vehicles or combustion power plants, can be utilized in conjunction with certain disclosed embodiments. V. Electrode Formats Using Ion-Conducting Polymers

[0143] The ion-conducting polymer is included along with other electrode components. Collectively, the polymer and the other components form an electrode. The ion-conducting polymer may be used in the anode, the cathode, or both. The ion-conducting polymer may be a cation conductor, an anion conductor, or a mixed anion and cation conductor. A. Anode Component

[0144] The anode comprises (a) an electrochemically active material that can be oxidized and release electrons during discharge, (b) an optional electronically conductive material, and (c) an optional ionically conductive material. The anode may be in electrical contact with a current collector, typically having a negative polarity. 1. Ion-conducting materials

[0145] Depending on the reaction occurring at the anode, the ion-conducting polymer may conduct anions, cations, or both. In certain embodiments using a metal-containing anode, the ionomer conducts hydroxide ions. For example, an aluminum metal electrode may use an ionomer that conducts hydroxide ions.

[0146] In certain embodiments, the ion-conducting polymer is a cation-conducting polymer. A cation-conducting polymer preferentially conducts cations (e.g., protons) over anions. In some embodiments, the cation-conducting polymer may conduct cations from the anode to the electrolyte. The cations depend on the type of electrode being used. Examples of conducted cations include hydrogen ions, aluminum ions, and zinc ions. Examples of conducted anions include hydroxide ions, bicarbonate ions, and bisulfate ions. Chemically, the ion-conducting polymer substrate may include an organic polymer backbone having pendant ionic groups, such as sulfonic acid groups, disulfide bonds; aromatic ring structures; methyl groups; phosphate groups; hydroxyl groups; carbonyl groups; aldehyde groups; nitroxyl groups; nitrosonium groups; or quaternary ammonium groups. The ion-conducting polymer may have a backbone that includes aromatic rings, double bonds, or aromatic rings and double bonds. Ion-conducting polymers with aromatic rings may have heteroatoms or no heteroatoms present. Ion-conducting polymers whose main chains contain aromatic rings but no heteroatoms include poly(fluorene), polyphenylene, polypyrene, polyazulene, and polynaphthalene. Ion-conducting polymers whose main chains contain aromatic rings and have nitrogen-containing heteroatoms in the aromatic rings include poly(pyrrole) (PPY), polycarbazole, polyindole, and polyazepine. Ion-conducting polymers whose main chains contain aromatic rings and have nitrogen-containing heteroatoms outside the aromatic rings include polyaniline (PANI). Ion-conducting polymers whose main chains contain aromatic rings and have sulfur-containing heteroatoms in the aromatic rings include poly(thiophene) (PT) and poly(3,4-ethylenedioxythiophene) (PEDOT). Ion-conducting polymers whose main chains contain aromatic rings and have sulfur-containing heteroatoms outside the aromatic rings include poly(p-phenylene sulfide) (PPS). Ion-conducting polymers whose main chains contain double bonds include poly(acetylene) (PAC). Ion-conducting polymers whose backbones contain both aromatic rings and double bonds include poly(p-phenylene vinylene) (PPV). The ion-conducting polymer may be a linear polysaccharide. The ion-conducting polymer may be an anionic copolymer polyelectrolyte.In some embodiments, anionic copolymer polyelectrolytes may aid in ion transport and support of biofilm structure. Ion-conducting polymers have a specific conductivity for anions and / or cations of at least 1 mS / cm. Anion-conducting polymers are ion-conducting polymers that conduct primarily anions (although there may still be some small amount of cation conduction). Cation-conducting polymers are ion-conducting polymers that conduct primarily cations (e.g., there may still be incidental amounts of anion conduction). In various embodiments, the ion-conducting polymer is an organic polymer with pendant ionic groups, such as sulfonic acid groups or quaternary ammonium groups.

[0147] In addition to the polymer or gel matrix material as described above, the electrolyte or separator may include water to hydrate and / or swell the matrix, one or more ionic species, one or more biocompatible agents, and combinations thereof. In one example, extracellular polysaccharides may be used.

[0148] In certain embodiments, the thickness of the polymer or gel electrolyte is between about 0.025 mm and 10 cm. The thickness of the electrolyte may depend on the microorganism used in the cell. In some embodiments, the thickness may be between about 25 μm and about 250 μm.

[0149] However, some microorganisms may use layers thicker than this range.

[0150] The polymer or gel electrolyte or electrode may have remarkable optical properties. In certain embodiments, the electrolyte is transparent or partially transparent to wavelengths in some or all of the UV, IR, and / or visible regions of the electromagnetic spectrum. This may be a beneficial property in systems that use photosynthetic microorganisms.

[0151] In various embodiments, the microorganisms are carried in and / or migrate through the anode and / or cathode of the bioelectrochemical energy conversion cell. As described elsewhere herein, certain microorganisms can facilitate the operation of the bioelectrochemical energy conversion cell. In some cases, the ion-conducting polymer includes pores or other openings that can accommodate the microorganisms within the electrodes. In some embodiments, the ion-conducting polymer includes pores having an average cross-sectional diameter or other cross-sectional dimension of approximately 0.1 to 10 μm. 2. Electronic Conduction Materials

[0152] Optionally, the anode includes an electronically conductive material as one component. Such a material may be mixed with other components, including electrochemically active materials and ionically conductive components. The electronically conductive material described herein is distinct from the electronically conductive current collector.

[0153] Examples of suitable electron-conducting materials include carbon, metals, organic electron-conducting materials, and conductive oxides, nitrides, and the like. When carbon is used, it can be in various forms, such as carbon black, graphite, graphene structures, fullerene structures, such as nanotubes, and the like. When a metal or metal-containing structure is used, it should be inert under the chemical and electrical conditions experienced at the anode. In certain embodiments, the electron-conducting material is a conductive oxide. In some cases, the conductive oxide is a transparent conductive oxide, such as indium tin oxide or fluorinated tin oxide.

[0154] In some embodiments, the electronically conductive material may include an insulating backing material, such as polyethylene terephthalate (PET) with PVC lead wires and a conductive plastic film having 25% carbon and 75% polyethylene. The material may further include a conductive hydrogel having 20% ​​high polymer material, 59% glycerin, 20% water, and 1% salt.

[0155] In some embodiments, the particles or other units of electronically conductive material have a shape or form that provides a path for electrons to move between locations within an electrode, or between the electrode and other bioelectrochemical energy conversion cell components, such as the biofilm, electrolyte, and / or current collector. In a separate section, the disclosure further describes the properties of such electronically conductive materials. Examples of such properties include the shape and dimensions of the electronically conductive particles. Furthermore, the location of the electronically conductive material may be selected based on the components between which it conducts electrons. 3. Biochemical and Electrically Active Materials

[0156] Many different types of electrochemically active materials can be used in the anode. Some of these are described elsewhere herein. In certain embodiments, the electrochemically active material is a metal, a metal oxide, and / or a metal chalcogenide. Examples of anode metals include aluminum, zinc, silver, silver / silver chloride, carbon, histidine-modified carbon, arginine or polyarginine-modified carbon, histidine or polyhistidine, and / or carbon modified with zwitterionic moieties.

[0157] In some embodiments, a fuel cell reductant, such as hydrogen or methanol, can be continuously fed to the anode where it is oxidized and donates electrons. Such implementations can utilize microorganisms that produce methanol or hydrogen. Methanol-oxidizing microorganisms are microorganisms that use methanol as a carbon source for energy. Some methanol-oxidizing microorganisms can be from the bacteria and eukaryote domains. Examples can include Pichia pastoris, Saccharomyces cerevisiae, Candida spp., Trichosporon spp. Further examples are described in Steffen Kolb, Aerobic methanol-oxidizing Bacterial in soil, 300 FEMS Microbiology Letters 1, Nov. 2009, pp. 1-10, available at https: / / academic.oup.com / femsle / article / 300 / 1 / 1 / 528736. Certain microorganisms can be utilized in a fuel cell reductant bilayer design using materials including Nafion with functionalized carbon-doped agar / sodium alginate biofilm.

[0158] Methanol fuel cells can be categorized in two general ways: indirect and direct methanol fuel cells. FIG. 1F shows a process flow that can be used to form a methanol fuel cell according to certain disclosed embodiments. In operation 120, methanol is produced via a microbial reaction. In operation 130, methanol is recovered and purified. In operation 140, water and purified methanol are combined in a solution, and the solution is fed to the fuel cell anode. Diagram 150 shows a direct or indirect methanol fuel cell with an anode, electrolyte, and cathode, where the electrolyte is sandwiched between the anode and the cathode. Indirect methanol fuel cells are also referred to as reformed methanol fuel cells (RMFCs), which have a methanol reformer upstream of the anode that reforms methanol into hydrogen and carbon dioxide. The H2 / CO2 mixture is then fed to the anode along with low concentrations of reformer contaminants such as carbon monoxide. H2 is the fuel at the anode in this case. On the other hand, in a direct methanol fuel cell (DMFC), a methanol / water solution is fed directly to the anode. The methanol / water solution then reacts at the fuel cell anode.

[0159] In some embodiments, metal-free polymer-based electrodes may be used. The polymer-based electrodes may be formed from polypeptides. The polypeptide-based electrodes may have a non-degradable aliphatic backbone with redox-active pendant groups. In some embodiments, the polypeptides are enzymes. In other embodiments, the polypeptide-based electrodes are protected from proteolytic and hydrolytic activity. In yet other embodiments, the polypeptide-based electrodes undergo hydrolysis to form amino acids, which serve both as an energy source to the device and also as a secondary nutrient source to the microbial population. In one example, the polypeptide backbone may be the cathodic conductive material, while the viologens and nitroxide radicals and other redox-active groups may be the anode conductive material. The polypeptide-based electrodes may have the added advantage of being easily degraded when necessary for a zero waste process. Additionally, the degraded amino acids and other components may be reused and / or resynthesized to form new electrodes. Polypeptide organic radical battery materials are further described in Nguyen et al., "Polypeptide organic radical batteries," Nature, Vol. 583, p. 61, May 6, 2021.

[0160] In another embodiment, metal-free polymer-based electrodes, which are nucleic acids in nature, can be formed on a receiving surface or as a layer on their own. The chirality of DNA organizes the negative charges of the DNA backbone and the hydroxyl moieties on the nucleic acid bases to provide for electron transfer. The orientation that creates the polarity of DNA can be advantageous in certain embodiments. DNA can also degrade over time and serve as a nutrient source for microbial populations. 2. Overall composition of the anode

[0161] As shown, the anode comprises an electrochemically active material, an optional electronically conductive material, and an optional ionically conductive material, such as an ionically conductive polymer. It may contain other components that do not contribute to the electronic or electrochemical properties of the anode. Examples of such other components include binders and wetting materials. The ranges or relative amounts of these components may of course vary depending on the cell design, the microorganisms used, and other factors.

[0162] In certain embodiments, the anode comprises an ionically conductive material at a concentration of about 5% to about 70% by weight, or about 15% to about 50% by weight, or about 25% to about 40% by weight. In certain embodiments, the anode comprises an electronically conductive material at a concentration of about 30% to about 95% by weight, or about 50% to about 85% by weight, or about 60% to about 75% by weight. In certain embodiments, the anode comprises an electrochemically active material at a concentration of less than about 40% by weight, or less than about 25% by weight, or less than about 10% by weight. In some embodiments, the anode comprises an ionically conductive material having a concentration of about 0.1% by weight / volume (w / v) to about 55% by weight / volume (w / v). Such embodiments may include microorganisms inside or outside the electrochemical cell. For example, a direct methanol fuel cell (DMFC) with methanol-producing bacteria has microorganisms outside the fuel cell. In some embodiments, the anode, separator, cathode, and surrounding materials may also provide a friendly environment for microorganisms that directly participate in the electrochemical cell reaction.

[0163] In certain embodiments, the anode comprises a microorganism-compatible material at a concentration of about 20% to about 80% by weight, or about 30% to about 70% by weight, or about 35% to about 60% by weight. In certain embodiments, the anode comprises electronically conductive particles at a concentration of about 20% to about 80% by weight, or about 30% to about 70% by weight. In certain embodiments, the anode comprises less than about 40% by weight of electrochemically active material, or less than about 25% by weight of electrochemically active material, or less than about 10% by weight of electrochemically active material. In some embodiments, the anode may be immersed in water and dissolved ions, such as NaCl, to provide some ionic conductivity. Such embodiments may include microorganisms inside the electrochemical cell. However, this does not exclude the possibility of also including microorganisms outside the electrochemical cell that provide additional functions (e.g., microorganisms outside the electrochemical cell may provide raw materials to the electrochemical cell and the microorganisms inside it). In some embodiments, anodes, such as voltaic cells including those described herein, may include additional layers, such as, but not limited to, a conductive carbon layer, a metal layer, a transparent conductive layer, and / or a gas diffusion layer (GDL).

[0164] For some implementations, only a relatively small amount of electrochemically active material is used compared to the electronically conductive material, for example, an anode may have a relatively low percentage of aluminum powder compared to the percentage of carbon, because only a relatively small amount of electrochemically active material is needed to set the potential of the electrode.

[0165] To allow the microorganisms to make a significant contribution to the electrochemical energy conversion process, rather than letting the material, e.g., aluminum, determine the energy conversion and electrical properties of the cell, only small amounts of inorganic electrochemically active material may be used. In certain embodiments, the weight ratio of electrochemically conductive material to electronically conductive material is about 1:1 to 1:5 or less.

[0166] The anode can have any of a variety of forms. For example, the anode can be a mixture of particles, a free-standing sheet, or a layer or coating on a substrate. In some cases, the sheet is a flexible structure, such as a film typically used in a membrane electrode assembly. In some cases, the layer or coating is applied as an ink. In some cases, the substrate is a current collector.

[0167] In some cases, the microorganism or microorganisms facilitate the operation of the anode. As described elsewhere herein, a biofilm may be provided on or within the anode.

[0168] In certain embodiments, the thickness of the anode is at least approximately 25 μm thick. The maximum thickness of the anode may depend on the microorganism used.

[0169] The anode may have noteworthy optical properties. In certain embodiments, the anode is transparent or partially transparent to wavelengths in some or all of the UV, IR, and / or visible regions of the electromagnetic spectrum. This may be a beneficial property in systems that use photosynthetic microorganisms. In some cases, the transparent electrode uses a transparent conductive oxide, such as indium tin oxide or fluorinated tin oxide.

[0170] In some embodiments, the anode is not transparent, hi some embodiments, the anode is opaque. B. Cathode Component

[0171] The cathode comprises (a) an electrochemically active material capable of being reduced during discharge and accepting electrons from a circuit, (b) an optional electronically conductive material, and (c) an optional ionically conductive material.

[0172] In certain embodiments, the cathode may be similar to a conventional air electrode, for example in a fuel cell. It may include, for example, carbon particles or fibers, and a gas diffusion layer having a hydrophobic material, for example, a fluorinated polymer (e.g., PTFE). It may also have a flow path for air to reach the electrode, which may have any of a variety of paths. In one example, the flow path includes multiple parallel paths. In some cases, the flow path follows a tortuous path. In some implementations, the cathode may be an air cathode and is opaque or otherwise non-transparent to a large portion of the solar spectrum. For this reason, bioelectrochemical energy conversion systems using phototrophs may be designed such that the cathode is not located between the area where the phototrophs are located and the direction in which solar radiation contacts the cell. In some embodiments, the cathode includes carbon particles and Nafion ionomer as an ink or paste. It may be manufactured by using a paste including carbon, Nafion suspension or solution, and platinum catalyst supported by a solvent, and removing the platinum to form a carbon-containing paste. The paste may be mixed in isopropyl alcohol. In some embodiments, Nafion may be replaced with a polymer or gel to alter the pH, provide a biocompatible environment, and reduce the cost of manufacturing. In some embodiments, microorganisms may be incorporated within the carbon layer. In some embodiments, the carbon layer may be composed of alternative conductive carbon materials, such as graphite, graphene, or carbon nanoparticles. In some embodiments, conductors may replace carbon in the cells, for example, by using metal particles (e.g., silver) and / or conductive metal oxides.

[0173] The air cathode requires access to air. In some embodiments, this is accomplished by a bioelectrochemical energy conversion cell design in which at least one surface or face of the cathode is exposed to air. In some horizontal configuration cell designs, a portion of the air electrode extends beyond the electrolyte or buffer and contacts the air. In some implementations, air is provided, for example, via a pump that pushes air to the cathode.

[0174] Water management may be a consideration in certain cell designs, especially those using air cathodes. Some electrode reactions consume water and some produce water. Additionally, water may evaporate from the cell. If there is a net water loss in the cell, some mechanism may be provided to provide refill water to the cell. In some embodiments, a pump is used for this purpose. C. Method of Fabricating Electrodes

[0175] In one embodiment, the anode is prepared as a carbon ink containing a liquid carrier, such as ethanol, carbon particles, a defined amount of aluminum powder or other electrochemically active material, and an ionically conductive polymer, and applied to the substrate. After the ink is applied, the liquid is evaporated, forming the anode on the substrate. By way of example, the substrate may include a current collector, a glass sheet, or a clear plastic sheet.

[0176] In another embodiment, liquid polypeptide or DNA is applied to the template and some or most of the water is removed by heating, freeze-drying, or evaporation. The resulting electrode can be a gel or a powder.

[0177] In another embodiment, the electrodes may be formed onto a backbone / surface having specific structural characteristics and may be sprayed or deposited onto the backbone structure / surface and cured.

[0178] Sonication or similar techniques may be used in some embodiments to aid in the dispersion of the carbon ink and aluminum powder.

[0179] In the manufacture of the aluminum anode, a separate current collector may not be used: the leads may be embedded directly into the gel during manufacture. VI. Electronic Conduction Pathways in the Electrodes

[0180] In some embodiments, bioelectrochemical energy conversion cells include structures within the electrolyte or electrodes that provide electronically conductive pathways. These pathways can be provided as structures that facilitate the transfer of electrons donated by the microbial species. The structures transfer the donated electrons to the electrode or a component associated with the electrode, such as a current collector. In some cases, the structures transfer the electrons to an electrochemically active redox material within the electrode.

[0181] The structures that provide the electron pathway can be naturally occurring or synthetic and / or provided to aid in the electron pathway. In some embodiments, the structures are purposefully formed to have specific properties specific to the selection of microbial populations and their corresponding growth environments. In some embodiments, the structures are mixed within the electrode itself, for example, within the anode that accepts electrons for the electrical circuit to which the bioelectrochemical energy conversion cell is coupled. Alternatively, these materials can be located at the interface of the electrode, for example, at the interface between the anode and the electrolyte.

[0182] The electron pathway may be composed of any number of electron conducting materials. Examples include carbon-containing materials, metals, such as copper, and transparent conducting materials, such as fluorinated tin oxide and indium tin oxide. As one example, the carbon-containing material may be a carbon fullerene structure, such as a nanotube. In some embodiments, the structure is an electron siphon. However, in preferred embodiments, the length may vary based on the design, ranging from 5 nm to 5 cm per unit, and may vary in shape such as coils, cylinders, points, wires, rods, piluses, and meshes, and may be composed of carbon, metals, and biopolymers.

[0183] In some embodiments, the electron pathways are provided as a powder or granular material. In some embodiments, they are provided on a continuous substrate, such as a sheet. The sheet may, for example, comprise a transparent conductive oxide. In some cases, the sheet-like structures are etched to thereby provide a larger surface area. The etching may create fingers or lines that aid in the movement or direction of the electrons.

[0184] The structures that provide the electron pathways are typically shaped to transfer electrons from one region to another. To this end, they can be wire-like or wire-shaped. In certain embodiments, they have, on average, an aspect ratio greater than 1, or greater than 3. The average particle length (in the direction of electron transfer) can be at least about 5 nanometers, or between about 5 nanometers and 500 millimeters.

[0185] Such structures may be in direct or indirect contact with the conductive components of the bioelectrochemical conversion cell, such as the electron conductive material, the anode conductive material, the cathode conductive material, and the ion conductive material. The structures are selected and formed to help facilitate the transfer of electrons from one or more of the microorganisms to the electrode without contact with the counter electrode, which would create a short circuit. The electron conductive pathway may be used in conjunction with an ion conductive polymer that helps to keep one or more microorganisms in a location close to the electrode where they can donate electrons, and avoid the transfer of electrons to the counter electrode. A. Methods for Integrating Electronic Conduction Pathways into Bioelectrochemical Energy Conversion Cells

[0186] An embodiment comprising mixing the electronic conduction pathway with one or more of an electrode, a biofilm, an electrolyte, or other components of a bioelectrochemical energy conversion cell.

[0187] In certain embodiments, an anode is prepared and applied to a substrate having an etched transparent conductive material, such as a transparent conductive oxide (e.g., indium tin oxide). Etching can be performed using chemical etchants, lasers, or other agents to partially remove material. VII. Microbial Confinement Enclosures (Cages)

[0188] In certain embodiments, bioelectrochemical energy conversion cells are manufactured using one or more microbial containment enclosures (sometimes referred to as "cages") to maintain the microorganisms in place during cell manufacture and operation.

[0189] FIG. 1G shows a microorganism containment enclosure 1800A in a perspective view. As shown, the structure completely encloses the area occupied by the microorganism 1810. Such an enclosure can be used during the manufacture of a bioelectrochemical cell. For example, one or more types of microorganisms in a liquid or gel medium can be provided in the enclosure, which is then positioned in a vessel of a bioelectrochemical energy conversion cell and then converted into a biofilm or a portion of a biofilm, optionally by adding a matrix-forming material. FIG. 1G shows a second microorganism containment enclosure 1800B with a second microorganism 1820A in a perspective view.

[0190] 1H shows microbial containment enclosure 1800A and microbial containment enclosure 1800B in situ within a multilayer biofilm 1801 of a bioelectrochemical energy conversion cell 1800 with electrode 1820B. Microbial containment enclosure 1800A may have walls constructed from a polypropylene material or borosilicate reinforced glass.

[0191] In certain embodiments, the enclosure has pores in its walls that allow water or other fluids to pass between the inside and outside of the enclosure. However, the pores are usually too small to allow microorganisms to pass from the inside to the outside of the structure. Such pores may be less than approximately 0.22 μm in diameter. The pores may also be sealed with filters that allow flexibility in pore size to accommodate water flow while preventing loss of microorganisms from the cells.

[0192] In some cases, a process for forming a biofilm begins with a microorganism containment enclosure having an open side. FIG. 1I shows a process flow diagram illustrating operations that may be performed according to such a process 2001. In operation 2003, a microorganism containment enclosure having an open side is provided, such as the microorganism containment enclosure illustrated in FIG. 1G. In operation 2005, a liquid or gel medium comprising microorganisms to be incorporated into a biofilm is poured into the open side of the microorganism containment enclosure. The enclosure encloses some or all of the microbial medium. Optionally, the microorganism containment enclosure is closed to keep the microorganisms inside during manufacture. In operation 2007, the restrained microorganisms in the enclosure are optionally mixed with and incorporated into a material that forms a substrate or matrix for the biofilm. For example, the microorganism containment enclosure may be mixed with or positioned into an alginate and / or agarose-containing matrix. In operation 2009, the resulting biofilm is then applied to a voltaic or bioelectrochemical energy conversion cell component. In operation 2011, components within the biofilm matrix may then be incorporated into a bioelectrochemical energy conversion cell. VIII. The Role of Biofilms

[0193] The disclosed voltacells include a biofilm at one or more locations, each of which may have one or more functions that facilitate energy production within the voltacell. The biofilm may be used as a stabilizing factor for the microbial community. The biofilm may also increase the efficiency of electron transfer by doing so on the solid surface. The biofilm in certain disclosed embodiments performs many different functions.

[0194] In some embodiments, the microorganisms may facilitate electron and / or ion conduction required by the cell, participate in energy generating redox reactions at the electrodes, and / or harvest energy from an external source. Any one or more of these roles may be performed better when the microorganisms are present in the biofilm compared to when the microorganisms are not present in the biofilm. For example, microorganisms suspended in an electrolyte or buffer medium may have reduced energy production, reduced electron and / or ion conduction, or slower redox reactions compared to the same microorganisms performing the same function when disposed in a biofilm.

[0195] Biofilms can provide a medium for a nutrient-supported symbiotic relationship when two or more microorganisms are present on the biofilm. For example, two microorganisms with complementary pathways on the same biofilm can collectively produce more energy together because waste products produced by the metabolic pathway of the first microorganism are more efficiently consumed by the second microorganism. Examples can include sulfur oxidizing bacteria paired with sulfur reducing bacteria; nitrogen oxidizing bacteria paired with nitrogen fixing bacteria; photosynthetic bacteria (carbon fixing bacteria) paired with organoheterotrophs. Additional examples of microorganisms with complementary pathways are discussed in U.S. Patent No. 10,090,113, issued October 2, 2018, which is incorporated by reference in its entirety for the purposes of providing examples of complementary pathway microorganisms. As another example, two organisms in a biofilm can interact mutually beneficially and become healthier, due to any of a variety of types of symbiosis, and the like.

[0196] Exemplary microorganisms that may be used within the biofilm include, but are not limited to, Bacillus spp., Rhodopseudomonas spp. (e.g., Rhodopseudomonas palustris), Geobacter spp. (e.g., Geobacter sulfreducens), Acidithiobacillus spp., Shewanella spp. (e.g., Shewanella oneidensis), Desulfobacteriales, Desulfovibrioles, Syntrophobacteriales, Desulfotomaculum spp., Desulfosporomusa spp., Desulfosporosinus spp., Thermodesulfovibrio spp., Thermodesulfobacteria spp., Thermodesulfobium ... spp.), Archaeoglobus, Thermocladium, Caldivirga, Proteus, Pseudomonas, Salmonella, Sulfurospirillum, Desulfomicrobium spp., Pyrobaculum, Chrysiogenes, Neisseria, Escherichia, Eikenella spp.), Corynebacterium, Rhodospirillum, Rhodobacter, Aquaspirillum, Pirellula, Nostoc, Helicobacter, Enterobacter, Photobacterium, Brucella, Borrelia, Azoarcus, Dinoflagellates, Zoanthella, Azotobacter, Parabasalia, Aeromonas, Thermococcus, Methanopyrus, Thermoplasma, Pyrococcus, Methanococcus, Desulfurococcus spp., Methanoculleus spp., Archaeoglobus, Thiobacillus, Synechococcus, Spirillum, Sphaerotilus, Ruminobacter spp.), Roseobacter spp., Streptomyces spp., Spirulina spp., Vorticella spp., Xanthophyceae spp., Propionibacterium spp., Leptothrix spp., Frankia spp., Pleurocapsa spp., Chloroflexus spp., Beggiatoa spp., Anabaena spp., Ustilago spp., Magnetospirillum spp., Moorella thermoacetica spp., Desulfobacter spp., Desulfococcus spp., Desulfovibrio spp., Erythrobacter spp., Thermotoga spp., Rhodoferax spp., Pelobacter spp., Carboxydothermus spp., Lawsonia spp., Thermodesulfobacteria spp., Desulfromonas spp., Methanofollis spp. spp.), Methanosarcina, Methanosphaera, Methanothermobaccaria, Crenarchaeota / Thaumarchaeota from group I (e.g., Duboscquellida) and group II (e.g., Syndiniales) alveolates, Euryarchaeota, Radiolaria-dominated plankton, and Opisthokonta and alveolates, Alteromonas macleodii "surface ecotype", Pelagibacter ubique, Nitrosopumilus maritimus, Chlorobium spp., Chloroherpeton spp., GSB1, Prochlorococcus spp., Porphyrobacter spp., Idyukogome spp., Phaeodactylum spp., Chromatium spp., Roseiflexus spp. spp.), and Porphyrobacter spp., among others, and other microorganisms that have type IV pili or electron-accepting outer membrane components (Reguera et al., 2006; Leang et al., 2010; Richter et al., 2012, which are incorporated herein by reference in their entireties).

[0197] Specific examples of microorganisms that can be used in the biofilm include, but are not limited to, Rhodoferax ferrireducens, Lactobacillus acidophilus, Rhodospirillum rubrum, Desulfovibrio desulfuricans subsp. desulfuricans, Peptostreptococcus anaerobius, Rhodospirillum centenum, Catonella morbii, Lachnospiraceae spp., Photobacterium leiognatii, Allochromatium vinosum, Lactobacillus casei, Fusobacterium nucleatum subsp. polymorphum, Helcococcus kunzii, Cutibacterium acnes, Rhodospirillum rubrum, Helcococcus kunzii, Allochromatium vinosum, and Ferrovum mixofaciens. Table 1 lists these examples and the properties of these microorganisms that, when formed into a biofilm, may facilitate bioelectrochemical voltaic cell function. [Table 1]

[0198] In another example, two microorganisms that utilize different regions of the solar spectrum can together produce more energy by converting energy without having to compete for resources.

[0199] In another example, two microorganisms that use different types of energy may benefit from the presence of a biofilm: for example, one microorganism may be a chemotroph that obtains energy by oxidizing electron donors from their environment, while another may be a phototroph that uses solar energy.

[0200] In another example, two microorganisms that use different sources as electron or hydrogen donors can be used when a biofilm is present, for example, one microorganism can be an organotroph while another can be an autotroph.

[0201] In another example, two microorganisms that use different organic compounds for energy can be used when a biofilm is present, for example, one microorganism can be a heterotroph while another can be an autotroph.

[0202] In another example, two microorganisms that obtain energy using metabolic pathways involving different catalysts can be utilized, for example, an anaerobic microorganism can be combined with an aerobic organism.

[0203] In another example, the electronic conducting structure is designed to ensure that electrons produced or consumed by the microorganisms contact the electrode and not the counter electrode, which could result in a short circuit of the cell. The electronic conducting pathway can be used to conduct the electrons using an ion-conducting polymer electrolyte that helps the microorganisms keep them in a location close to the electrode where they can donate electrons, avoiding the transfer of electrons to the counter electrode.

[0204] The two or more microorganisms that may be used include two or more of the following types of microorganisms: phototrophs, photoorganotrophs, photolithotrophs, photoorganoheterotrophs, photoorganoautotrophs, photolithoheterotrophs, photolithoautotrophs, chemotrophs, chemoorganotrophs, chemolithotrophs, chemoorganoautotrophs, chemolithotrophs, chemoorganoautotrophs, chemolithoheterotrophs, chemolithoautotrophs, and mixotrophs.

[0205] Specific combinations of microorganisms that can be utilized with biofilms can be selected based on complementarity, ability to grow under the same or similar conditions, e.g., temperature, pH, salinity, predominant gas species, hydrodynamic flow, resistance to chemical species, amount of exposure, type of exposure, biofilm environment (e.g., having a common extracellular matrix), or combinations thereof.

[0206] Additional examples of biofilms with a syntrophic relationship are when two or more microorganisms are present on the biofilm.

[0207] In some embodiments, the biofilm may include features that may maintain a suitable environment for a selected microorganism. For example, gels with sustained release nutrients (carbohydrate disks, nano- and micro-pelletized amino acids, CO2 / O2 gas cartridges), buffering agents (citric acid, acetic acid, potassium phosphate, CHES, borates), acids (hydrochloric acid, perchloric acid, glacial acetic acid, phosphoric acid, nitric acid), or bases (sodium hydroxide, sodium bicarbonate, calcium carbonate, potassium hydroxide) may be used to optimize a suitable environment for a particular microorganism. Additional examples include, but are not limited to, sustained release pH adjusters (e.g., zwitterionic compounds, e.g., histidine, and buffering materials, e.g., boric acid) and sustained release nutrients. Boric acid may be added into the biofilm composition to provide a long-term acidic environment suitable for acidophilic microorganisms. In some embodiments, polyhistidine tags may be used.

[0208] In some embodiments, the microorganisms may form morphological structures within the biofilm that would not normally be expected to form if the microorganisms were not within the biofilm. The morphological structures used may improve the efficiency of improving the function of the microorganisms, for example, by increasing the electron and / or ion conductance required by the cell, by increased reaction rate or catalysis of energy-generating redox reactions at the electrodes, and / or by increased energy harvested from an external source. In some embodiments, the morphological structures formed are electronic, chemical, or ion-transmitting filaments that facilitate transfer between the electrolyte and the electrodes.

[0209] In some embodiments, the biofilm comprises a single species of microorganism. In some embodiments, the biofilm comprises two or more species of microorganism.

[0210] Microorganisms within a biofilm may take on any particular shape or topography. In some embodiments, the microorganisms grow randomly on the biofilm surface. In some embodiments, the microorganisms grow in a preferential configuration on the biofilm surface. In some embodiments, the microorganisms grow on top of each other on the biofilm surface. In some embodiments, the microorganisms grow preferentially along a common axis of the surface. In some embodiments, the microorganisms are arranged such that they associate with each other to maximize nutrient utilization for each of them.

[0211] In some embodiments, the microorganisms are suspended within the bioelectrochemical voltaic cell.

[0212] In some embodiments, methanol-producing microorganisms (e.g., "methanogens"), or methanol-consuming microorganisms, or both, may be used in methanol fuel cell embodiments. In some embodiments, a substrate may be made from pectin or added to agar, agarose, and / or polyacrylamide, and methanol-producing microorganisms may be grown to produce methanol in methane fuel cell embodiments.

[0213] Exemplary methylotrophic microorganisms include, but are not limited to, members of the phyla Alpha-, Beta-, and Gammaproteobacteria, Verrucomicrobium, Firmicutes, and Actinomycetes; and of the classes Actinomycetes, Spirochaetes, Alpha-, Beta-, Gamma-, and Deltaproteobacteria, of the phyla Firmicutes, Bacteroidetes, Chloroflexus, Acidobacteria, Nitrospira, Verrucomicrobium, Cyanobacteria, and Planctomycetes, and / or the domain Archaea.

[0214] Methanogens can be grown by electrosynthesis. In some embodiments, methanogens can be grown along or on electrically conductive nanowires or pili, by direct membrane or electrode contact with an anode or cathode, or by diffusion of extracellular electron carriers. Methanogens can be grown using photons to store energy or to photocatalyze certain metabolic reactions. In some embodiments, methanogens can synthesize photoactive cofactors that can function as transmembrane ion pumps or chromophores for photocatalytic redox reactions.

[0215] Exemplary Methanogens include the orders Methanopylidales (e.g., Methanopylus kandleri), Methanococcales (e.g., Methanococcus maripaludis), Methanobacteriales (e.g., Methanobacterium thermoautotrophicum), Methanosarcinales (e.g., Methanosarcina mazei), Methanomicrobiales (e.g., Methanospirillum hyungatei), Methanocellales (e.g., Methanocella paldicola), Methanomassiliicoccales (e.g., Methanomassiliicoccus ruminiensis), Halobacteriales (e.g., Halobacterium salinarum), Thermoplasmales (e.g., Thermoplasma vulcanium), and Archaeoglobiales (e.g., Archaeoglobus fulgidus). The methanogenic pathway may be hydrogenotrophic, methylotrophic, carboxydotrophic, or acetolytic. In some embodiments, the particular methanogen order may be an aerobic halophilic heterotroph (e.g., Halobacteriales), a thermophilic heterotroph (e.g., Thermoplasmales), or an anaerobic sulfate reducer (e.g., Archaeoglobulares). Exemplary hydrogenotrophic methanogen orders include Methanopyrales, Methanococcales, Methanobacteriales, Methanosarcinales, Methanomicrobiales, and Methanocellales. Exemplary methylotrophic methanogen orders include Methanosarcinales and Methanomassiliicoccales. Exemplary carboxydotrophic methanogen orders are Methanosarcinales. Exemplary acetolytic methanogen orders are Methanosarcinales.

[0216] FIG. 2A shows one example of a filament 265 a within a biofilm 299 a having a microbial population 260 .

[0217] FIG. 2B shows an example of pili 280, microorganisms 265b contained within a biofilm 299b on a surface 201b. The example shows an example of connectivity between microorganisms of the same type on an electrode surface in a biofilm configuration where the microorganisms are arranged in an organized manner. The pili 280 between the microorganisms may represent a connection, e.g., a physical bond, an electron sink, an electron transfer, or other material transfer. In various embodiments, the pili are conductors, may store an electric charge, may perform redox reactions, or any combination thereof. In some embodiments, the pili may provide structure to the biofilm. Other proteins may be present instead of or in addition to the pili.

[0218] 2C shows an example of a biofilm 299c that includes two microorganisms, a first microorganism 265c having pili 281 and a second microorganism 267a having filaments 282. This example shows the generally amorphous irregular structure of the microorganisms, which in some cases may be present within the biofilm 299c. Other proteins, including but not limited to pili, flagella, and fimbria, may be used in place of or in addition to pili 281 and filaments 282.

[0219] FIG. 2D shows an example of a biofilm 299d having two microorganisms, where the microorganisms are oriented such that filaments 283 and 284 preferentially connect to a particular side of microorganisms 265d and 267b. This may be used in embodiments where one microorganism produces waste that can be consumed by a second microorganism, such that the preferential orientation allows waste released from a particular side of the first microorganism to be efficiently transferred to the second microorganism for consumption. In such embodiments, each of the first microorganisms and / or each of the second microorganisms may be spaced apart from other microorganisms of the same species generally so as not to compete for the same resources. Other proteins, including but not limited to pili, flagella, and fimbria, may be used in place of or in addition to filaments 283 and 284.

[0220] Biofilms can include various types of microorganisms, some of which have one or more pili (e.g., shown in pili 284 of microorganism 269 in FIG. 2E), and some of which have flagella (e.g., shown in flagellated microorganism 270 with filament 285 in FIG. 2F). One example where pili are pilA polymers. Pili and filaments can act as electron sinks, allow for electronic conductivity, and act as physical anchoring points for binding to surfaces or nearby microorganisms.

[0221] In some cases, the filaments may provide a conductive connection between the microorganisms. Figure 2G shows two microorganisms, a first microorganism 271 and a second microorganism 272, where a filament 286 connects the first microorganism 271 and the second microorganism 272. Although three filaments are illustrated in Figure 2G, it will be understood that one or more filaments may connect the microorganisms within various regions of the microorganisms.

[0222] Biofilms allow for electron conductivity to move more efficiently across a population of microorganisms. Electron flow in a cell with microorganisms that have pili or filament connections may flow more efficiently if the microorganisms are arranged in a biofilm. An exemplary electron flow diagram is illustrated diagrammatically in FIG. 2H. In FIG. 2H, electrons flow from electron transport chain 210 to binding site 220. Excess electrons are stored in filament 240. As electrons flow from electron transport chain 210 to binding site 220, they can flow down filament 230 and be stored in filament 240. IX. Biofilm Location

[0223] The biofilm may be located at any of a variety of locations within the voltacell. The location may depend on the configuration of the voltacell itself (see, for example, the different configurations of voltacells in FIG. 1A and FIG. 1B). In general, the biofilm may be attached to any surface within the voltacell. Examples include electrodes, container walls, filters, and barriers. In some cases, the biofilm may form on or near a surface where a positive species is donated, or a surface where a positive species is received, or a surface where a negative species is donated, or a surface where a negative species is received, or any combination thereof. In some embodiments, the type of biofilm selected for use with the energy conversion cell is selected for its adhesive properties to the surface of the energy conversion cell.

[0224] One or more biofilms may be formed on the surface of the anode, such as biofilm 199a shown on anode 115 in FIG. 1A. The biofilm formed on the surface of the anode may be grown directly on the surface. The anode may be textured or treated to improve adhesion to the surface. In some embodiments, an adhesion layer is formed on the anode prior to growing the biofilm to promote attachment of the biofilm onto the anode. In some embodiments, the biofilm is formed directly on the anode without texturing, treating, or otherwise modifying the surface of the anode. The composition of the biofilm may be selected depending on the material of the surface of the anode.

[0225] One or more biofilms may be formed on the surface of the cathode, such as biofilm 199b shown on cathode 117 in FIG. 1A. The biofilm formed on the surface of the cathode may be grown directly on the surface. The cathode may be textured or treated to improve adhesion to the surface. In some embodiments, an adhesion layer is formed on the cathode prior to growing the biofilm to promote attachment of the biofilm onto the cathode. In some embodiments, the biofilm is formed directly on the cathode without texturing, treating, or otherwise modifying the surface of the cathode. The composition of the biofilm may be selected depending on the material of the surface of the cathode. In some embodiments, a biologically active biofilm on the surface of the electrode is formed by adding microorganisms to the film component. For example, microorganisms may be added directly onto a molten hydrogel, which is then poured directly onto the surface of the electrode.

[0226] One or more biofilms may be formed on the surface of the permeability barrier and exposed to the interior of the energy conversion cell facing a surface of a biofilm exposed to a microbial population (e.g., biofilm 199d on the surface of the permeability barrier 111 in FIG. 1A) or to a microbial population in another compartment (e.g., biofilm 199c on the permeability barrier 111 exposed to compartment 113 in FIG. 1A). The biofilm formed on the surface of the permeability barrier may directly contact the barrier. The permeability barrier may be textured or both treated to improve adhesion onto the barrier surface. In some embodiments, an adhesion layer is formed on the permeability barrier prior to growing the biofilm to promote attachment of the biofilm onto the permeability barrier. In some embodiments, the biofilm is formed directly on the permeability barrier without texturing, treating, or otherwise modifying the surface of the permeability barrier. The composition of the biofilm may be selected depending on the material of the surface of the permeability barrier.

[0227] One or more biofilms may be formed on a spacer used in a compartment of the energy conversion cell, for example, spacer 143 in FIG. 1B. The biofilm formed on the surface of the spacer may be grown directly on the surface. The spacer may be textured or treated to improve adhesion of the surface. In some embodiments, an adhesion layer is formed on the spacer prior to growing the biofilm to promote attachment of the biofilm onto the spacer. In some embodiments, the biofilm is formed directly on the spacer without texturing, treating, or otherwise modifying the surface of the spacer. The composition of the biofilm may be selected depending on the material of the surface of the spacer.

[0228] One or more biofilms may be formed on any other surface of the energy conversion cell, for example on one or more areas of the bottom of the energy conversion cell or on an optional cover element of the energy conversion cell, so long as the biofilm is capable of contacting one or more microbial populations, which may be grown on the bottom of the energy conversion cell or may be floating or suspended in the liquid.

[0229] One or more biofilms may be formed on a surface of the energy conversion cell that is in contact with the microbial population but not with the ionically conductive medium, for example, on the anode plate 137 of FIG. 1B facing the microbial population 145. The biofilm formed on this surface may be grown directly on the surface. The surface may be textured or both treated to improve adhesion onto the surface. In some embodiments, an adhesion layer is formed on the surface prior to growing the biofilm to promote attachment of the biofilm onto the surface. In some embodiments, the biofilm is formed directly on the surface without texturing, treating, or otherwise modifying the surface of the surface. The composition of the biofilm may be selected depending on the surface material of the surface.

[0230] One or more biofilms may form on the surface of a liquid that may contain one or more microbial populations. For example, a biofilm formed on the surface of a liquid may form when microbial populations bind or interact with each other to produce a thin matrix of microorganisms. In some embodiments, a biofilm formed on the surface of a liquid is held together by some molecular and / or intercellular bonds.

[0231] The biofilm may form over the entire surface of the solid surface of the energy conversion cell, or may form in clusters or irregular or shaped areas on the surface of the energy conversion cell. The biofilm may form thicker on one surface of the energy conversion cell, but thinner on another surface of the same energy conversion cell.

[0232] Different biofilms or biofilms with different properties may form on the same surface of the energy conversion cell and may be spaced apart from each other, separated from each other, or in contact with each other.

[0233] One consideration in placing biofilms and a property of biofilms is the possibility of electrode fouling. Electrode fouling occurs when a biofilm blocks or otherwise disables a portion of an electrode, making it less effective or not effective at the blocked portion. For example, a biofilm may block the transmission of species to and / or from the electrode surface or across an ion-permeable separator. Such species may participate in or facilitate electrochemical reactions occurring at the electrode surface. Examples of such species include cations, anions, uncharged chemical species, water molecules, and the like. In some implementations, electrode fouling is reduced or avoided by using a biofilm that receives the species. As one example, metal-reducing bacteria microorganisms can regenerate metals that have been electrochemically oxidized at the anode. And as explained below, a biofilm can be restricted to a specific area on a surface, such as the active surface of the anode or cathode or the surface of an ion-permeable voltaic cell separator. In other words, the biofilm occupies only a portion of the affected surface.

[0234] Biofilm may also be present on some areas of the electrode surface or adjacent to the electrode surface, but excluded from other areas of the electrode surface. FIGS. 3A-3D provide examples of biofilm on specific areas of the electrode surface from the perspective of the surface of the electrode where the surface in contact with the electrolyte or buffer faces the viewer, while not providing examples of other areas of the electrode surface. While the biofilm illustrated in FIGS. 3A-3D is continuous across the surface of the electrode, it will be understood that in some embodiments, the biofilm may form on various spaced apart areas of the electrode surface. FIG. 3A shows a biofilm 399a on electrode 315a, where the biofilm occupies a designated half area of ​​the electrode surface. FIG. 3B shows a biofilm 399b on electrode 315b, where the biofilm occupies a corner area of ​​the electrode surface. FIG. 3C shows a biofilm 399c on electrode 315c, where the biofilm 399c assumes a free-form shape over an area of ​​electrode 315c. 3D shows a biofilm 399d on an electrode 315d where the biofilm 399d assumes an approximately circular shape over the area of ​​the electrode 315d. It will be understood by one of skill in the art that the biofilm configuration and area of ​​occupation on the surface of an electrode is not limited to these examples and may vary depending on the organisms in the biofilm, the material of the electrode, and the general configuration of the biofilm.

[0235] As shown, the biofilm is provided or grown on a substrate within the voltaic cell. In some embodiments, the substrate is the electrode or the cell separator itself. In some embodiments, the substrate is a separate intermediate structure, which in some embodiments may be sandwiched between the electrode and the biofilm.

[0236] Porous or speckled surfaces increase the surface area and provide more binding sites for the biofilm to occupy. Materials include nanoparticles (metal, silica), porous hydrogels (agarose, agar, nitrocellulose, methylcellulose, gelatin, alginate) and mucus (polysaccharides).

[0237] 4A illustrates a cross-sectional view of one example of an electrode surface with an intermediate substrate 480 between the electrode 415 and the biofilm 499. The substrate can be used to help support and anchor the biofilm while allowing electrolyte access to the electrode or cell separator. In some embodiments, the intermediate substrate 480 can be porous such that openings in the biofilm 499 and / or intermediate substrate 480 allow electrolyte access to the electrode 415 even while the biofilm 499 is attached to the intermediate substrate 480.

[0238] The intermediate substrate may have any suitable thickness. On the substrate, the biofilm may be grown to any suitable thickness. The porous intermediate substrate may have a particular porosity.

[0239] An exemplary porous structure is shown in FIG. 4B. This view is from the angle illustrated in FIG. 4A. An intermediate substrate 480 is sandwiched between the biofilm 499 and the electrode 415. Pores 470 provide access through which the electrolyte can contact the electrode 415. In this example, the pores 470 extend through both the biofilm 499 and the electrode 415. However, it will be understood that in certain disclosed embodiments, pores may appear only on the biofilm 499, or only on the electrode 415, or on both the biofilm 499 and the electrode 415, but may not form completely overlapping and continuous pores between the electrolyte and the electrode.

[0240] If the substrate is not an electrode, the substrate can be a biocompatible polymer, ceramic, or metal.

[0241] In some embodiments, when the substrate to which the biofilm is attached is an electrode, the electrochemical role of the electrode may limit its structure and composition. However, the electrode may have certain properties that make it compatible with directly attached biofilm. For example, the electrode may have a porous structure. Exemplary electrode structures include carbon, metal, or ceramic materials with foam, woven, felt, mesh, perforated, or similar morphology. X. Horizontal electrode cell design

[0242] Some microorganisms naturally separate to the bottom of a container under the influence of gravity. In some cases, these organisms are amotile; in other words, they do not have the ability to move around under their own motive force. Non-motile microorganisms often do not contain structures that facilitate motive forces. Examples of such structures in motile organisms include pili and flagella.

[0243] To the extent that non-motile microorganisms are required at a specific location within the bioelectrochemical energy conversion cell, that location may be provided at the bottom of a horizontally oriented cell. For example, if non-motile microorganisms are used at the anode of such a cell, the anode may be substantially horizontally oriented and positioned at the bottom of the cell. In this manner, the organisms will preferentially reside near the electrodes where they facilitate the operation of the bioelectrochemical energy conversion cell. Note that the organisms may naturally be attracted by gravity to the bottom of the cell.

[0244] Another feature or potential benefit of horizontally oriented cells is that if some of the liquid electrolyte evaporates (e.g., by contact with air swept at the cathode), the electrodes, or at least the electrodes at the bottom of the cell, are not even partially exposed to air. Among other difficulties resulting from exposure of the electrodes to air is that the surface area exposed to air is not available for use in electrochemical energy conversion. In some embodiments, horizontally oriented cells may take advantage of the vertical state of the cell to expose the biofilm to air, while preventing the electrodes from being exposed to air.

[0245] Exemplary non-motile classes of microorganisms include Coccida and non-motile Bacillario. Specific examples of non-motile microorganisms include Staphylococcus, Streptococcus, Bacillus, Pseudomonas, Chlorella, Pseudomonas, and others.

[0246] FIG. 5A shows a schematic diagram of a cross section of a horizontally oriented cell 505. The energy conversion cell 505 includes a containment vessel 407 that holds a fluid, e.g., a buffer, in its interior 509. The cell 505 also includes an optional cover element 531 fitted over the vessel 507. The element 531 may be transparent to radiation in the wavelength range to which the photosynthetic microbial population responds. The cell 505 includes an anode 515 and a cathode 517 that are electronically separated from each other, in this example, by a biofilm 599. In some embodiments, the biofilm 599 serves as an electrolyte that allows direct contact between the interfaces of the microorganisms and the electrodes. In some embodiments, an ionically conductive medium may separate the anode 515 from the cathode 517. During operation, the microbial population in the biofilm 599 may generate electrons that are collected at the anode 515. These electrons flow through a load 519 in a circuit connecting the cathode 517 and the anode 515. The cell 505 may include a process controller 525, and a fluid system 521 coupled to the vessel 507, optionally with a separate port for the compartment 509. The cell 505 may also be interfaced with a controller 525 that controls the fluid system 521. The controller 525 may have one or more other functions. For example, it may receive inputs from various components of the system, such as sensors 527 and 529 located in the circuit coupling anode 515, cathode 517, fluid system 521, and / or compartment 509. The sensors 527 and 529 may monitor any one or more relevant operating parameters of the cell 505. Examples of such parameters include temperature, chemical properties (e.g., constituent concentrations and pH), optical properties (e.g., opacity), electrical properties (e.g., ionic conductivity), and the like.

[0247] The vessel 507 includes an electrode, for example, an anode 517 positioned at the bottom of the vessel 507. The anode 517 can be a continuous sheet disposed at the bottom of the vessel 507. The end of the anode 517 can be spatially separated from the counter electrode or cathode 515 by a barrier or other non-conductive medium. The counter electrode (or cathode) 515 can be disposed in a variety of locations. One location for the counter electrode is around the perimeter of the vessel 507. The surrounding counter electrode 515 can itself be oriented horizontally or vertically. So, for example, a copper cathode can be a vertical sheet of copper that lines the perimeter of the cell, which can be a disk or cylindrical shaped enclosure. In another example, the surrounding cathode includes a carbon material and a catalyst that allows for the reduction of oxygen (e.g., in air). As shown, the anode 517, which can be an aluminum-containing material, can be oriented and disposed horizontally at the bottom of the vessel 507. For example, if the vessel 507 is cylindrical in shape, such that the bottom of the vessel 507 is round, oval, or circular in shape and the walls of the vessel 507 are vertical, the cathode 515 is positioned at the bottom of the vessel 507, but can also be cylindrical with walls extending parallel to the walls of the vessel 507.

[0248] FIG. 5B is an example of a voltaic cell with a three-layer horizontal design: anode layer 565 / biofilm 599 layer / cathode layer 567. Any or all of these layers may be in the form of a gel. In some embodiments, the first layer is the anode layer 565. The anode layer 565 may include aluminum powder 560 in an ionically conductive gel. The next layer is the biofilm layer 599, which may include alginate and / or agarose. An optional third layer (not shown) includes an ionically conductive layer, such as an ionically conductive polymer, which may function to prevent the transfer of electrons and hold the microorganisms in place. The final layer is the cathode layer 567. The cathode layer 567 may be a carbon layer, a metal-containing layer, such as a copper mesh, a felt layer, or an ionically conductive gel matrix.

[0249] In some embodiments, the anode includes an electropositive material (relative to the electrochemically active material in the cathode), such as a metal. In some cases, the anode includes aluminum, optionally in powder form. The aluminum or other electropositive material may be disposed in a gel, which may be an ion-conducting polymer. In some embodiments, the aluminum or other electropositive material may be distributed evenly or as a gradient within a layer. In some embodiments, the cathode composition includes copper (optionally in mesh form) or carbon (optionally in powder form).

[0250] 5C is an example of a horizontal configuration of a "pack" form vessel 597 including a cathode layer 525, an ionically conductive polymer, gel, or liquid (not shown), a biofilm 599, and an anode layer 577 in a perspective view. In some embodiments, a liquid buffer 509 may be used. The buffer may be a liquid electrolyte in some embodiments. In some cases, a liquid electrolyte may be used without an ionically conductive medium 525. In some embodiments, the biofilm 599 has sufficient gel volume to act as a barrier between the anode 577 and the cathode 525.

[0251] Certain disclosed embodiments can be used to increase the surface area for microbial proliferation and growth in biofilms. Leads can be embedded directly into the gel medium or by physically bonding the ends to an aluminum anode.

[0252] FIG. 5D shows another side view of an exemplary voltaic cell with carbon cloth 760 separated by a layer of sterile gauze 799 over an aluminum anode 767 coated with gel media. The leads (aluminum lead 775 and carbon lead 765) are connected to the carbon cloth / aluminum anode and threaded through small openings. This allows better air access for the carbon cloth while utilizing the gel media as both a trapping mechanism to ensure microbial growth along the surface of the anode, and also provides a structural network for the bacterial species to facilitate and contribute to the voltage output, which is the desired analytical measurement of bacterial activity. Modulated parameters and features of this voltaic cell include voltage output noise reduction, signal attenuation, solution / buffer reintroduction, and bacterial incorporation.

[0253] Several exemplary horizontal cell formats are now described. 1. A horizontal design with an anode in the central region and a cathode around the periphery may include: a. the anode optionally comprises a metal, where the metal is oxidized; b. the cathode can be an air reduction electrode; and c. Biofilm on the anode, optionally with phototrophs and / or optionally with two or more sub-layers. In this embodiment, the anode and cathode may be separated physically by their structure, by a physical separator that may function as an electrolyte, or by both. 2. A horizontal design with a cathode in the center and an anode around the perimeter may include: a. the anode optionally comprises a metal, where the metal oxidizes; b. the cathode can be an air reduction electrode; and c. Biofilm on the anode, optionally with phototrophs and / or optionally with two or more sub-layers. In this embodiment, the anode and cathode can be physically separated by their structure, by a physical separator that can function as an electrolyte, or by both. Designs 1 and 2 allow the biofilm to be exposed to solar radiation because there is no cathode above it to block the solar radiation. 3. A horizontal design with a three-layer stack may include: a. Cathode on the bottom of the stack; b. a biofilm as an intermediate layer; optionally with phototrophs and / or optionally with two or more sublayers; and c. Anode above the biofilm. The anode may be at least partially transparent to solar radiation, thereby allowing the radiation to reach the phototrophic organisms. XI. Biofilm composition

[0254] The composition of a biofilm may include one or more microorganisms and a substrate, which may include water and one or more other materials. In some embodiments, the substrate includes a naturally occurring polymer. In some embodiments, the substrate includes a synthetic polymer. In various embodiments, the substrate includes any one or more materials, such as a hydrate of a nucleic acid, a protein, a carbohydrate, or any combination thereof. In a naturally occurring substrate, one or more components of the substrate may be secreted or otherwise produced by the microorganisms of the biofilm. Exemplary nucleic acid substrate materials include RNA and DNA. Exemplary protein substrate materials include PilA, fimbria, proteinases, and metabolic enzymes. Exemplary carbohydrate substrate materials include dextran and other polysaccharides. Another example is an aromatic dye molecule.

[0255] In some embodiments, the biofilm substrate is provided in the form of a hydrogel. Examples of components that can be used to form the hydrogel include pectin and alginates (e.g., sodium alginate). In some implementations, the biofilm substrate comprises approximately 5-15% pectin by weight, approximately 1-8% sodium alginate by weight, and water. In some embodiments, the biofilm substrate comprises approximately 2-7% pectin by weight, using an agar composition having approximately 2-5% agar by weight. Biofilm synthesis can also include trapping specific bacterial strains within the layer and introducing growth-promoting materials (e.g., tryptic soy agar (TSA), HCl, H3BO3, etc.) according to defined ATCC growth conditions.

[0256] The gel can be viscous, semi-solid, or solid. In various embodiments, the gel comprises a matrix with porosity that allows for ion and nutrient flow but does not allow migration of microorganisms out of the gel matrix.

[0257] The gel may include one or more additives. Examples of additives include, but are not limited to, salt (e.g., sea salt medium), DNA (e.g., salmon sperm DNA), and saline solutions, such as phosphate buffered saline (PBS). One exemplary additive is a protein source. An exemplary protein source may be a protein hydrolysate, such as a peptone.

[0258] In some embodiments, the bacterial specific composition of the gel medium used for certain disclosed embodiments may include the use of sodium alginate powder, TSA, and hydrochloric acid in the synthesis of a gel medium for acidophilic bacteria.

[0259] In some embodiments, the bacterial specific composition of the gel medium used for certain disclosed embodiments may include the use of TSA powder in the composition of the gel medium for neutrophils.

[0260] The following is an exemplary composition of the gel: Example 1. 1.25% agarose + 0.5% alginate gel. Example 2. 0.75% gelatin + 0.5% alginate + 0.15% pectin gel. Example 3. 2% agarose gel. Example 4. 0.5% agarose + 1% cellulose + 0.25% alginate gel. Example 5. 1% salmon sperm DNA + 1.25% agar gel. Example 6. 1% salmon sperm DNA + 3.5% polyacrylamide gel. Example 7. 8% hydrogel. Example 8. 5% w / v polypeptide containing an optimal number of zwitterionic amino acids, eg, histidine, in 1x phosphate buffered saline (PBS). Example 9. 5% w / v salmon sperm DNA in 1.25x PBS Example 10. 5% sea salt medium in water Example 11. 50 mM sodium chloride (NaCl) in Roswell Park Memorial Institute (RPMI) medium Example 12. 50 mM NaCl in Dulbecco's Modified Eagle Medium (DMEM) Example 13. Brain Heart Infusion (BHI) medium Example 14. Lysogeny (LB) medium Example 15. 0.2 g ammonium sulfate ((NH4)2SO4), 0.5 g manganese(II) sulfate (MgSO4), 0.25 g calcium chloride dihydrate (CaCl2·2H2O), 3.0 g potassium phosphate (KH2PO4), and 2.0 g yeast extract in 1.0 L distilled water is adjusted to a final pH of 0.7 using sulfuric acid (H2SO4) and autoclaved at 121 °C for 15 minutes. Example 16. Prepare 1.5 g ammonium chloride (NH4Cl), 0.6 g sodium phosphate (NaH2PO4), 0.1 g potassium chloride (KCl), 2.5 g sodium bicarbonate (NaHCO3), 0.82 g sodium acetate, 10.0 mL Wolfe's vitamins, 10.0 mL Wolfe's metals in 976 mL distilled water with 4 mL 0.025% resazurin, dispense into fumarate-free medium anaerobically under 80% nitrogen (N2) and 20% carbon dioxide (CO2) and autoclave for 15 min at 121 °C in two tubes of 2 mL each of 1 M (16 g / 100 mL) sodium fumarate.

[0261] In some embodiments, the individual biofilm layers are thin, and the film is introduced in a step-by-step layering manner by adding bacterial species, media, and other growth factors to obtain a suitable structure.

[0262] In some embodiments, the components of the matrix are provided in cross-linked form. The cross-links can be in the form of covalent bonds, electrostatic bonds, van der Waals bonds, hydrogen bonds, or any combination thereof.

[0263] Typically, a biofilm as used herein includes some amount of water. Water may be produced by microorganisms within the biofilm, added during construction of the voltacell, and / or incorporated from electrolytes or buffers present in the voltacell. In some embodiments, water confers mechanical, biological, and / or electrical properties to the biofilm. For example, ionically conductive water may facilitate ionic transfer between the electrolyte and the electrode on which the biofilm resides. In some cases, water confers adhesion, mechanical strength, or other physical properties to the biofilm substrate material. In some embodiments, water is present in the biofilm at a concentration of approximately 60-90% by weight. A. Multilayered Biofilm

[0264] In certain embodiments, the voltacell comprises a multi-layered biofilm having different layers containing different microorganisms. In some cases, the microorganisms in the different layers are complementary to one another. Complementary microorganisms are described elsewhere herein.

[0265] In some embodiments, the biofilm is provided as a laminate or other multi-layer structure. In some cases, the biofilm has two different sub-layers, one configured to incorporate one type of microorganism and another sub-layer configured to incorporate a different type of microorganism. Such an embodiment may be suitable when the two different microorganisms have complementary properties, but are not suitable when they are intimately mixed or in direct contact with each other. 1. Structure of multilayer biofilms

[0266] The physical, chemical, and biological properties of the individual layers may vary from layer to layer.

[0267] To minimize the resulting resistance from the biofilm itself, the substrate can have a much thinner thickness than normal, e.g., less than about 0.5 cm. Furthermore, because of this functional aspect of the fuel cell, the sodium alginate biofilm substrate preparation maintains flexibility and structure over an extended period of time, e.g., about 3 to about 4 weeks. In some embodiments, the addition of DMEM+CaCl2 to a 2% sodium alginate / pectin (1:1) solution over a 30 minute period results in crosslinking.

[0268] Compositions vary primarily in additional growth factors for strain-specific microbial growth. Some examples include, but are not limited to, the addition of 10 mL of 0.1 M HCl and DMEM for certain microorganisms, and the addition of Van Neel's yeast agar for Rhodospirillum rubrum microorganisms.

[0269] Multiple biofilms may be in direct physical contact over large horizontal surface areas. The stacking / organization of the biofilm depends on the relationship of the multiple organisms used for the cell. One example includes a photosynthetic bacterial biofilm on top of a conductive filamentous biofilm.

[0270] In some embodiments, one of the microorganisms is a photosynthetic organism and the other is not. In such cases, the photosynthetic organism may be located closer to the light source. In some implementations, this means that the photosynthetic microorganism should be in the upper layers of the multi-layer biofilm stack.

[0271] The thickness and / or composition of each layer of a biofilm may vary from layer to layer. 2. Porosity of multilayered biofilms

[0272] In some cases, the sublayers are manufactured separately and different microorganisms are incorporated into the layers separately. In other embodiments, one sublayer may have a different pore size range than the pore size range in another sublayer. In some cases, one sublayer has a pore size that accommodates the microorganisms themselves, while another sublayer has a pore size that accommodates the microbial vesicles, i.e., the other sublayer has a smaller pore size than the first sublayer. The different pore sizes may aid in ion flow and stability.

[0273] Microorganisms within a biofilm may be located or distributed in any of a variety of regions of the biofilm. Examples of such regions include portions of a two-dimensional surface of a substrate on which a biofilm resides, embedded within a matrix within a biofilm, sandwiched between two or more biofilms, or suspended in a liquid between successive regions of the biofilm. In some embodiments, a biofilm comprises one or more layers. In some embodiments, microorganisms are preferentially located within a subset of layers. For example, microorganisms may be preferentially located within one of one or more layers. In some embodiments, microorganisms are located in two or more layers. For example, FIG. 5E shows an example of one microbial layer 570 within a biofilm 599. While one layer is shown, it will be understood that there may also be two or more layers. In some embodiments, the layers vary in number or size depending on the location across the entire surface of the biofilm.

[0274] The microorganisms in the biofilm are 3 It is characterized by its density, measured in microorganisms per cubic metre. 3. Method for manufacturing multi-layer stacks

[0275] In some implementations, the individual layers are fabricated separately and then assembled. In some implementations, the first layer, e.g., the layer closest to the vessel wall or electrode, is fabricated in place. The second and subsequent layers are then fabricated on the first layer. In certain embodiments, one or more layers of the multi-layer biofilm are fabricated using a microbial enclosure, as described elsewhere herein.

[0276] Each layer is manufactured individually in situ. In some embodiments, post-setting stacking may not be used due to structural concerns. Once the matrix gel is synthesized, a molten solution is poured directly onto the previous layer at specific process conditions. For example, the solution is an acidic solution. In some embodiments, the solution is an alkaline solution. Bacteria layer capture occurs during the cooling phase of the gel prior to casting. Sonication may be used to aid in complete dispersion of added material components. Sonication is not used if bacterial strains are present. B. Biofilm Microbial Composition

[0277] Biofilms can contain various types of microorganisms. Exemplary classes of microorganisms include anaerobic, aerobic, and facultative anaerobic microorganisms.

[0278] Biofilms can contain combinations of complementary microorganisms. Two microorganisms can be complementary if the combination of the two microorganisms has certain complementary properties. For example, one example of a complementary property is a sulfur-oxidizing microorganism and a sulfur-reducing microorganism. 1. Types of microbial complementation

[0279] In some cases, the complementation is based on metabolic pathways, such that a metabolic pathway of one microorganism produces a product that is consumed by a different microorganism. In some cases, the complementation is based on one microorganism producing and maintaining an environment that is beneficial to a different microorganism. The beneficial environment can be a particular range of pH values ​​or other conditions described herein. In some cases, multiple microorganisms produce an environment in which multiple organisms can grow.

[0280] In certain embodiments, the microorganisms in at least one of the layers generate electrons or are otherwise actively involved in the bioelectrochemical energy conversion process.

[0281] Complementary microorganisms that can advantageously utilize biofilm structures are described above in connection with Section VIII. XII. Biofilm characteristics 1. Selection of Microorganisms

[0282] The choice of biofilm type may depend on the bioelectrochemical cell and its purpose. Other factors that may determine the choice of microorganisms used in the biofilm include, but are not limited to, the microbial growth conditions, the surface on which the biofilm grows, the configuration of the energy conversion cell, and the metabolic pathways for each microorganism in the bioelectrochemical cell. 2.Electrical characteristics

[0283] Biofilms can exhibit a variety of electrical properties. In some embodiments, biofilms have a net charge on the bound surfaces. Charged biofilms can facilitate the attraction and / or transport of oppositely charged mobile species, e.g., ions in electrolytes. In some embodiments, the substrate of the biofilm comprises a charged component. In some embodiments, naturally occurring charged substrate proteins, or other polymers, have a net positive charge by virtue of positively charged monomers. Exemplary charged polymers include lysine-rich, histidine-rich, and / or arginine-rich proteins or peptides. Examples of negatively charged substrate components include negatively charged monomers, e.g., aspartic acid and / or glutamic acid-rich proteins or other polymers. 3. Mechanical properties

[0284] Biofilms have mechanical properties, some of which may affect their role in biochemical voltaic cells. For example, in some embodiments, biofilms in bioelectrochemical energy conversion cells may strongly adhere to the substrate surface. Adhesion may be measured by standard tests for coating adhesion, such as the scratch test.

[0285] The dimensions and other physical characteristics of the biofilm may also depend on the microorganism, the substrate, and other components of the biofilm. The thickness of each layer of the biofilm layer in the multi-layer structure may be at least approximately 0.5 cm thick. In some embodiments, the biofilm has a specific surface roughness, where surface roughness refers to the roughness measured on the exposed surface in contact with the electrolyte. In some embodiments, the thickness of the biofilm is approximately 10 to 300 micrometers. 4.Porous

[0286] Biofilms may also have a particular topography and / or porosity. Porous biofilms may have pores or openings of a particular maximum cross-sectional dimension. In certain embodiments, the pores in the biofilm matrix have an average or median pore size of approximately 10 nm to 10 μm. Functionally, the pore size of the biofilm may match the size or size range of the microorganisms incorporated within the biofilm. In some embodiments, the pore size matches the size of the vesicles produced by the microorganisms within the biofilm. In certain embodiments, the pore size for accommodating the microorganisms may be approximately 1 to 100 micrometers on average. In certain embodiments, the pore size for accommodating the microbial vesicles may be approximately 10 to 100 nanometers on average. XIII. How to Create Biofilms

[0287] Various methods can be used to create biofilms. Biofilms can be produced on or using gel-like substances. Gel-like substances include alginate, agar, agarose, pectin, gelatin, and Sephadex. In one example, biofilms are formed by producing hydrogels using hydrogel-forming molecules. For example, algae cultures and sodium alginate can be homogenized and mixed in a wet state, then filtered and collected to form an alginate hydrogel. The hydrogel can be rinsed and filtered to form a biofilm pad. In some embodiments, the biofilm pads have microorganisms trapped within them. In some embodiments, the biofilm pads form a structure on which microorganisms can grow. Alternatives to alginate include, but are not limited to, gelatin and pectin. In some embodiments, a mixture of alginate and pectin can be used. For example, a biofilm pad can be formed having between about 3% and about 15% pectin, and about 3% to about 7% sodium alginate. In some embodiments, the pad can be coated onto an aluminum anode. In some embodiments, the homogenized liquid mixture can be used for deposition onto a separate mold to form a transferable biofilm pad. The biofilm can be applied directly onto the surface of the bioelectrochemical voltacell, either in layers next to each other, or on top of each other, or a combination thereof. In some embodiments, the microorganisms can be applied to the biofilm pad using an aerosol suspension, for example by powder coating.

[0288] 6 shows a process flow 601 including multiple steps that may be used to create a biofilm for use in a voltaic cell. The illustrated process begins with operation 603, where one or more precursor or component materials are provided for a biofilm substrate. In some embodiments, the precursor is a polymer and / or gel-forming material. In some embodiments, the precursor has properties that allow it to form a porous substrate.

[0289] In some embodiments, the process modifies the chemical or physicochemical properties of one or more precursors to form a biofilm matrix. In FIG. 6, this is illustrated by optional operation 605. In some embodiments, the modification includes reacting one or more polymer precursors to form a crosslinked matrix. The crosslinking can be via electrostatic forces, e.g., ionic or van der Waals forces, or it can be via covalent bonds. In some embodiments, operation 605 includes changing a morphological property, e.g., porosity or surface roughness. In some embodiments, operation 605 includes changing a physical property, e.g., wettability and / or adhesion. In some embodiments, operation 605 includes changing a biological property, e.g., compatibility (or lack of compatibility) with one or more types of microorganisms. As one example, microbial nutrients can be incorporated into the matrix. The modification can include applying a physical effect, e.g., heat, pressure (or vacuum), or radiation (e.g., UV radiation). In one embodiment, the modification includes first heating the precursor to a temperature of approximately 80-110° C. and then cooling the composition to a temperature of approximately 20-50° C. Such heating and cooling operations may serve to crosslink certain precursors, e.g., hydrogel precursors. Note that in some implementations, operation 605 is not performed, e.g., because the precursor is provided in a form that does not require modification to perform the role of a biofilm.

[0290] After the biofilm substrate component is provided and optionally modified, process 601 optionally forms the substrate into a biofilm shape that can be used in a voltacell. See operation 607. This operation is optional since in certain embodiments the substrate is formed directly onto the voltacell component without first forming the biofilm substrate component into a suitable shape. When operation 607 is performed, it may include forming the biofilm substrate via melt processing, solution processing, or solid state processing. In some embodiments, the biofilm shape is formed by molding, spraying the biofilm onto a substrate, pouring a molten or solubilized biofilm substrate onto a substrate, extruding the biofilm into a sheet or other shape, compressing a solid biofilm substrate, and the like.

[0291] Process 601 applies the biofilm substrate to a structural component to be used in a voltaic cell. See operation 609. Examples of such structural components are described elsewhere herein. Electrodes and electrolyte separators are examples of structural components that may be used. Applying the biofilm substrate to the structural component may include adhering the biofilm substrate to a surface of the component. Adhering may be accomplished, for example, by applying an adhesive to the biofilm and / or the component surface, contacting the component surface with a melted or solubilized biofilm substrate, etc. In some embodiments, a preformed biofilm substrate (e.g., a substrate resulting from optional forming operation 607) is first aligned with the structural component and then adhered.

[0292] In some embodiments, operations 607 and / or 609 are performed to create a laminate or other multi-layer structure having two or more biofilm matrix sub-layers. As described elsewhere, such sub-layers may have different morphologies, chemical compositions, biological compositions, harbor different types of microorganisms, etc.

[0293] In process 601, the final illustrated operation is to mount or otherwise provide the component with its biofilm matrix in a voltace cell, where during normal operation, the microorganisms in the biofilm facilitate electrochemical energy conversion to produce electricity, see operation 611. If the component is an electrode or electrolyte separator, the electrode or separator, along with its biofilm matrix, is mounted within a container that defines the boundaries of the voltace cell.

[0294] The illustrated process 601 does not show the process of incorporating microorganisms into a biofilm matrix. Generally, microorganisms can be incorporated at any point in the process as long as subsequent operations in the process do not kill or substantially harm the microorganisms.

[0295] In some embodiments, one or more microorganisms are provided to the precursor in operation 603. In some embodiments, one or more microorganisms are provided during substrate modification operation 605. For example, the polymer mixture may be spiked with the microorganisms while cooling from the heating or radiation induced crosslinking operation. In some embodiments, one or more microorganisms are provided during biofilm substrate formation operation 607. In some embodiments, one or more microorganisms are incorporated into the substrate when the substrate is applied to the component in operation 609. In some embodiments, one or more microorganisms are provided to the substrate during or after the component is mounted in the voltaic cell in operation 611. As one example, after the substrate-containing component is mounted in the voltaic cell, the microorganisms are incorporated into the substrate by contacting the substrate with a buffer or other medium containing the microorganisms. Contacting may be accomplished by flowing the medium over the component, spraying the medium over the component, or the like.

[0296] In some embodiments, the microorganisms are applied in more than one phase, for example, some microorganisms may be applied during operation 605 and some other microorganisms may be applied during operation 611.

[0297] Biofilms can include materials that increase the surface area that microorganisms contact with the surface of a bioelectrochemical voltaic cell. For example, the cell can utilize carbon paint on certain surfaces to increase the surface area on which microorganisms can grow biofilms. Carbon paint is a relatively low resistance material, but allows a large surface area for biofilms to grow on. Biofilms can be grown on surfaces that have low resistance compared to the internal resistance of the battery.

[0298] An example is provided in Figure 7. Figure 7 shows a structure in which an aluminum-based paint and a conductive carbon paint are mixed to form an anode in which the carbon paint contains aluminum particles. In this example, the carbon paint and aluminum composite serves as the anode, and filter paper separates it from the copper cathode. Although copper is shown in this example, it will be understood that other materials and metals can be used for the cathode material. As shown, the fungus or microorganisms may be in contact with the cathode and / or the carbon / aluminum composite material, and in some embodiments, may be between the carbon / aluminum composite and the filter paper separator, or may be between the cathode and the filter paper separator. In some embodiments, the microorganisms may be in solution before, during, or after biofilm formation.

[0299] FIG. 8 shows another example where carbon paint is used, but instead of mixing it with an aluminum-based paint as in FIG. 7, in this example the carbon paint is coated on both sides of an aluminum sheet and separated from the copper cathode by a filter paper separator as in FIG. 7. It will be understood that although both sides of the aluminum sheet are coated with carbon paint, in some instances only one side of the aluminum sheet is coated, e.g., the side exposed to the microorganisms. Without being bound to a particular theory, it is believed that a cell such as the one illustrated in FIG. 8 can be utilized for lower current density embodiments. Both the embodiment and its variations may be suitable for forming biofilms thereon and for scaling to larger industrial size tools for generating stable power.

[0300] FIG. 9 shows another example in which a horizontally oriented voltaic cell is illustrated, including a window layer (e.g., glass) exposed to sunlight or artificial light. Beneath the window layer are an anode agar layer; an anode layer, which may be aluminum nanoparticles or microparticles, transparent conductor particles, hydrophilic polymers or gels; a separator or electrolyte layer; a cathode agar layer; a cathode gas diffusion layer (GDL); and cathode airflow hardware. The layers may be rearranged, and the materials for each layer may vary depending on the particular application and microorganisms used. The microorganisms may be present in one or more of the anode agar layer, the anode layer, the separator, and the cathode agar layer. XIV. Methods for maintaining biofilms.

[0301] Various methods can be used to maintain the biofilm during normal operation of the bioelectrochemical energy conversion cell and ensure a working supply of the non-microbial building blocks of the biofilm.

[0302] In various embodiments, biofilms formed on electrodes, such as those described in Figures 7 and 8 and variations thereof, can be utilized after rehydrating the surface. In various embodiments, the bioelectrochemical voltacells described herein are free-standing.

[0303] In some embodiments, certain acidophilic energy-producing microorganisms can be used as biofilms: the microorganisms alkalize their environment and cease to be metabolically active, so adding acid to the system rejuvenates the microorganisms and maintains their function.

[0304] Figure 10 shows the results from an experiment measuring the electrical energy of acidophilic energy-producing microorganisms over time. As shown, when acid was reintroduced, a spike in the current was seen, indicating that the maintenance of acidophilic energy-producing microorganisms can be activated by maintaining the pH in the cell, which can be done by introducing acid or by utilizing a buffer suitable for the microorganisms. XV. Features of containers for voltaic cells

[0305] In one embodiment, the key function of the voltaic cell is to harvest photons and utilize excited electrons contained within the cell to generate an electric current using a population of photosynthetic microorganisms and photosynthetic microbial membranes. The cell may include a leak-proof container or housing for the microbial energy conversion cell medium and the microbial population. In some embodiments, the microbial energy conversion cell further includes electrodes, sensors, semi-permeable barriers, ionically conductive materials, wires, and the like.

[0306] Typically, cells utilizing photosynthetic microorganisms should be designed to accept external radiation and convert the energy therein into excited electrons in the light-harvesting antennae of the microbial membrane, and to provide conductive materials for utilization of the resulting high-energy electrons generated by the electron transport chain within each membrane of the microorganism.

[0307] The microbial energy conversion cells of the disclosed embodiments may have full access to the environment and may be constructed to allow photon conversion in temperatures ranging from -20° C. to 65° C. and weather ranging from completely sunny to cloudy or foggy. The microbial energy conversion cells of the disclosed embodiments may also be portable and have variable access to the environment as determined by the user.

[0308] In certain embodiments, the container can withstand high temperatures (e.g., above about 50° C.) and internal pressures (above atmospheric pressure) of about 50 Pa to about 10 kPa; about 500 Pa to about 3 kPa; about 800 Pa to about 1.5 kPa. It is noted that some embodiments employ microorganisms whose natural habitat is a high pressure environment, e.g., deep sea vents.

[0309] In some embodiments, the cell is a closed system with no flow of fresh buffer or other solutions into the system and no exposure to atmospheric gas exchange. In other embodiments, it is a semi-closed system, including, for example, a system of tubing, valves, and ports, allowing the entry of fresh buffer, control elements, fresh microbial antenna populations, and / or atmospheric gases into the system. The ports include 0.22 μm filters to prevent contamination of the system with airborne microbial contaminants. In other aspects, the ports include 0.45 μm filters to prevent contamination of the system with larger airborne microbial contaminants.

[0310] In yet other embodiments, the cell is an open system with full access to the environment. In some cases, the open system is a body of water such as a pond, lake, river, reservoir, stream, or other open body of water. The open system may also include a system of tubing, valves, and ports to allow for the circulation of the inherent, unused microbial antenna population into the open system microbial energy conversion cell.

[0311] A submersible open system may have an anode and a cathode, and a semi-permeable barrier that allows ionic conduction but blocks microbial transmission. The barrier may be an antimicrobial coating (e.g., silver). There may be conductive electrical leads from the anode and cathode. The system may include components that are part of a circuit, part of a mechanical support structure, or both.

[0312] The container adjacent to the voltacell may be constructed from any of a number of materials, including, by way of example, a polymer, such as polyethylene, polypropylene, or polyurethane, glass, metal, or a combination thereof, In various embodiments, the container material is a gas and liquid impermeable material.

[0313] The container may include a multi-layer unit including an outermost layer and one or more inner layers. The outer layer may include clear plastic, glass, metal, or other materials to provide protection against the environment. In some embodiments, the container has an outermost layer that allows various spectral wavelengths of electromagnetic radiation to pass through. In some embodiments, the outermost layer may be transparent to most spectral wavelengths of light energy. In some embodiments, a portion of the container may include an outermost layer that may be impermeable to most spectral wavelengths of light energy, and a second portion of the container that includes an outermost layer that may be transparent to most spectral wavelengths of light energy.

[0314] In some embodiments, the container that defines the outer boundary of the microbial energy conversion cell is rigid. The rigid enclosure may comprise glass or polymer with a stiffness of greater than approximately 1.3 GPa and has a shape resembling a cube, cuboid, sphere, cylinder, cone, frustum, pyramid, or prism. The thickness of the walls of the enclosure may range from approximately 1 mm to 20 cm. Enclosures with wall thicknesses ranging from approximately 5 mm to 25 mm are preferred.

[0315] The volume, shape, and dimensions of the vessel may be selected to complement the overall structure of the energy conversion system in which it resides. In some embodiments, the volume of the vessel is approximately 0.0000001 m 3 ~Approximately 3m 3 ;approximately 0.000001m 3 ~ approx. 2m 3 ;Approximately 0.0001m 3 ~ approx. 1.5m 3 ; approx. 0.01m 3 ~Approximately 1m 3 or approximately 0.1m 3 ~ approx. 0.5m 3 The range may be:

[0316] The containers may be manufactured by standard methods including part molding, injection molding, extrusion, laser etching, gluing, soldering, caulking, and other suitable techniques.

[0317] In some embodiments, the container that defines the outer boundary of the microbial energy conversion cell is a frame that has electrical insulating properties. In some aspects of the present disclosure, the framed enclosure has thermal insulating properties and is filled with a foam. The frame of the disclosed embodiments includes fiberglass, aluminum, stainless steel, graphite, polycarbonate, carbon fiber, polystyrene, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polychlorotrifluoroethylene, polyethylene terephthalate, meta-aramid polymer, or copolyamide.

[0318] In other embodiments, the enclosure defining the outer boundary of the microbial energy conversion cell is flexible. Examples of flexible enclosures include one or more transparent polymers having a stiffness of less than about 1.2 GPa and having an amorphous shape or a shape resembling a cube, a rectangular parallelepiped, a sphere, a pillar, a cylinder, a cone, a frustum, a pyramid, or a prism. Examples of suitable polymers include polypropylene, polystyrene, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polychlorotrifluoroethylene, polyethylene terephthalate, a meta-aramid polymer, or a copolyamide. The thickness of the enclosure wall can range, for example, from about 0.5 mm to 25 mm. In some embodiments, the enclosure has a wall thickness in the range of about 1 mm to 10 mm.

[0319] In some embodiments, a window is included within the microbial energy conversion cell for photon energy entry into the energy conversion cell. The window can be transparent to light in the range between approximately 100 nm and 1060 nm and can include glass, crystalline composites and polymers, such as poly(3,4-ethylenedioxythiophene), poly(3,4-ethylenedioxythiophene), poly(styrenesulfonate), poly(4,4-dioctylcyclopentadithiophene) or other transparent polymers. In certain embodiments, the window can be approximately 1 mm to 30 cm thick. In some cases, the window ranges from approximately 5 mm to 25 mm thick.

[0320] In some embodiments, gaskets or seals may be included within the microbial energy conversion cell and used to provide leak-proof seals between the cell's frame and windows, and between the cell's enclosure and ports or tubing. Suitable gaskets or seals may include UV resistant silicone, cure-in-place resin, ethylene-propylene diene, closed cell nitrile, or other UV resistant gaskets or sealants.

[0321] In one example, the containment chamber includes a glass panel juxtaposed to a UV resistant gasket that is fitted onto a continuous injection molded polymer sidewall and backing unit having inlets and / or outlets and / or 0.22 μm filter gas exchange ports for fluids and fitted electronic flow conduit plates that are connected to electrical wiring for centralized flow of DC to the AC converters of the solar panels.

[0322] In another example, the vessel shape is a hollow polymer tube. In some embodiments, the vessel is shaped as a cylinder, a rectangle, a square, a sphere, a cylindrical object, or a planar object. In some embodiments, the vessel is designed as a fermenter, growth chamber, or other cell culture device.

[0323] In certain embodiments, the cell system includes a housing frame, a photoconversion system adapter, an AC adapter, and an electrical cord. In some embodiments, the system can accommodate an array of photoconversion systems. In other embodiments, the solar panel can be manufactured in such a manner that the housing frame can allow for removal and replacement of the photoconversion system. Cells as disclosed herein can serve a functional role and can be used in solar panels to provide current to a dedicated external electrical load (e.g., the grid), while other aspects of the present disclosure use portable photovoltaic cells to provide current to devices.

[0324] In some embodiments, the cell housing is a rigid system and serves a structural role in addition to its radiant energy receiving role.

[0325] In certain embodiments, the volta cells can be used in structural and functional roles, such as in automobiles and aircraft as hoods, roofs, sunroofs, moonroofs, trunks, frames, wings, windows, etc. Additionally, the cells can be used in buildings as walls, wall curtains, roofs, windows, doors, walkways, patios, driveways, decks, fences, etc.

[0326] In other embodiments, the cell housing is a flexible system that may serve a physical role in addition to the energy conversion role. Examples of uses for flexible microbial energy conversion cells are: retractable elements such as awnings, sails, covers, tarps, cloaks, capes; and foldable elements such as blankets, visors, umbrellas, parasols, fans, and clothing.

[0327] The cell may also include an electro-siphon, examples of which are discussed in U.S. Patent No. 10,090,113, issued October 2, 2018, which is incorporated by reference in its entirety for the purposes of providing examples of electro-siphons. XVI. Conclusion

[0328] Although the above embodiments have been described in some detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be implemented within the scope of the appended claims. It should be noted that there are many alternative ways to implement the process, system, and apparatus of the present embodiments. Thus, the present embodiments should be considered as illustrative rather than restrictive, and the embodiments are not limited to the details given herein.

Claims

1. (a) an anode for receiving electrons and providing the electrons to an external circuit or load; (b) a cathode for donating electrons to the electrochemical reaction; (c) a biofilm having microorganisms, said biofilm being in electrical contact with said anode or cathode; (d) a buffer having an ionically conductive medium in contact with the anode and the cathode; and (e) a container for at least partially containing the biofilm and the buffer. A voltaic cell comprising:

2. 10. The voltaic cell of claim 1 further comprising an ion-permeable, electron-donor-impermeable barrier separating the buffer into an anode compartment and a cathode compartment, thereby preventing an electron donor population from contacting the cathode.

3. The voltaic cell of claim 1 , wherein the biofilm is in contact with at least one of the anode and the cathode.

4. The voltaic cell of claim 2 , wherein the biofilm is in contact with at least one of the anode, the cathode, and the ion-permeable, electron donor-impermeable barrier.

5. The voltaic cell of claim 1 , wherein the biofilm comprises two or more microorganisms.

6. The voltacillus of claim 1 , wherein the biofilm is formed on a substrate within the voltacillus.

7. The voltaic cell of claim 6 , wherein the substrate is either the anode or the cathode.

8. The voltaic cell of claim 6 , wherein the substrate is in contact with a surface of the anode or the cathode.

9. The voltaic cell of claim 1 , wherein the biofilm has positively charged moieties.

10. The voltaic cell of claim 1 , wherein the biofilm comprises negatively charged moieties.

11. The voltaic cell of claim 1 , wherein the biofilm has a synthetic portion.

12. The voltaic cell of claim 1 , wherein the biofilm has a non-synthetic portion.

13. The voltaic cell of claim 1 , wherein the biofilm has one or more filamentous appendages.

14. The voltaic cell of claim 1 , wherein the biofilm comprises one or more microbial classes selected from the group consisting of anaerobic, aerobic, and facultative anaerobic microorganisms.

15. The voltaic cell of claim 1 , wherein the biofilm comprises sulfur-oxidizing and sulfur-reducing microorganisms.

16. 2. The voltacell of claim 1, wherein the biofilm comprises one or more microorganisms selected from the group consisting of Rhodoferax ferrireducens, Lactobacillus acidophilus, Rhodospirillum rubrum, Desulfovibrio desulfuricans subsp. desulfuricans, Peptostreptococcus anaerobius, Rhodospirillum centenum, Catonella morbi, Lachnospiraceae species, Photobacterium leiognatii, Allochromatium vinosum, Lactobacillus casei, Fusobacterium nucleatum subsp. polymorpham, Hercococcus kunzii, Cutibacterium acnes, Rhodospirillum rubrum, Hercococcus kunzii, Allochromatium vinosum, and Ferrovum mixofaciens.

17. The voltaic cell of claim 1 , wherein the biofilm has a substrate comprising a natural polymer, a synthetic polymer, a DNA hydrate, a protein hydrate, or a carbohydrate hydrate.

18. The voltaic cell of claim 2 , wherein the ion-permeable, electron donor-impermeable barrier is electronically conductive.

19. The voltaic cell of claim 2 , wherein the ion-permeable, electron donor-impermeable barrier contacts the anode.

20. The voltaic cell of claim 1 further comprising a current collector in electrical communication with the anode.

21. 2. The voltacell of claim 1, wherein the ionically conductive medium comprises a first species of microorganism and a second species of microorganism, and the first species of microorganism and / or the second species of microorganism have a light-harvesting antenna.

22. 22. The voltaic cell of claim 21 , wherein the first species of microorganisms is excited by electromagnetic radiation in a first band and at least one other species of microorganisms in the buffer is excited by electromagnetic radiation in a second band, and the first band and the second band do not substantially overlap.

23. 22. The voltacillus cell of claim 21, wherein the first species of microorganisms comprises phototrophic or chemotrophic microorganisms.

24. 22. The voltacillus of claim 21, wherein the first species of microorganism is a chemotroph and the second species of microorganism is a phototroph.

25. 2. The voltac cell of claim 1, wherein the ionically conductive medium comprises a first species of microorganism having a first primary metabolic pathway and a second species of microorganism having a second primary metabolic pathway, the first primary metabolic pathway oxidizing compounds containing carbon, nitrogen, phosphorus, or sulfur to form oxidized compounds, and the second primary metabolic pathway reducing the oxidized compounds produced by the first primary metabolic pathway.

26. 2. The voltacell of claim 1, wherein the ionically conductive medium comprises a first species of microorganism, the first species of microorganism having a fimbrial, fibrous, flagellar, and / or filamentous shape.

27. The voltacell of claim 1 , wherein the ionically conductive medium comprises a first species of microorganism, the first species of microorganism having a plurality of metabolic pathways.

28. 10. The voltacell of claim 1, wherein the ionically conductive medium comprises a first species of microorganism, the first species of microorganism being a naturally occurring microbial species.

29. 29. The voltacell of any one of claims 1 to 28, wherein the ionically conductive medium comprises a first species of microorganism having a first major metabolic pathway and a second species of microorganism having a second major metabolic pathway, each of the first and second major metabolic pathways involved in cellular respiration.

30. (a) Cathode airflow hardware; (b) a cathode gas diffusion layer; (c) cathode agar layer; (d) an electrolyte layer having an ionically conductive medium in contact with the anode and the cathode; (e) an anode layer for receiving electrons and providing the electrons to an external circuit or load; (f) anode agar layer; (g) a window layer; and (h) Biofilms with Microorganisms A voltaic cell comprising:

31. 31. The voltaic cell of claim 30, wherein the microorganisms are present in one or more of the cathode gas diffusion layer, cathode agar layer, electrolyte layer, anode layer, anode agar layer, and window layer.

32. 31. The voltaic cell of claim 30, wherein the anode layer comprises a material selected from the group consisting of aluminum nanoparticles, aluminum microparticles, transparent conductor particles, hydrophilic polymers, and hydrophilic gels.

33. 33. The voltaic cell of any one of claims 30 to 32, wherein the window layer comprises glass.

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